PI3K/AKT/mTOR signalling inhibitor chrysophanol ameliorates neurobehavioural and neurochemical defects in propionic acid-induced experimental model of autism in adult rats

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder marked by social and communication deficits as well as repetitive behaviour. Several studies have found that overactivation of the PI3K/AKT/mTOR signalling pathways during brain development plays a significant role in autism pathogenesis. Overexpression of the PI3K/AKT/mTOR signalling pathway causes neurological disorders by increasing cell death, neuroinflammation, and oxidative stress. Chrysophanol, also known as chrysophanic acid, is a naturally occurring chemical obtained from the plant Rheum palmatum. This study aimed to examine the neuroprotective effect of CPH on neurobehavioral, molecular, neurochemical, and gross pathological alterations in ICV-PPA induced experimental model of autism in adult rats. The effects of ICV-PPA on PI3K/AKT/mTOR downregulation in the brain were studied in autism-like rats. Furthermore, we investigated how CPH affected myelin basic protein (MBP) levels in rat brain homogenate and apoptotic biomarkers such as caspase-3, Bax, and Bcl-2 levels in rat brain homogenate and blood plasma samples. Rats were tested for behavioural abnormalities such as neuromuscular dysfunction using an actophotometer, motor coordination using a beam crossing task (BCT), depressive behaviour using a forced swim test (FST), cognitive deficiency, and memory consolidation using a Morris water maze (MWM) task. In PPA-treated rats, prolonged oral CPH administration from day 12 to day 44 of the experimental schedule reduces autistic-like symptoms. Furthermore, in rat brain homogenates, blood plasma, and CSF samples, cellular, molecular, and cell death markers, neuroinflammatory cytokines, neurotransmitter levels, and oxidative stress indicators were investigated. The recent findings imply that CPH also restores abnormal neurochemical levels and may prevent autism-like gross pathological alterations, such as demyelination volume, in the rat brain.


Introduction
Autism spectrum disorder (ASD) is a severe neurodevelopmental illness characterised by stereotyped or repetitive behaviour, memory and cognitive dysfunctions associated with sensory and motor function impairments (Gvozdjáková et al. 2014;Neumeyer et al. 2019). Early childhood ASD symptoms appear shortly after birth, resulting in lifelong disabilities (Jin et al. 2015). Neurochemical alterations trigger Autism-like repetitive and stereotypical behaviours in specific brain regions, including the hippocampus, cerebellum, amygdala, and cerebral cortex (Khera et al. 2022a(Khera et al. , 2022bLee et al. 2016;Morimoto et al. 2020;Sacai et al. 2020). Previous research has found that a subgroup of autistic children has gastrointestinal symptoms and aberrant gut microbiota Finegold et al. 2012).
Autistic patients have increased numbers of the bacteria Clostridium and Desulfovibrio, which are known to produce short-chain fatty acids such as Propionic acid (PPA) (Finegold 2011). Propionic acid (PPA) is a short-chain fatty acid that readily crosses the gut-blood barrier and can enter the CNS via the monocarboxylate transporter system (Mirza and Sharma 2019;Shams et al. 2019). PPA accumulates inside cells, causing intracellular acidification, mitochondrial malfunction , increased oxidative stress markers , altered neurotransmitters (Tiwari et al. 2021), and impaired synaptic transmission in autistic rat brains (Meeking et al. 2020). Sharma et colleagues found that stereotaxic PPA injection into the neocortex area promotes stereotyped behaviour and neurochemical changes in experimental models of autism in adult rats .
The PI3K/AKT/mTOR signalling pathway is primarily involved in neural activities such as synaptic plasticity, neuronal development, memory consolidation, and protein synthesis (Kassai et al. 2014;Takei and Nawa 2014). It also affects a variety of physiological and biological processes, such as neuronal growth, axon guidance, cell proliferation, and differentiation (Rai et al. 2019;Jafari et al. 2019).
The PI3K/AKT/mTOR pathways are activated in the development of several neurodegenerative illnesses, including Huntington's disease (Abd-Elrahman and Ferguson 2019), Alzheimer's disease (Hodges et al. 2018), and brain trauma . Furthermore, motor neuron illnesses such as Multiple Sclerosis (Giacoppo et al. 2017) and Parkinson's disease are caused by the overactivation of the PI3K/AKT/mTOR pathway (Chen et al. 2019).
Chrysophanol (CPH) is a 1, 8-dihydroxy-3-methyl derivative of the 9, 10-anthracenedion ring identified in rheum rhubarbarum, a herbaceous perennial plant in the Polygonaceae family (Singh et al. 2013). CPH has a wide range of pharmacological effects and biological activities, including anti-depressant, anti-bacterial, and anti-cancer properties (Rokaya et al. 2012;Su et al. 2020). CPH also has antimicrobial, anti-inflammatory, and antiviral effects (Lian et al. 2017) and is used to treat a variety of neurological dysfunctions (Chae et al. 2017;Jeong et al. 2018). CPH has been shown in preclinical investigations to alleviate cognition deficits and neuronal death in streptozotocin-induced diabetic encephalopathy (Chu et al. 2018). CPH has been demonstrated to protect against neurodegenerative diseases affecting the motor neurons, including multiple sclerosis (MS) (Lee et al. 2016) and Parkinson's disease (PD) (Chae et al. 2017). A recent preclinical study has investigated the neuroprotective potential of CPH via PI3k/Akt/mTOR pathway in intracerebral haemorrhage (Jadaun et al. 2022a(Jadaun et al. , 2022b. To summarise CPH's relationships with possible targets, Wang and Lv validated CPH's interaction with mTOR against malignant meningioma by blocking mTOR signalling and increasing NF2 signalling (Wang and Lv 2021). Previous research on colorectal cancer (Deng et al. 2020) and colon cancer (Lee et al. 2011) found that CPH reduced PI3K/AKT/mTOR levels, reducing pathological conditions.
Based on the findings, we hypothesise that CPH can downregulate the abnormal PI3K/AKT/mTOR signalling mechanisms, reducing the neuropathological abnormalities in an ICV-PPA-induced experimental model of autism in adult rats. As a result, the current study looked at the overexpression of PI3K/AKT/mTOR, which is involved in the pathogenesis of autism. We investigated the neuroprotective effect of CPH on behavioural, neurochemical, and morphological characteristics in an ICV-PPA-induced experimental model of autism in adult rats. Thus, CPH provides neuroprotection in autistic rats by downregulating the PI3K/AKT/mTOR signalling pathway, which was proven by examining neurochemical parameters in biological samples such as CSF, blood plasma, and brain homogenates.

Experimental animals
A total of 36 rats were used in the current study. All experiments were conducted on six-month-old adult Wistar rats' weight 250-300 g. Each group contains six rats, either sex; they were obtained from the Central Animal House, ISF College of Pharmacy, Moga, Punjab. India. Animals were housed in an acclimatized environment with a 12-hour light-dark cycle with food and water at room temperature at 23 ± 2 °C. The Institute for Animal Ethics Committee (IAEC) approved the project as 816/PO/ReBiBt/S/04/CPC-SEA as IAEC/CPCSEA/Meeting No: 27/2020/Protocol No. 454, following the guidelines provided by the government of India. The rats were randomly divided into six groups, and the sample size was based on a validated animal sampling method suggested by Charan and Kantharia 2013. Animals were acclimatized to laboratory conditions before experimentation.

Drugs and chemicals
PPA was purchased from Sigma-Aldrich (USA). CPH was provided as an ex-gratia sample from BAPEX, India. All other chemicals utilized in the experiments are of analytical grade. Before using the drugs and chemicals, fresh solutions were prepared. CPH was given orally by dissolving in an aqueous solution of 2% ethanol (Chu et al. 2018). The dosing of chrysophanol was determined based on previous research findings in various brain diseases, including ischemic brain injury (Zhao et al. 2016a(Zhao et al. , 2016b, learning and memory deficits (Dong et al., 2010), cognition deficits and neuronal loss (Chu et al. 2018), and Cerebral Ischemia (Zhang et al. 2014a(Zhang et al. , 2014b.

Experimental grouping of animals
The total duration of the experimentation was of 44 days. Propionic acid (PPA) was injected from day 1st to day 11th into the intracerebroventricular (ICV) region of the rat brain to induce autism. CPHwas administered orally from day 12th to day 44th. Animals were randomly assigned into six groups. Group 1-vehicle control; Group 2-sham control; Group 3-Chrysophanol perse (20 mg/kg., p.o.); Group 4-PPA (10 μl/0.26 M, i.c.v.); Group 5-PPA (10 μl/0.26 M, i.c.v.) + Chrysophanol (10 mg/kg., p.o.); Group 6-PPA (10 μl/0.26 M, i.c.v.) + Chrysophanol (20 mg/kg., p.o.). The present study was unblinded, and the experimenter was known regarding the care of animals. All behavioral parameters were conducted from day 1 to day 44th. On day45 th , after completing the protocol schedule, the blood plasma, CSF was collected from adult Wistar rats. Besides, Sodium pentobarbital 270 mg/ml, i.p., was used to anaesthetize the animals deeply. After anesthetization, the fresh brain was removed and preserved with ice-cold PBS (0.1 M) PBS for further neurochemical analysis. The experimental protocol is summarized in (Fig. 1).

ICV-PPA induced experimental model of autism in adult rats
The PPA-induced experimental model of Autism in rats was established and validated by Tiwari et al. 2021. Experimental rats were treated with a PPA-ICV injection of 10 μl/0.26 M for consecutive 11th days. The study by Rahi et al. 2021 suggests that PPA causes behavioral and neurochemical alteration similar to an experimental animal model of autism and is regarded as a validated experimental model for researching the pathophysiological changes identical to those seen in Autistic patients.
Rats were allowed to be habituated in a laboratory environment. Eventually, rats were anaesthetized by intraperitoneal injection of 75 mg/kg ketamine. Then, the rats were placed on the stereotaxic instrument (Stoelting Co., Wood Dale, IL, USA) in a skull-flat position. The positioning of the head was adjusted prior to the surgery to ensure that the bregma and lambda coordinates were similar and at the same level. The rats' heads were shaved, the scalp had been cleaned with 70% ethanol and incised with a blade (mid-sagittal), the skin was removed, and the skull was exposed to spot bregma and lambda that was indicated to assist in defining ICV injection coordinates. Wet cotton swabs were put on rat eyes to prevent dehydration, and cotton buds were used to stop bleeding. A hole was drilled in the skull (Stereotaxic coordinates: AP = −1.3 mm; ML = −1.8 mm; DV = −3.0 mm), a cannula inserted in the burr hole, and the cannula was closed using a plastic ear-pin. The dental For post-operative care, rats were kept independently in a polyacrylic cage that contained warm cloth. Special care was needed until they restored spontaneous movement, which occurred approximately 2-3 hours after anaesthesia. The room temperature was set at 25 ± 3 °C. For 2-3 days, milk and glucose water are provided inside the cages to avoid physical trauma after surgery. To prevent sepsis, gentamycin (35 mg/kg) was given intraperitoneally for three days, and lignocaine gel was applied to the sutured area to relieve the pain. Neosporin powder was sprinkled on them to prevent bacterial infection of the skin. After surgery, the body's overall health and clinical symptoms such as dehydration, body weight, infection, and other physical changes were closely monitored.

Measurement of weight variations
Assessment of body weight The body weight was measured on the 1st, 13th, 23rd, 33th, and 43rd days of the experiment protocol schedule Sahu et al. 2022).

Measurement of relative brain-body weight ratio
The relative brain-body weight ratio was calculated on the 45th day of the experimental protocol schedule (Khera et al. 2022a(Khera et al. , 2022bGopi et al. 2019).

Behaviour parameters
Morris water maze task (MWM) The Morris water maze test was conducted to evaluate memory and cognitive impairment (Morris 1984). Escape latency time (ELT) was measured using MWM on the protocol schedule's 40th, 41st, 42nd, and 43rd. Time (seconds) taken by rats to reach the target platform was considered as escape latency. On day 44th , rats were exposed to swim in the pool containing a hidden platform; for 120 seconds, and time spent in the target quadrant (TSTQ) was recorded. The TSTQ represents the degree of memory consolidation, which occurred after learning (Yadav et al. 2022;Duggal et al. 2020).
Locomotor activity The locomotor activity was performed on the 1st, 13th, 23rd, and 43rd days of the experimental protocol schedule using an actophotometer (INCO Group of Companies Dubai, United Arab Emirates). The behaviour parameter was evaluated using the method described by Mehan et al. 2018. The animal was placed in a digital actophotometer equipped with infrared photocells. They are then observed for five minutes in a square, the closed arena. The value of a digital actophotometer begins as counts per 5 minutes (Tiwari et al. 2021;Mehan et al. 2018).

Beam crossing task (BCT)
The beam crossing task was conducted on days 1st, 13th, 23rd, and 43 rd of the experimental protocol schedule to evaluate motor coordination. During each trial, the number of foot slips was recorded, and additionally, the direction of an animal's fall was observed against the cut-off time of five minutes (Khera et al. 2022a(Khera et al. , 2022bSharma et al. 2019).

Forced swim test (FST)
The forced swim test was used to measure the depressive-like behaviour of rats on the 1st, 13th, 23rd, and 43rd days. The first exposure of rats in the tank during the training phase is for 15 minutes, and the second is after 24 hours later for 5 minutes. A single sixminute exposure is used during the testing session. The first two minutes serve as a habituation period, with the final four minutes serving as the test itself, which determines the length of immobility (Minj et al. 2021).

Collection and preparation of biological samples
Blood plasma collection and separation On day 45th of the protocol schedule, anaesthetized the rats with the chloroform before sample collection. Immediately after anesthetization, a capillary tube is placed at the medial canthus of the eye, and then the sinus is ruptured. Instantly 1-2 ml of blood was collected from the rats through retro-bulbar puncture (Sahu et al. 2022;Kumar et al. 2017). The obtained blood samples were then cold centrifuged at 10,000×g for 15 minutes to separate the plasma. Then separated plasma was carefully stored at −80 °C deep freezer for biochemical analysis.
CSF collection Rats were deeply anaesthetized with 270 mg/ml sodium pentobarbital through i.p. injection. The rats' head was fixed using a holder to reveal the Arachnoid membrane and a skin incision was made, and a translucent dura mater was exposed. A maximum volume of 100 μL CSF was obtained by direct inserting a 30-gauge needle at a 30° angle into the cisterna magna. Within 20 minutes after collection, the sample was centrifuged at 2000 g for 10 minutes at 4 °C. After centrifugation, the supernatant was stored at −80 °C until further analysis (Kozler et al. 2015).
Brain homogenate preparation Rats were sacrificed by decapitation on the 45th day of the treatment schedule. The whole fresh brain was removed, washed with ice-cold isotonic saline solution, and homogenized with 0.1 M (w/v) of chilled phosphate buffer saline (pH = 7.4). The rat brain homogenate was then centrifuged at 10,000×g for 15 minutes, the supernatant was separated, and the aliquots were preserved. The samples were stored in a deep-freezer at −80 °C to be used as and when the need for various neurochemical estimations ).

Estimation of MBP level
The MBP levels were assessed in rat brain homogenate using an ELISA kit (E-EL-R0642/ MBP; Elabsciences, Wuhan, Hubei, China). The values were expressed in μg/mg protein ).

Assessment of apoptotic markers
Caspase-3 concentrations were assessed in brain homogenate ) and blood plasma (Guo and Li 2018) using an ELISA kit. Bax protein level was determined in brain homogenate (Tiwari et al. 2021) and blood plasma . The anti-apoptotic protein such as Bcl-2 levels was estimated in brain homogenate ) and blood plasma ) using ELISA commercial kits (E-EL-R0160/Caspase-3; E-EL-R0098/ Bax/Bcl2Elabsciences, Wuhan, Hubei, China). The values are expressed in ng/g protein in brain homogenate and ng/ ml in blood plasma.

Assessment of neurotransmitter levels
Measurement of acetylcholine (ach) level Acetylcholine level was measured using a diagnostic kit (E-EL-008Ach; ELabSciences, Wuhan, Hubei, China). All samples and reagents were freshly prepared as per manual instruction provided by the kit's. The optical density of the reaction mixture was measured at 540 nm. The neurotransmitter in the supernatant was estimated and the value expressed as ng/ mg protein .

Measurement of dopamine level
The dopamine levels in the striatal tissue sample were determined. Dopamine levels in the striatum are a sign of neural excitability, which leads to mood changes. The electrochemical detector was used to assess dopamine levels in the brain homogenate using the HPLC technique. The level of dopamine in brain homogenates is expressed as ng/mg protein (Gupta et al. 2022;. Estimation of glutamate level Glutamate was assessed after derivatization with o-phthalaldehyde/−mercaptoethanol (OPA/−ME), and quantitative analysis in tissue samples using HPLC was carried out according to Rahi and her coworker's method. The glutamate level in rat brain homogenate is expressed as ng/mg protein .

Measurement of serotonin level
Serotonin level was measured in the brain homogenate sample by HPLC using an electrochemical detector and C18 reverse-phase column. The mobile phase consisted of sodium citrate buffer (pH 4.5) -acetonitrile (87:13, v/v). The supernatant was filtered through 0.22 mm nylon filters before being injected into the sample injector. The serotonin concentration was assessed from the standard curve generated using a standard with a concentration of 10-100 mg/ml (Khera et al. 2022a(Khera et al. , 2022bSharma et al. 2019).

Evaluation of oxidative stress markers
Estimation of acetylcholinesterase (AChE) level Acetylcholinesterase concentration was estimated using spectrophotometrically. The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01 M sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide, and 0.10 ml DTNB (Ellman reagent). The change in absorbance was recorded instantly at 412 nm spectrophotometrically. The enzymatic activity in the supernatant was expressed as μM/mg protein (Deshmukh et al. 2009).
Measurement of superoxide dismutase (SOD) enzymatic activity SOD activity was measured using spectrophotometrically by auto-oxidation of epinephrine at pH 10.4. The supernatant (0.2 ml) of the brain homogenate was mixed with 0.8 ml of 50 mM glycine buffer, pH 10.4, and the reaction was initiated with the addition of 0.02 ml epinephrine. After 5 minutes, the absorbance was spectrophotometrically measured at 480 nm. SOD activity was quantified as nM/mg protein (Sahu et al. 2022;Mehan et al. 2017).

Estimation of GSH level
The level of reduced glutathione in the brain was assessed using the method described by Ellman et al. 1959. 1 ml supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4 °C for one hour. The samples were centrifuged at 1200×g for 15 min. To 1 ml of the supernatant, 2.7 ml of phosphate buffer (0.1 M, pH 8) and 0.2 ml of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were added. The yellow colour appeared immediately measured with a spectrophotometer at 412 nm. The glutathione concentration in the supernatant was expressed as μM/mg protein (Yadav et al. 2022;Bala et al. 2015).

Estimation of nitrite level
The nitrite concentration in the supernatant, indicating the formation of nitric oxide (NO) is evaluated by a colorimetric assay using a Greiss reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% phosphoric acid) as described by Green et al. 1982. Equal volumes of supernatant and Greiss reagent are mixed, the mixture incubated for 10 min at room temperature in the dark, and the absorbance determined spectrophotometrically at 540 nm. The amount of nitrite in the supernatant is determined from a sodium nitrite standard curve and expressed as μM/mg protein ).

Estimation of malondialdehyde (MDA) level
The quantitative determination of malondialdehyde (MDA) was performed in brain homogenate. After its reaction with thiobarbituric acid, the concentration of MDA was measured at 532 nm using a spectrophotometer and expressed as nM/mg protein ).

Protein estimation
The protein content was quantified by using the Coral protein estimation kit (Biuret method).

Gross pathological examination of rat brains
On the 43rd day, rats were sacrificed by decapitation; brains were removed for gross pathological analysis performed. After analyzing the whole brain, coronal sections were taken (Tiwari et al. 2021). Sectioned 2-mm thick brain pieces (coronally from the anterior pole to the posterior poles of the cerebral cortex) were placed on glass slides. A digital camera (Fujix digital camera, Fujifilm, Japan) was used to visualize all the brain regions. The demyelination region (mm) in each brain segment was measured on day 43rd after completing the procedure through MOTICAM-BA310 image plus 2.0 analysis software. The demyelination scale (mm) volume was calculated for each coronal brain segment by converting the demyelination region (mm). The demyelination size (mm 3 ) in each brain section was measured from the dark greyish area near the striatum by image analysis on the 43rd day. The injury's size was calculated in each coronal 2-mm-thick brain section by calculating the demyelination area (l × b × h) (Khera et al. 2022a(Khera et al. , 2022bRahi et al. 2021).

Statistical analysis
Data were analyzed using two-way ANOVA followed by Post hoc test Bonferroni and one-way ANOVA repeated measures followed by Post hoc test Tukey's multi comparison test. P < 0.001 was considered statistically significant. Data was found to be normalized, and the sample size was calculated by checking the normality distribution by the Kolmogorov Smirnov test. All statistical results were performed out by GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA, USA). Statistical results were expressed as the mean ± standard deviation (SD).

Restoration of body weight after chronic administration with chrysophanol
Body weight was measured on the 1st, 13th, 23rd, 33th, and 43rd days of the protocol schedule. There was no significant difference in body weight between any treatment groups prior to treatment beginning. Rats who received daily PPA injections for 11 consecutive days had lower body weight on the 13th day as compared to the vehicle, sham, and CPH 20 perse treated groups. Prolonged oral administration of CPH 10 mg/kg and CPH 20 mg/ kg restored body weight significantly on the 23rd, 33rd, and 43rd days when compared to PPA-treated autistic rats [two-way ANOVA: F(20,120) = 157.72, p < 0.001]. On the 43rd day, CPH 20 mg/kg was found to be more effective than CPH 10 mg/kg in successfully restoring body weight. (Fig. 2a).

Restoration of relative brain-body weight ratio after chronic administration with chrysophanol
On the final day of the procedure, the relative brain-body weight ratio was assessed. On the 43rd day, there was no significant difference in the relative brain-body weight ratio among all groups. When compared to the vehicle, sham, and CPH 20 perse treatment groups, chronic ICV injection of PPA for 11 days resulted in a substantial reduction in the relative brain-body weight ratio. Long-term CPH 20 mg/kg and CPH 10 mg/kg administration led to an increase in relative brain-body weight ratio on the 43rd day when compared to PPA-treated autistic rats [one-way ANOVA: F(5,25) = 1.218, p < 0.001]. CPH 20 mg/kg, on the other hand, significantly restored the relative brain-body weight ratio as compared to the CPH 10 mg/kg treated group (Fig. 2b).

Improvement in memory and cognition after chronic administration with chrysophanol
To assess memory and cognitive impairment, the Morris water maze test was used. The escape latency was measured on the 40th, 41st, 42nd, and 43rd days of the experiment protocol schedule. PPA-treated rats have a progressive increase in escape latency time (ELT) as compared to vehicle, sham, and CPH 20 perse treatment rats. Longterm oral CPH 10 mg/kg and CPH 20 mg/kg administration significantly reduced ELT in a dose-dependent manner when compared to the PPA-injected group [two-way  (Values expressed as mean ± SD (n = 6 rats per group). *p < 0.001v/s vehicle control, sham control, and CPH20Perse; @ p < 0.001 v/s PPA; @# p < 0.001 v/s PPA + CPH10 (two-way ANOVA followed by post hoc multiple comparison test Bonferroni). (b) Effect of chrysophanol on relative brain-body weight in ICV-PPA induced experimental model of autism in adult rats. (Values expressed as mean ± SD (n = 6 rats per group). *p < 0.001v/s vehicle control, sham control and CPH-20Perse; @ p < 0.001 v/s PPA; @# p < 0.001 v/s PPA + CPH10 (oneway ANOVA followed by Tukey's multiple comparison test). (c) Effect of chrysophanol on escape latency time in ICV-PPA induced experimental model of autism in adult rats. (Values expressed as mean ± SD (n = 6 rats per group). *p < 0.001v/s vehicle control, sham control, and CPH20Perse; @ p < 0.001 v/s PPA; @# p < 0.001 v/s PPA + CPH10 (two-way ANOVA followed by post hoc multiple comparison test Bonferroni). (d): Effect of chrysophanol on TSTQ in ICV-PPA induced experimental model of autism in adult rats. (Values expressed as mean ± SD (n = 6 rats per group). *p < 0.001v/s vehicle control, sham control and CPH20Perse; @ p < 0.001 v/s PPA; @# p < 0.001 v/s PPA + CPH10 (one-way ANOVA followed by Tukey's multiple comparison test). (e): Effect of chrysophanol on locomotor activity in ICV-PPA induced experimental model of autism in adult rats. (Values expressed as mean ± SD (n = 6 rats per group). *p < 0.001v/s vehicle control, sham control, and CPH20Perse; @ p < 0.001 v/s PPA; @# p < 0.001 v/s PPA + CPH10 (two-way ANOVA followed by post hoc multiple comparison test Bonferroni). (f): Effect of chrysophanol on neuromuscular coordination in ICV-PPA induced experimental model of autism in adult rats. (Values expressed as mean ± SD (n = 6 rats per group). *p < 0.001v/s vehicle control, sham control, and CPH20Perse; @ p < 0.001 v/s PPA; @# p < 0.001 v/s PPA + CPH10 (two-way ANOVA followed by post hoc multiple comparison test Bonferroni). (g): Effect of chrysophanol on immobility time in ICV-PPA induced experimental model of autism in adult rats. (Values expressed as mean ± SD (n = 6 rats per group). *p < 0.001v/s vehicle control, sham control, and CPH20Perse; @ p < 0.001 v/s PPA; @# p < 0.001 v/s PPA + CPH10 (two-way ANOVA followed by post hoc multiple comparison test Bonferroni) ANOVA: F(15,90) = 19.48, p < 0.001]. Furthermore, CPH 20 mg/kg administered to rats reduced ELT more effectively than CPH 10 mg/kg administered to animals (Fig. 2c). On the 44th day of the protocol schedule, the time spent in the target quadrant (TSTQ) was measured. TSTQ in chronic PPA-infused rats is considerably lower than in a vehicle, sham, and CPH 20 perse treated rats. Long-term CPH 10 mg/kg and 20 mg/kg administration enhances TSTQ in a dose-dependent manner compared to PPA-treated autistic rats [one-way ANOVA: F(F (5,25) = 25) =6.594, p < 0.001]. CPH 20 mg/kg-treated rats improved TSTQ and memory consolidation more than CPH 10 mg/kg-treated rats (Fig. 2d).

Improvement in locomotion after chronic administration with chrysophanol
Rat locomotion was observed using locomotor activity. The actophotometer device was used to perform the test on the1st 13th, 23rd, and 43rd days. On the first day of the protocol schedule, there were no significant differences between the treatment groups. PPA-injected rats had significantly lower locomotion on the 13th day compared to the vehicle, sham, and CPH 20 perse treated groups. In comparison to PPA-treated autistic rats, persistent oral treatment of CPH 10 mg/kg and CPH 20 mg/kg significantly improved locomotion on the 23rd and 43rd days [two-way ANOVA: F(15,90) = 644.72, p < 0.001]. CPH 20 mg/kg was more effective than CPH 10 mg/kg in improving locomotor activity on the 43rd day than CPH 10 mg/kg (Fig. 2e).

Improved motor coordination after chronic administration with chrysophanol
The beam crossing task was used to assess rats' motor coordination abilities. The task was completed on the 1st, 13th, 23rd, and 43rd days. There were no significant changes between treatment groups on the first day. On the 13th day, chronic PPA-treated rats experienced considerably more slips than the vehicle, sham, and CPH 20 perse treated groups. Prolonged oral treatment with CPH 10 mg/kg and CPH 20 mg/kg on days 23 and 43 significantly reduced the number of slips in a dose-dependent manner compared to the PPA treatment group [two-way ANOVA: F(15,90) = 35.18, p < 0.001]. CPH 20 mg/kg was substantially more effective than CPH 10 mg/kg in reducing slip count and enhancing beam efficiency on the 43rd day (Fig. 2f).

Reduced depression-like behavior after chronic administration with chrysophanol
The forced swim test was used to assess rats' depressivelike behaviour. The immobility time was recorded on the 1st, 13th, 23rd, and 43rd days. There was no significant difference between treatment groups on the first day. PPA-injected rats show longer immobility time significantly on the 13th day of the protocol schedule compared to the vehicle, sham, and CPH 20 perse treatment groups. Long-term oral administration of CPH 10 mg/kg and CPH 20 mg/kg to rats on the 23rd and 43rd days significantly reduces immobility time in a dose-dependent manner when compared to PPA-treated rats [two-way ANOVA: F(15,90) = 910.07, p < 0.001]. On the other hand, CPH 20 mg/kg was found to be more effective than CPH 10 mg/kg in considerably reducing immobility time and restoring depressive-like behaviour on the 43rd day (Fig. 2g).

Decreased PI3K level after chronic administration with chrysophanol
The PI3K protein level was evaluated in rat brain homogenate and CSF samples at the end of the experimental protocol. When compared to the vehicle, sham, and CPH20 perse treated groups, PPA-treated rats have a significant increase in PI3K protein levels in rat brain homogenate and CSF samples. Long-term oral treatment of CPH at doses of 10 mg/kg and 20 mg/kg for 44 days consistently lower PI3K levels as compared to PPA-treated rats. CPH 20 mg/kg treatment group was shown to be more efficient than CPH 10 mg/kg treatment group in reducing PI3K levels in rat brain homogenate [one-way ANOVA: F(5,25) = 1.136, p < 0.001] and CSF samples [one-way ANOVA: F(5,25) = 0.256, p < 0.001]. (Table 1a.)

Decreased AKT level after chronic administration with chrysophanol
The AKT level was determined using an ELISA kit in rat brain homogenate and CSF samples. Chronic PPA injection significantly elevated AKT levels in rat brain homogenate and CSF when compared to the vehicle, sham, and CPH20 treatment groups. Prolonged oral CPH 10 and 20 mg/kg therapy significantly lowered AKT levels in brain homogenate and CSF compared to PPA-injected groups. Furthermore, CPH 20 mg/kg is shown to be more efficient than CPH 10 mg/kg in reducing AKT levels in brain homogenate [one-way ANOVA: F(5,25) = 1.209, p < 0.001] and CSF samples [one-way ANOVA: F(5,25) = 0.151, p < 0.001] (Table 1b).

Decreased mTOR level after chronic administration with chrysophanol
The level of mTOR was determined in rat brain homogenate and CSF samples. Long-term PPA-treated rats have higher mTOR levels in rat brain homogenate and CSF samples than the vehicle, sham, and CPH20 groups. Continuous oral administration of CPH 10 and 20 mg/kg significantly reduces mTOR levels in rat brain homogenate [one-way ANOVA: F(5,25) = 1.212, p < 0.001] and CSF [one-way ANOVA: F(5,25) = 0.551, p < 0.001] compared to PPA treatment groups. CPH 20 mg/kg was found to be more efficient in lowering mTOR levels in brain homogenate and CSF samples (Table 1c).

Restoredmyelin basic protein level after chronic administration with chrysophanol
The level of myelin basic protein (MBP) was determined in rat brain homogenates using an ELISA kit. PPA-injected rats have significantly lower MBP levels when compared to vehicle, sham, and CPH20 perse treated groups. Long-term oral treatment of CPH 10 and 20 mg/kg causes a significant increase in MBP levels as compared to PPA-injected rats [one-way ANOVA: F(5,25) = 1.687, p < 0.001]. CPH 20 mg/ kg was more efficient than CPH 10 mg/kg in restoring MBP levels in rat brain homogenate (Table 1d).

Reduction in caspase-3, Bax, and increased Bcl-2 levels after chronic administration with chrysophanol
Neuronal apoptotic markers such as Caspase-3, Bax, and Bcl-2 were measured in rat brain homogenate and blood plasma. Prolonged PPA exposure resulted in significant elevations in Caspase-3 and Bax protein levels in rat brain homogenate and blood plasma. Furthermore, when compared to the vehicle, sham, and CPH20 perse groups, ICV-PPA treated rats have a significant drop in anti-apoptotic Bcl-2 levels in brain homogenate and blood plasma. Caspase-3 levels in brain homogenate [one-way ANOVA: F(5,25) = 0.210, p < 0.001] and blood plasma [one-way ANOVA: F(5,25) = 1.052, p < 0.001] are significantly reduced by persistent oral CPH therapy at 10 and 20 mg/kg.

Restoration of neurotransmitters level after chronic administration with chrysophanol
Neurotransmitters such as serotonin, dopamine, acetylcholine, and glutamate were assessed in rat brain homogenate at the end of the treatment schedule. ICV-PPA injections resulted in a significant decrease in dopamine, serotonin, and acetylcholine levels, as well as an increase in glutamate levels, in rat brain homogenate, as compared to vehicle, sham, and CPH20 perse-treated rats. Long-term oral CPH 10 and 20 mg/kg treatment significantly increases dopamine [one-way ANOVA: F(5,25) = 2.546, p < 0.001], serotonin [one-way ANOVA: F(5,25) = 0.228, p < 0.001], and acetylcholine levels while decreasing glutamate Table 1 Effect of chrysophanol on PI3K, AKT, mTOR and myelin basic protein level in ICV-PPA induced experimental model of autism in adult rats Values expressed as mean ± SD (n = 6 rats per group). *p < 0.001v/s vehicle control, sham control and CPH20Perse; @ p < 0.001 v/s PPA; @# p < 0.001 v/s PPA + CPH10 (one-way ANOVA followed by Tukey's multiple comparison test Among these, CPH 20 mg/kg was more effective than CPH 10 mg/kg in restoring neurotransmitter levels in rat brain homogenate (Table 3).

Decreased inflammatory cytokines level after chronic administration with chrysophanol
Using an ELISA kit, inflammatory cytokines such as TNF and IL-1β were quantified in rat brain homogenate and blood plasma samples. Chronic ICV-PPA treatment results in significant increases in proinflammatory mediators such as TNF and IL-1β1 in rat brain homogenate and blood plasma as compared to the vehicle, sham, and CPH20 treated groups. TNF levels in rat brain homogenate [one-way ANOVA: F(5,25) = 0.357, p < 0.001] and blood plasma [one-way ANOVA: F(5,25) = 1.180, p < 0.001] are significantly reduced by continuous oral administration with CPH 10 and 20 mg/kg. Similarly, long-term treatment with CPH10 and 20 mg/kg significantly reduced IL-1β levels in rat brain homogenate [one-way ANOVA: F(5,25) = 4.582, p < 0.001] and blood plasma [one-way ANOVA: F(5,25) = 0.782, p < 0.001] compared to PPA treated rats. CPH 20 mg/kg treatment, on the other hand, was found to be more effective in lowering inflammatory cytokines when compared to CPH 10 mg/kg treated rats (Table 4).

Amelioration of oxidative stress markers level after chronic administration with chrysophanol
In rat brain homogenate, the levels of oxidative stress markers AchE, LDH, SOD, GSH, nitrite, and MDA were evaluated. Chronic PPA administration rats showed a significant increase in AchE, LDH, nitrite, and MDA levels in rat brain homogenate compared to the vehicle, sham, and CPH 20 perse treatment groups. ICV-PPA injected groups showed a significant reduction in anti-oxidant enzyme levels such as SOD [one-way ANOVA: F(5,25) = 0.968, p0.001] and GSH [one-way ANOVA: F(5,25).
Prolonged oral treatment with CPH 10 and 20 mg/ kg for 44 days resulted in significantly lower levels of AchE [one-way ANOVA: F(5,25) = 1.266, p0.001], LDH  Similarly, continuous oral CPH 10 and 20 mg/kg treatment results in a significant rise in SOD and GSH levels in rat brain homogenate. Likewise, CPH 20 mg/kg is more effective than CPH 10 mg/kg in restoring anti-oxidant enzyme levels in rat brain homogenate ( Table 5).

Restoration of whole-brain alterations after chronic administration with chrysophanol
The normal, vehicle, and CPH 20 perse treated groups all showed normal brain size and morphology. In comparison to the vehicle, sham, and CPH 20 perse treatment groups, the ICV-PPA-treated rat brains had a disrupted clotted outermost layer with rupture meninges. Prolonged oral administration of CPH at 10 mg/kg and 20 mg/kg doses repaired the morphological changes and aided the rat brain's recovery from subsequent injury. Similarly, animals given CPH 20 mg/kg showed considerable recovery in the damaged area of the brain as well as recovery of brain injury when compared to rats given CPH 10 mg/kg (Fig. 3a).

Reduction of pathological changes in brain sections after chronic administration with chrysophanol
Brain sections of rats treated with vehicle, sham, and CPH 20 mg/kg perse were structurally intact and undamaged, with clearly visible basal ganglia, cortex, and hippocampal tissue. The brain sections of the ICV-PPA treated rats showed cortical and hippocampus shrinkage, as well as atrophy in subcortical areas such as the medial thalamus, putamen, caudate nucleus, and internal medullary lamina, as compared to the vehicle, sham, and CPH 20 perse, treated rats. The pathological abnormalities in rat brain slices were reversed by long-term oral treatment of CPH 10 and 20 mg/kg (Fig. 3b).

Reduction in demyelination volume after chronic administration with chrysophanol
The normal, vehicle, and CPH20 perse treated groups all demonstrated no significant change in demyelination volume. However, when compared to the normal, vehicle, and CPH20 treatment groups, long-term administration of the neurotoxin PPA for 11 days significantly increased the area of demyelination. Long-term oral CPH administration at dosages of 10 mg/kg and 20 mg/ kg significantly reduced demyelination volume in autistic-like rats compared to PPA-treated autistic-like rats. As a result, CPH 20 mg/kg had a dose-dependent effect on demyelination volume reduction when compared to CPH 10 mg/kg treated rats [one-way ANOVA: F(5,25) = 0.241, p0] .001] (Fig. 4).

Discussion
The current study used a PPA-induced experimental adult rat model to evaluate neurobehavioral and neurochemical abnormalities in autistic-like animals. Several investigations have found that ICV-PPA injection plays a significant role in developing autistic-like behaviour in adult rats (Khera et al. 2022a(Khera et al. , 2022bRahi et al. 2021;Tiwari et al. 2021;Sharma et al. 2019). PPA-exposed rats exhibit behavioural and neuropathological abnormalities that are comparable to those seen in ASD patients, including hyperactivity, poor social interaction, stereotypic and repetitive movements, and have been recognized as a viable adult ASD model in rodents (Nemechek and Moore 2020; Chow et al. 2012). PPA, as a weak organic acid, can passively accumulate within CNS cells, resulting in a fall in intracellular pH, which has several physiological implications (Thomas et al. 2010). PPA exerts a range of physiological effects on the brain, corresponding to the increased locomotion found in experimental rats (Lobzhanidze et al. 2020). As a result, it can alter neurotransmission in brain regions related to locomotor behaviour, such as the hippocampus and prefrontal cortex (Meeking et al. 2020;MacFabe et al. 2007). PPA-treated rat brains had higher amounts of cytokines and oxidative stress markers, as well as other abnormalities associated with ASD (MacFabe et al. 2007;Bhandari and Kuhad 2017).
CPH was investigated to see whether it may protect against the behavioural changes caused by neurotoxin PPA in an experimental model of autism in adult rats. PPA infusion causes ASD-like behavioural and neuroinflammatory responses in adult rats. It is a neurotoxin that alters rat behaviour by impairing learning and memory, disrupting social interactions, and causing anxiety (Shultz et al. 2008;Ku et al. 2016;Wu et al. 2017). Additionally, we measured the protein levels of various cellular and molecular markers, apoptotic markers, neurotransmitters, inflammatory cytokines, and oxidative stress parameters in rat brain homogenate, blood plasma, and cerebrospinal fluid (CSF) samples. Long-term oral treatment of CPH at two different doses showed a neuroprotective effect against neurobehavioral and neurochemical changes in the ICV-PPA induced experimental model of autism in adult rats.
Bodyweight measurements taken on several days revealed that rats significantly decreased bodyweight after 11 days of ICV-PPA infusions. Prior research has shown that PPA, a short-chain fatty acid, influences weight loss in both animal and human populations due to altered fatty acid metabolism and increased gluconeogenesis after entering the citric acid cycle (Nankova et al. 2014). (Choi et al. 2018). In the current investigation, this weight loss increased in a dosedependent manner after CPH administration. The brain-body weight ratio was also calculated by dividing the fresh brain weight by the bodyweight at the end of the experimental treatment schedule. The brain-body weight ratio was significantly lower in ICV-PPA-treated autistic rats, although it increased dose-dependently with CPH therapy. During the forced swim test, the immobility time was used to assess depressive-like behaviour in ICV-PPA induced experimental model of autism in adult rats. A previous study found that PPA therapy had an increased depressed effect (Bhandari and Kuhad 2017). The current study's findings revealed an increase in immobility time after PPA injection that was mitigated by continuous CPH treatment.
The PI3K/Akt signaling pathway have an important role in regulating neuronal metabolism, neuoinflammation, cell development and survival Lin et al. 2020). Moreover, PI3K/Akt/ mTOR signaling pathway is demonstrated to be correlated to neuroprotection and is overexpressed at the occurrence of neurodegeneration (Fakhri et al. 2021). Various studies have implied that PI3K is involved in synaptic plasticity, learning, memory, and major depression (Budni et al. 2012;Horwood et al. 2006). Formation of lipid products followed by PI3K activation, act as second messengers by employing proteins, such as protein kinase B (PKB/Akt) that results in the activation of its downstream kinases and consequently increasing mTOR phosphorylation (Hoeffer and Klann 2010). It has been demonstrated that Akt is a central regulator of disease progression due to its vast signaling in the ASD brain (Gazestani et al. 2019).
Activation of mTOR signaling stimulates messenger RNA (mRNA) translation and protein synthesis by activating p70S6 kinase (p70S6K). This produces quick and sustained elevation of synapse-associated proteins, including postsynaptic density-95 protein (PSD95) responsible for scaffolding, organization of receptors, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, modulation of the voltage-gated ion channels expression and alterations in the neurotransmitters expression levels (Hoeffer and Klann 2010;Niere and Raab-Graham 2017). The various GPCRs found to activate mTOR in neurons includes the glutamate metabotropic mGlu1/5 receptors (Banko et al. 2006;Ronesi and Huber 2008), the μ-opioid receptor (Polakiewicz et al. 1998), the dopaminergic D 1 (Santini et al. 2009) and D 3 receptors (Salles et al. 2013), the amino acid/glutamate T1R1-T1R3 receptors (Wauson et al. 2012), the serotonin 5-HT 6 receptor (Meffre et al. 2012), the GABA B receptors (Workman et al. 2013) and the cannabis (CB 1 ) receptor (Meffre et al. 2012). The CB 1 receptors activates mTOR indirectly which depends on an inhibition of GABA release from inhibitory GABAergic interneurons (Puighermanal et al. 2009). This leads to an enhanced activity of the excitatory networks including glutamate NMDA receptor activation and, consequently, mTOR activation (Puighermanal et al. 2009). This causes hyperexcitability in ASD-like conditions. It has been previously reported that the upregulation of mTOR increases the levels of pro-inflammatory cytokines and decreases the anti-inflammatory cytokines production (Jadaun et al. 2022a(Jadaun et al. , 2022b. Moreover, pro-inflammatory cytokine increases glutamate release from microglial cells via upregulation of glutaminase, stimulating AMPA receptor expression, and inducing GABA receptor endocytosis, resulting in changes in the neuronal excitability (Sanz and Garcia-Gimeno 2020). Previous research has linked the PI3K/AKT/GSK3/mTOR/ BDNF pathway to the development of mood-related disorders and has also been linked to the adaptive stress response . Upregulated PI3K-AKT/mTOR signaling pathway activity in neurons is linked with stereotypically autistic behaviors, including memory and learning changes, serotonergic impairment, epilepsy, and changes to both structural and synaptic plasticity (Costa-Mattioli and Monteggia 2013; Hutsler and Zhang 2010). Numerous research has revealed an association between PI3K/AKT/mTOR and depression and anxiety (Leibrock et al. 2013;Moretti et al. 2014). In a chronic stress model of depression in mice, CPH treatment promotes antidepressant efficacy by blocking the mTOR signalling pathway ). All of these data confirmed our study's findings that CPH reduced depression symptoms in the PPA-induced experimental model of autism in adult rats.
Additionally, the hyperactivity and repetitive behaviours associated with autistic individuals have been highlighted as primary symptoms (Kong et al. 2021). As a result, PPA and other short-chain fatty acids increase glial and intracellular neuronal acidification and calcium levels, affecting neurotransmitter release, including serotonin, dopamine, glutamate, and norepinephrine (Daghestani et al. 2017;Thomas et al. 2012).
PPA has also been shown to enhance glutamatergic transmission, which causes excitability in brain areas related with locomotor activity.o Our locomotor activity results showed increased locomotion after ICV-PPA injection, which is consistent with the findings of our previously reported studies (Tiwari et al. 2021;Sharma et al. 2019). There was a significant and dose-dependent improvement in hyperactive and repetitive behaviour after CPH treatment at doses of 10 mg/kg and 20 mg/kg. The beam crossing task assessed balance and motor coordination by counting the number of slips while moving across a wooden beam. Our results show that PPA autistic rats had a higher number of slips, indicating poor motor coordination, which was reduced in a dosedependent way after CPH treatment.
Autistic children are reported to have poor spatial memory, cognitive impairments, and intellectual deficiencies ). The Morris water maze was used to test rats' long-term memory and spatial learning abilities. Mepham et al. found that intracerebroventricular PPA injection decreased spatial cognition in adult rats (Mephem et al., 2019). Our results demonstrated that rats treated with PPA had severe memory loss as a result of increased ELT and decreased TSTQ. Additionally, CPH treatment reduces the escape latency time (ELT), even though increased TSTQ in MWM indicates enhanced spatial memory.
In order to investigate a cellular signalling mechanism, we also examined the effect of CPH on the PI3K/AKT/ mTOR protein levels. Upregulation of PI3K/AKT/mTOR has been linked to the onset and progression of neurological dysfunctions (Bozdagi et al. 2013;Chen et al. 2014). Recently, this signaling pathway overactivation has been linked to pathological processes that may be responsible for autism (Kwon et al. 2006). In comparison, inhibiting the PI3K/AKT/mTOR signalling pathway resulted in a neuroprotective effect that was employed as a diagnostic marker in autistic patients (Yeung et al. 2017). As a result, we found elevated PI3K/AKT/mTOR levels in the CSF and brain homogenate of ICV-PPA induced experimental model of autism. However, CPH therapy decreases the PI3K/AKT/mTOR protein levels, which protects against autism by lowering apoptosis, neuroinflammation, oxidative stress in CSF and rat brain homogenate.
Researchers used new techniques to detect abnormalities in the white matter region of the brain that is connected with autistic dysfunction. A previous study revealed that MBP levels were lower in the brains of autistic patients (Gonzalez-Gronow et al. 2015). The current study found decreased MBP levels in ICV-PPA rats' brain homogenate. Furthermore, CPH administration restored MBP levels in autistic rats' brain homogenates. Apoptotic cell death impairs brain maturation and is thought to be a risk factor for the development of autism (Eftekharian et al. 2019). PPA exposure elevated the levels of apoptotic indicators such as Bax and Caspase 3, while decreasing the levels of anti-apoptotic marker Bcl-2. Long-term CPH therapy has been demonstrated to protect cells against cell death by decreasing Bax and Caspase-3 levels while increasing Bcl-2 levels.
The neurochemical analysis in our study provides a clear indication of CPH neuroprotective potential. Neurotransmitters, which regulate memory, emotion, and behaviour, must balance normal function and neuronal development. Neurotransmitter disruption was identified to be one of the primary causes of the onset of behavioural traits. One of the most prominent aspects of autism is neurotransmitter imbalance (Kuo and Liu 2018). The most investigated neurotransmitters in autism include serotonin, dopamine, glutamate, and Ach. As a result of serotonin's role in the brain's development, autistic individuals exhibit socially impaired, repetitive, and depressive behaviour (Kane et al. 2012;Amodeo et al. 2021).
Furthermore, a decrease in dopamine, Ach, and an increase in glutamate were linked to autistic behaviour (Drenthen et al. 2016;Karvat and Kimchi 2014;DiCarlo et al. 2019). Increased glutamate levels activate microglia and promote neuroinflammation, whereas decreasing dopamine and acetylcholine levels influence neuronal excitability and cause mood abnormalities (Acharjee et al. 2018). Our results show that repeated ICV-PPA injections considerably affect the amount of neurotransmitters in rat brain homogenates. Dopamine, serotonin, and acetylcholine levels in ICV-PPA-treated rats decreased, whereas glutamate levels significantly increased, indicating neuronal excitotoxicity. CPH treatment restores neurotransmitter levels in a dosedependent manner and improves autistic-like behaviour.
TNF and IL-1 are significant mediators of oxidative stress, and neuroinflammation are implicated in neurodegenerative diseases (Saghazadeh et al. 2019;Mirza and Sharma 2019). Clinical studies on autistic children clearly showed higher inflammatory cytokine levels in CSF, resulting in immunological response and brain damage (Chez et al. 2007). according to our findings, PPA infusion elevated inflammatory cytokines such as TNF and IL-1β in blood plasma and brain homogenate. CPH treatment significantly decreases inflammatory cytokine levels in blood plasma and brain homogenate, resulting in anti-inflammatory actions. Previous research has identified elevated oxidative stress as one of the pathogenic characteristics of autism (Morimoto et al. 2020;Abruzzo et al. 2019;El-Ansary et al. 2012). We measured oxidative stress markers in the brain homogenates of PPA and CPH-treated rats to characterize the severity of the disease and the preventive effects of CPH against oxidative stress. Our data clearly reveal an increase in AchE, LDH, nitrite, and MDA, as well as a decrease in antioxidants, particularly SOD and GSH, in an ICV-PPA induced experimental model of autism in adults rats. PPA-induced autistic rats treated with CPH showed significantly reduced levels of oxidative stress markers, and showing antioxidant properties.
This study looks at the morphological structure of the brain, whole-brain sections, and demyelination volume. Previous research has indicated that the hippocampus is relatively sensitive to PPA exposure. The gross pathological and morphological findings show that PPA-treated rats' brains differ in size and shape . Prolonged CPH administration improved morphological abnormalities in ICV-PPA-treated autistic rats, such as damaged meninges and constricted prefrontal cortex. Coronal sections of ICV-PPA-treated rats exhibited malformed basal ganglia, a defragmented hippocampal area, and degraded white matter. Furthermore, the measurement of demyelination volume in rat brains revealed a significant decrease in white matter volume following PPA injections . Continuous CPH therapy was found to lessen the severity of pathological and morphological changes. Long -term treatment of CPH10 mg/kg and CPH20 mg/kg to autistic rats recovered abnormalities in brain sections and enabled remyelination of damaged areas as compared to ICV-PPA treated autistic rats.
Our research primarily focuses on CPH's neuroprotective potential by decreasing the PI3K/AKT/mTOR protein levels, alleviating behavioural, neurochemical, morphological, and gross pathological abnormalities in the ICV-PPA induced experimental model of autism in adult rats. Concurrent studies, such as Western Blot and immunohistochemistry, are also required to provide molecular support for this hypothesis. Despite these limitations, CPH's neuroprotective potential in resolving or downregulating aberrant PI3K/AKT/ mTOR signalling pathways in the CNS appears promising.

Conclusion
Based on the findings, we conclude that PPA injection causes considerable changes, including irregular neural cell structure, autism-like neurobehaviors, and neurochemical abnormalities. To date, there have been no pre-clinical investigations on the neuroprotective effect of CPH via PI3K/ AKT/mTOR signalling downregulation in an ICV-PPA generated experimental model of autism in adult rats. It has been observed that PPA can cause autistic-like behaviour in experimental rats. Our findings suggest that CPH substantially improves social interaction, learning, and memory deficiencies in PPA-exposed rats. CPH significantly lowered oxidative stress, neuroinflammation, and apoptotic cell death in autistic rats by reducing the PI3K/AKT/mTOR protein levels in brain homogenate, blood plasma and CSF samples. Furthermore, the recovery of gross pathological defects in the whole brain and brain sections demonstrates CPH's potential to protect against PPA-induced neurological impairments. However, additional genetic study and immunohistochemical investigation are necessary to explain the underlying pathways governing such interactions. Indeed, PI3K/AKT/ mTOR can be used as a therapeutic target in combination with other standard pharmacological interventions.