Neuroprotective Effects of Berbamine, Bergepten, and Carveol on Paclitaxel-Induced Peripheral Neuropathy

doi:10.21203/rs.3.rs-1433882/v2

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Neuroprotective Effects of Berbamine, Bergepten, and Carveol on Paclitaxel-Induced Peripheral Neuropathy

https://doi.org/10.21203/rs.3.rs-1433882/v2

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Background: The current study investigates the effects of natural chemicals like berbamine (BBR), bergepten (BRG), and carveol (CAR) on paclitaxel (PTX) associated neuro-inflammatory pain.

Methods: The induction was carried out by administering 2 mg/kg PTX for a week with a day gap. On 7th, 14^th and 21^st days, behavioral testing was executed, and tissue was removed for molecular analysis.

Results: Treatment attenuates thermal hyperalgesia, improved latency of falling, normalized the changes in body weight and increased threshold for pain sensation. The drugs increased the protective GSH and GST levels in the sciatic nerve and spinal cord, while lowering iNOS and LPO. H and E and IHC examinations confirmed that the medication reversed the abnormal alterations.

Conclusion: The aforementioned natural substances inhibit COX-2, TNF-α and NF-κB over-expression, as evidenced by enzyme-linked immunosorbent assay (Elisa) and western blot, and hence provide neuroprotection in chronic constriction damage.

In-silico

chronic constriction injury

hyperalgesia

neuroprotective

Paclitaxel is known for its effects against various tumors [1]. Chemotherapy is one of the preferable tools in combating with cancer and improving the quality of life with consequent reduction in number of deaths worldwide. Researcher expects that there will be 35% increase in cancer survivors till 2022 with effective use of chemotherapy [2]. Peripheral neuropathy is one of the most common dose limiting adverse effect, associated with the utilization of anticancer drugs [3]. Texane is an important class of chemotherapeutic agents clinically utilized in diverse group of cancers since decades. Paclitaxel is a drug belongs to a class texane which is approved by the regulatory authority for lung cancer, breast cancer, prostate cancer and ovarian cancer [4]. Its utilization is associated with development of dose dependent neurotoxicity. Development of cold allodynia, mechanical allodynia, and burning, shooting and tingling sensation are the reason of discontinuation of therapy [5]. The current research project is aimed to investigate the therapeutic potential of natural compounds including berbamine, bargepten and carveol in paclitaxel-induced neuropathic pain. Herbal constituents have been extensively researched for their potential in preventing the paclitaxel-induced pain. Different natural agents like resveratrol [6], gallic acid [7], puerarin [8] and goshajinkigan [9] possess potential to attenuates paclitaxel-induced painful symptoms. Chemically berbamine bisbenzyl isoquinoline alkaloid obtained from the Chinese medicinal plant Berberis amurensis Rupr. It various potential including anticancer, anti-inflammatory and multi drug resistance or synergistic affect when used with other drugs [10]. Psoralen is a class of natural compounds obtained from plant Ammi majus collectively called as furocouamrin. Furocouamrin comprised of methoxsalen. Bergepten is one of the 5-methoxsalenpsoralen which has been previously investigated in various cancer, vitiligo and psoriasis [11]. Carveol is obtained from a plant named as Caraway. It is cultivated throughout the world and is well known source of essential oil. In addition, pinene, thujene, phellandrene, camphene, limonene, and carvone are also the constituents of Caraway [12].

2.1. Chemicals

Proteinase K, PBS tablets, hydrogen peroxide (H₂O₂), formaldehyde, GSH, DTNB), CDNB), Mouse monoclonal anti-p-NF-κB, TNF-α, COX-2, Avidin-biotin complex kit, DAB, trichloroacetic acid (TCA), horse radish peroxidase-conjugated secondary antibodies, mounting media, COX-2, p-NF-κB, TNF-α elisa and protien assay kit, skim milk and Bolt Mini Gels, X-ray film.

2.2. Animals

Adult male rats weighing 220–250 g (age 10–12 weeks) were procured from Riphah International University's animal house kept under of control environment temperature (25-30^oC) and humidity. The experimental protocols for handling and dosing of animal were priorly by Riphah Institute of Pharmaceutical Sciences (Ref. No. REC/RIPS/2019/28).

2.3. Paclitaxel-induced neuropathic pain

To develop paclitaxel-induced peripheral neuropathic pain, adult male Sprague Dawley rats were treated with paclitaxel. The available pharmaceutical grade of paclitaxel is 6 mg/ml which is further diluted (1:1 cremaphor: ethanol) to 1 mg/ml and will be injected intraperitoneally (i.p) at a dose of 2 mg/kg every other day (1, 3, 5, 7) with a total of four injections and final total dose of 8 mg/kg to develop pain. Paclitaxel was administered i.p earlier on day 1, 3, 5, and 7 to develop peripheral neuropathy. After one week on day 14th compounds was administered i.p and behavioral testing (thermal hyperalgesia and latency of falling) was be performed on the same day 30 minutes after treatment with compound. Behavioral test including body weight and mechanical hyperalgesia was determined on day 8th, 12th, 16th, and 20th and sciatic nerve (SN) and spinal cord (SC) were extracted [13].

2.4. Thermal pain sensation

Acclimatized animal’s paw sensitivity to heat was determined on hot plate (54 ± 1 ⁰C) with cut off time of time of 15 and 60 seconds. Thermal hyperalgesia was determined on day 14th after a single dose of compound. Three reading were collected at 90, 150 and 270 minutes after administration of compound [14, 15].

2.4.1. Latency of falling

The latency of falling was recorded through rotarod apparatus. Animals were first trained for 3 consecutive days to be able to remain on rod for 60 seconds with 5 minutes cut off time before starting the experimentation. After recording the baseline reading (day 0) the latency of falling was recorded. Latency of falling was also determined at 60, 120, 180 and 240 minutes after treatment with compound [16].

2.4.2. Body weight analysis

The animals were first weight at baseline before starting the experimentation. The weight of each animal was recorded after therapy on day 8, 12, 16 and 20 and the results were interpreted [16].

2.4.3. Pain threshold

Von-Frey filament apparatus was used to analyze pressure induced-pain threshold [17]. After its perpendicularly application on subplanter region, the response of the animal was noticed.

2.4.4. Oxidative stress markers

The samples after homogenization in phosphate buffer containing phenylmethylsulfonyl fluoride as protease inhibitor, was centrifuged at 4000×g for 10 minutes at 4 ^OC and supernatant was collected and processed for determination of GSH [18] and GST [19], lipid peroxidation [20] and Nitric oxide NO [21].

2.6. Hematoxylin and eosin staining

De-paraffinization was performed through xylene (100%), ethanol (95 and 70%) and distilled water. Washed with PBS and treated with hematoxylin for 10 minutes. Slides dipped in 1% each HCl, immersed in eosin for 5–10 minutes, washed, dried and fixed in xylene [22].

2.7. Immuno-histopathological evaluation

Paraffinized slides were washed with xylene, ethanol, distal water and then in PBS. Proteinase K was applied for antigen retrieval. Endogenous peroxidases were blocked by hydrogen peroxide. Normal goat serum (5%), primary antibody secondary antibody and ABC were applied followed. Finally slides were exposed to 0.1% DAB (diaminobenzidine peroxidase) solution and dehydrated by dipping in xylene/ethanol and dried in open air [23].

2.8. ELISA

TNF-α COX-2 and NF-κB were determined by using ELISA kits as explained by [24, 25]. Tissue was treated with designated antibodies expression was determined by using ELISA micro-plate reader.

2.9. Western blot assay

Equal amounts of protein (15–30 µg) were electrophoresis using 4–12% bolt Mini Gels 5% (w/v) skim milk to block the membrane and reduce nonspecific binding and incubated with primary antibodies at 4°C for 12 to 16 hours. After reaction with a horseradish peroxidase (HRP)-conjugated secondary antibody and proteins were detected using enhanced chemiluminescence detection. The X-ray films were scanned, and the optical densities of the bands were analyzed through densitometry using the computer-based Sigma Gel program, version 1.0 [26].

2.10. Statistical analysis

Data are presented as mean ± SEM. H and E Staining behavioral data, oxidative stress data were analyzed using One-Way ANOVA, followed by post hoc Tukey’s test using GraphPad Prism-6 software. The P value was calculated through GraphPad Instat software. ImageJ software was used to analyze the morphological data. One-Way ANOVA followed by post hoc Tuckey’s test was performed for ELIZA and western blot. Symbols ^# or * represents significant difference values p < 0.05, ^## or ** represent p < 0.01 and ^### or *** represents p < 0.001 values.

3.1. Effect on thermal pain sensation

Berbamine at 5 mg/Kg elevate (P < 0.01) heat latency (HL) on day 14th and 21st vs. PTX. At 15 mg/Kg, it improved (P < 0.001) HL on day 14th and 21st vs. PTX. Bergepten at 50 mg/Kg enhanced (P < 0.01) HL on day 14th and 21st vs. PTX. At 100 mg/Kg, HL was increased (P < 0.001) HL on day 14th and 21st vs. PTX. Carveol at 10 mL/kg increased (P < 0.01) HL on day 14th and 21st vs. PTX. At 20 mL/Kg, carveol increased (P < 0.001) HL on day 14th and 21st vs. PTX. PTX at 2 mg/Kg decreased HL on day 7th, 14th and 21st with P < 0.001 vs. control presented in Fig. 1.

3.2. Effect on latency of falling

Latency of falling (LF) or motor coordination was determined by rotarod apparatus. Berbamine at 5 mg/Kg augment (P < 0.001) LF on day 14th and 21st vs. PTX. At 15 mg/Kg, it shot up (P < 0.001) LF on day 14th and 21st vs. PTX. Bergepten at 100 mg/Kg, enhanced (P < 0.001) LF on day 14th and 21st vs. PTX. Carveol at 10 and 20 mL/kg raised LF at P < 0.01 and P < 0.001 LF on day 14th and 21st respectively vs. PTX. PTX at 2 mg/Kg decreased LF on day 7th, 14th and 21st with P < 0.001 vs. control presented in Fig. 2.

3.3. Effect on changes in body weight

Changes in body weight were also determined by utilizing weighing balance. Berbamine at 5 mg/Kg increased (P < 0.05) body weight on day 14th and 21st vs. PTX. At 15 mg/Kg, it increased (P < 0.01) body weight on day 14th and 21st vs. PTX. Bergepten at 50 mg/Kg, increased (P < 0.05) body weight on day 21st vs. PTX. Bergepten at 100 mg/Kg, increased (P < 0.01) weight on day 14th and 21st vs. PTX. Carveol at 10 mL/kg increased (P < 0.05) body weight on day 14th and 21st vs. PTX. Carveol at 20 mL/kg increased (P < 0.01) body weight on day 14th and 21st vs. PTX. PTX at 2 mg/Kg decreased latency of falling on day 7th, 14th and 21st with P < 0.001 vs. control presented in Fig. 3.

3.4. Effect on von-frey-induced pain

Berbamine at 5 mg/Kg increased (P < 0.05) PWT on day 21st vs. PTX. At 15 mg/Kg, berbamine improved (P < 0.01) PWT on day 14th vs. PTX. Berbamine at 15 mg/Kg, enhanced (P < 0.001) PWT on day 21st vs. PTX. Bergepten at 50 mg/Kg, hike (P < 0.05) PWT on day 21st vs. PTX. Bergepten at 100 mg/Kg, elevate (P < 0.01) PWT on day 14th vs. PTX. Bergepten at 100 mg/Kg, augment (P < 0.001) PWT on day 21st vs. PTX. Carveol at 10 mL/kg raised (P < 0.001) paw PWT on day 14th and 21st vs. PTX. Carveol at 20 mL/kg increased (P < 0.001) PWT on day 14th and 21st vs. PTX. PTX at 2 mg/Kg decreased PWT on day 7th, 14th and 21st with P < 0.001 vs. control presented in Fig. 4.

3.5. Effect on oxidative stress enzyme

Berbamine, bergepten and carveol were scrutinized for effect against enzymes contributing oxidative stress. PTX significantly down regulate the level of GSH and GST with P < 0.001 in PTX-induced neuropathic pain group. Berbamine significantly increased GSH (P < 0.01) and GST (P < 0.01) in SN. It also increased protective markers GSH (P < 0.01) and GST (P < 0.01) in SC. Bergepten improved GSH (P < 0.01) and GST (P < 0.01) in SN. It promotes GSH (P < 0.01) and GST (P < 0.01) in SC. Carveol diminished free radical formation by improving the GSH (P < 0.01) and GST (P < 0.01) in SN and in addition enhanced GSH (P < 0.01) and GST (P < 0.01) in SC presented in Table 1 and Table 2. The effect of berbamine, bergepten and carveol on iNOS and LPO was explored. It was found that destructive oxidative agents including iNOS and LPO were seen raised (P < 0.001) in PTX group. Berbamine notably minimized iNOS (P < 0.05) and LPO (P < 0.05) in SN. It also decreased iNOS (P < 0.01) and LPO (P < 0.01) in SC. Bergepten attenuated iNOS (P < 0.05) and LPO (P < 0.05) in SN. It also reduced the level of iNOS (P < 0.01) and LPO (P < 0.001) in SC. Carveol reduced iNOS (P < 0.05) and LPO (P < 0.05) in SN and in same fashion down regulate iNOS (P < 0.01) and LPO (P < 0.001) in SC as Table 1 and Table 2.

Table 1

Effect of berbamine (BBM), bargepten (BRG) and carveol (CAR) on expression of GSH, GST, iNOS, and LPO in sciatic nerve. Data is expressed as mean ± SEM, n = 6. One way ANOVA with post-hoc Tukey test. ^**P < 0.05, ^**P < 0.01 indicates significant difference vs. PTX and ### P < 0.001 indicates significant difference vs. saline.
Group	GSH	GST	iNOS	LPO
Saline (10 ml/Kg)	48.22 ± 2.1	43.88 ± 1.5	34.22 ± 3.1	62.43 ± 1.8
PTX (2 mg/kg)	7.22 ± 1.7^###	10.53 ± 2.6^###	105.32 ± 3.2^###	286.66 ± 2.2^###
PTX + BBM (15 mg/kg)	30.29 ± 2.2**	36.10 ± 3.4**	66.21 ± 1.6*	112.36 ± 2.8*
PTX + BRG (100 mg/kg)	24.24 ± 2.2**	29.14 ± 2.4**	78.11 ± 2.2*	128.10 ± 1.8*
PTX + CAR (20 mg/kg)	28.14 ± 1.2**	33.50 ± 1.4**	56.11 ± 2.6*	132.16 ± 3.8*

Table 2

Effect of berbamine (BBM), bargepten (BRG) and carveol (CAR) on expression of GSH, GST, iNOS, and LPO spinal cord. Data is expressed as mean ± SEM, n = 6. One way ANOVA with post-hoc Tukey test. ^**P < 0.01, ^***P < 0.001 indicates significant difference vs. PTX and ### P < 0.001 indicates significant difference vs. saline.
Group	GSH	GST	iNOS	LPO
Saline (10 ml/Kg)	43.22 ± 1.8	35.71 ± 2.1	41.35 ± 1.2	62.33 ± 1.3
PTX (2 mg/kg)	9.41 ± 1.5^###	7.53 ± 3.4^###	98.32 ± 2.3^###	195.68 ± 1.6^###
PTX + BBM (15 mg/kg)	33.21 ± 1.4**	26.14 ± 3.2**	52.41 ± 2.5**	76.66 ± 1.0**
PTX + BRG (100 mg/kg)	25.21 ± 2.6**	26.12 ± 3.2**	73.41 ± 1.5**	113.22 ± 1.8***
PTX + CAR (20 mg/kg)	29.14 ± 0.6**	22.22 ± 1.2**	63.11 ± 1.5**	103.36 ± 2.0***

3.7. H and E staining examination

In the saline group, H and E staining revealed assembled cellular pattern, no infiltration, and intact intracellular spaces without any signs of edema. PTX-induced pathological alterations, injury of the SN and SC with various forms of neuronal injuries in the form of increased intracellular spaces, infiltration, and edema disorderly pattern in the PTX group (P < 0.001) compared to the saline group. Treatment with berbamine, bergepten and carveol reversed the PTX-induced injury and pathological progression significantly in SN (P < 0.001) and SC (P < 0.001) vs. PTX group as shown in Fig. 5.

3.8. IHC analysis

The findings of IHC staining are presented in Figs. 6 and 7. COX-2 (P < 0.001), TNF-α (P < 0.001), and NF-κb (P < 0.001) notably seen raised in PTX group compared to the saline group in SN and SC. Berbamine attenuated COX-2 (P < 0.05), TNF-α (P < 0.05), and NF-κb (P < 0.01) significantly in SN. Bergepten vanished COX-2 (P < 0.05), TNF-α (P < 0.05), and NF-κb (P < 0.01) significantly in SN. Carveol suppressed COX-2 (P < 0.01), TNF-α (P < 0.01) and NF-κb (P < 0.01) significantly in SN presented in Fig. 6. Berbamine attenuated COX-2 (P < 0.01), TNF-α (P < 0.05) and NF-κb (P < 0.05) significantly in SC. Bergepten down regulates COX-2 (P < 0.01), TNF-α (P < 0.05), and NF-κb (P < 0.01) significantly in SC. Carveol reduced COX-2 (P < 0.01), TNF-α (P < 0.05), and NF-κb (P < 0.01) significantly in SC presented in Fig. 7.

3.9. Effects on inflammatory marker (ELIZA)

As shown in Fig. 8 we studied the effects of berbamine, bergepten, and carveol on the expression of COX-2, TNF-, and NF-b. All the three mediators including COX-2, TNF-α and NF-κb were found raised in PTX group vs. saline in SN (P < 0.01) and SC (P < 0.01). Berbamine at 1 mg/kg minimize expression of COX-2 with P < 0.05 in SN and SC. At 5 mg/kg, it suppressed COX-2 expression in SN and SC with P < 0.01, at 15 mg/kg, it reduced COX-2 expression in SN and SC with P < 0.001. Bergepten at 25 mg/kg decreased COX-2 with P < 0.05 in SN. At 50 mg/kg it down regulates COX-2 with P < 0.001 in SN, and at 50 mg/kg it reduced COX-2 with P < 0.05 in SC. Bergepten at 100 mg/kg minimize COX-2 with P < 0.001 in SN and SC. Carveol at 10 mg/kg decline COX-2 with P < 0.01 in SN and SC. At 20 mg/kg it subsides COX-2 with P < 0.01 in SN and SC as shown in Fig. 8a. Berbamine at 1 mg/kg decreased TNF-α with P < 0.05 in SN and SC. At 5 mg/kg it down regulates TNF-α with P < 0.001 in SN and SC, and at 15 mg/kg it diminishes TNF-α with P < 0.001 in SN and SC. Bergepten at 25 mg/kg decreased TNF-α with P < 0.01 in SN and SC. At 50 mg/kg it down regulates TNF-α with P < 0.01 in SN and SC. Bergepten at 100 mg/kg drop TNF-α with P < 0.001 in SN and SC. Carveol at 10 mg/kg decreased expression of TNF-α with P < 0.05 in sciatic nerve. At 20 mg/kg it down regulates TNF-α with P < 0.001 in SN and SC as shown in Fig. 8b. Berbamine at 1 mg/kg decreased NF-κb with P < 0.05 in SN and SC. At 5 mg/kg it down regulates NF-κb with P < 0.01 in SN and SC, and at 15 mg/kg it reduced NF-κb with P < 0.001 in SN and SC. Bergepten at 25 mg/kg decreased NF-κb with P < 0.05 in sciatic nerve. At 50 mg/kg it down regulates NF-κb with P < 0.001 in SN and with P < 0.05 in SC. Bergepten at 100 mg/kg decreased expression of NF-κb with P < 0.001 in SN and SC. Carveol at 10 mg/kg decreased NF-κb with P < 0.01 in SN and SC. At 20 mg/kg it down regulates NF-κb with P < 0.01 in SN and SC as shown in Fig. 8c.

3.10. Western blot findings

The inflammatory markers were further investigated in both sciatic nerve and spinal cord through western blot analysis and the results are presented in Fig. 9. The results revealed that all the inflammatory markers were significantly noticed raised with P < 0.001 in collected samples vs. saline. Berbamine down regulate COX-2 with P < 0.01, TNF-α with P < 0.05 and NF-κb with P < 0.05. Bergepten attenuate COX-2 with P < 0.01, TNF-α with P < 0.01 and NF-κb with P < 0.001. Carveol decreased COX-2 with P < 0.01, TNF-α with P < 0.01 and NF-κb with P < 0.01) in sciatic nerve sample of vs. PTX group. Berbamine down regulate COX-2 with P < 0.01, TNF-α with P < 0.01 and NF-κb with P < 0.01. Bergepten attenuated COX-2 with P < 0.01, TNF-α with P < 0.01 and NF-κb with P < 0.01. Carveol decreased expression of COX-2 with P < 0.01, TNF-α with P < 0.05 and NF-κb with P < 0.01 in spinal cord sample vs. PTX group as shown in Fig. 9.

Paclitaxel induced peripheral neuropathic pain (PIPNP) is one of the most serious and debilitating adverse drug reaction in more than 75% of cancer therapies [27]. In literature, various protocols are available for induction of paclitaxel-induced peripheral neuropathy in rats like preventive approach (neuropathy is established in the presence of drug) and pre- established neuropathy (neuropathy is established in the absence of drug). The later model was utilized to securitize the potential three compounds natural compounds including berbamine, bergepten and carveol in chemotherapy-induced pain. Paclitaxel, at 2 mg/Kg on day 1, 3, 5 and 7 to develop neuropathy and the administration of compound was hold till confirmation and development of neuropathic pain. The confirmation of pain threshold was analyzed by various behavioral protocols like thermal hyperalgesia, latency of falling, body weight and mechanical allodynia. As the cancer cases has been increasing and there is an intense need for development of drugs which are associated with less toxic and more efficacious profile to refine patient quality of life and reverse PIPNP. Many drugs have been researched for this purpose like berberine [28], icariin [29], and melatonin [30]. Several types of targets were also investigated including nuclear factor-2 and poly ADP-ribose polymerase involved in anticancer-induced neuropathic pain [31, 32]. It has been also known that NF-κB, nitric oxide, COX-2, IL-1β, IL-6, and TNF-α are seen over expressed in CIPNP/PIPNP [33]. Some receptors and channels have been identified confirming their active participation in CIPNP including Toll like receptors (TLR-1 TLR-2 and TLR-4) and transient receptor potential family of ion channels like (TRPV1 and TRPV4) [13, 34, 35]. It is well known that all the neuropathies are associated with oxidative stress. Paclitaxel induced oxidative stress is well reported mechanism of development and maintenance of PIPNP [36]. Its utilization results in depletion of GSH and GST which are involved in inhibiting the metabolites of toxic compounds in chemotherapy induced pain [37, 38]. In addition to GSH and GST, some damaging factors are also seen to be elevated during PTX therapy like LPO and iNOS [39], and play their role in promoting pain sensation. We also perform paclitaxel-induced peripheral neuropathy to evaluate the effect three natural compounds. Theme of scheme of the present research work is presented in Fig. 10. One of the important aspects of paclitaxel-induced neuropathy is the disturbance in latency of falling [40], body weight [41], and mechanical allodynia [42]. The behavioral analysis of our compounds reveals that berbamine, bergepten and carveol are corrected the behavioral deficit in paclitaxel-induced peripheral neuropathic pain as is shown in Fig. 1 to 4. The results obtained are complying with the previously literature. Many researchers have focused and reported morphological changes at neuronal level of sciatic nerve and spinal cord in paclitaxel-induced neuropathy[43]. We also perform morphological studies of two tissues, and it has been found that PTX was involved in promoting the disease progression by mediating neuronal degeneration, disturb myelin sheath formation and creating vacuolation. Treatment with the mentioned natural compounds reversed PTX-induced damages as shown in Fig. 5 to 7 and thus plays their role to be declared as neuroprotective agents. Molecular study is primarily playing role in determining the expression of various type of inflammatory mediators or transcription factors. These molecular techniques involved enzyme linked immunosorbant assay and western blot which are utilized for detecting the expression levels of endogenous markers [44]. Our study also progresses with the evaluation of different types of inflammatory markers including COX-2, TNF-α and NF-κB who’s up regulation is most commonly associated with chemotherapy-induced neuropathic pain. PTX increased the expression of all the aforementioned pathological markers were confirmed by both ELISA and western blot assay. Treatment according to the mentioned protocols down regulated and decreased the expression of COX-2, TNF-α and NF-κb and thus result in attenuation of paclitaxel induced neuropathic pain in animal model.

The overall finding of the study revealed that berbamine, bergepten and carveol reversed paclitexal-induced neuropathic pain by correcting behavioral deficit, improving the level of protective markers like GSH and GST, diminishing the damaging factors like LPO and iNOS, and mean while down regulated the over expressed level of inflammatory mediators like COX-2, TNF-α and NF-κB. These findings explain the neuroprotective effects of berbamine, bergepten and carveol. Further studies are needed to more clearly elaborate the pharmacokinetics and pharmacodynamics profile, stability and dosage form to expand the list of agents effective in management of neuropathic pain.

Berbamine (BBR)

Bergepten (BRG)

Carveol (CAR)

Paclitaxel (PTX)

Glutathione (GSH)

Glutathione S-transferase (GST)

Inducible nitric oxide synthase (iNOS)

Lipid peroxidase (LPO)

Hematoxylin and eosin (H and E)

Immunohistochemistry (IHC)

Cyclooxygenase (COX-2)

Tumor necrosis factor-alpha (TNF-α)

Nuclear factor kappa B (NF-κb)

Enzyme-linked immunosorbant assay (Elisa)

Phosphate buffer saline (PBS)

Hydrogen peroxide (H₂O₂)

5, 5’-dithio-bis-2-nitrobenzoic acid (DTNB)

2, 4-Dinitrochlorobenzene (CDNB) Diaminobenzidine peroxidase (DAB)

Trichloroacetic acid (TCA)

Research and ethical committee (REC)

Intraperitoneally (i.p)

Sciatic nerve (SN)

Spinal cord (SC)

Nitric oxide (NO)

Horseradish peroxidase (HRP)

Heat latency (HL)

Falling (LF)

Paw withdrawal threshold (PWT)

Paclitaxel induced peripheral neuropathic pain (PIPNP)

Chemotherapy-induced peripheral neuropathic pain (CIPNP)

Toll like receptors (TLR)

Transient receptor potential (TRP)

Not applicable

Ethical approval

The experimental protocols for handling and dosing of animal were priorly by Riphah Institute of Pharmaceutical Sciences (Ref. No. REC/RIPS/2019/28).

Consent for publication

All authors are agree to submit the manuscript

Availability of data and material

All the data is incorporated in the manuscript

Competing interest

No competing interest

Funding

Not applicable

Author Contributions

M.F. collected the data and carried out the research; wrote the manuscript and performed the bioinformatics part; A.-u.K., conceptualized and supervised the study; M.U.I and F.A.S. helped in performing the molecular study and act as a co-supervisor in the research. S.L and A.W.K edited and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

The auhors are grateful to Institite of pharmaceutical science, Riphah International University Islamabad, for providing lab fascility to conduct this research work

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No competing interests reported.

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Neuroprotective Effects of Berbamine, Bergepten, and Carveol on Paclitaxel-Induced Peripheral Neuropathy

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Abstract

Figures

1. Introduction

2. Material And Methods

2.1. Chemicals

2.2. Animals

2.3. Paclitaxel-induced neuropathic pain

2.4. Thermal pain sensation

2.4.1. Latency of falling

2.4.2. Body weight analysis

2.4.3. Pain threshold

2.4.4. Oxidative stress markers

2.6. Hematoxylin and eosin staining

2.7. Immuno-histopathological evaluation

2.8. ELISA

2.9. Western blot assay

2.10. Statistical analysis

3. Results

3.1. Effect on thermal pain sensation

3.2. Effect on latency of falling

3.3. Effect on changes in body weight

3.4. Effect on von-frey-induced pain

3.5. Effect on oxidative stress enzyme

3.7. H and E staining examination

3.8. IHC analysis

3.9. Effects on inflammatory marker (ELIZA)

3.10. Western blot findings

4. Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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