Effect of butyrate, a short-chain fatty acid, in repairing brain injury in neonatal rats

doi:10.21203/rs.3.rs-4681954/v1

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Effect of butyrate, a short-chain fatty acid, in repairing brain injury in neonatal rats

https://doi.org/10.21203/rs.3.rs-4681954/v1

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Butyrate, as a microbial metabolite, is considered to have an effect on improving the intestinal microenvironment. At present, it is shown that the brain and gut interact with each other through the gut-microbiota-immune-brain axis. An aberrant gut-microbiota-immune-brain axis in premature infants may aggravate brain injury. However, whether sodium butyrate can improve the microbial-intestinal-brain axis to repair the brain injury in premature infants remains unclear. In this study, we established a neonatal rat hypoxic-ischemic brain injury model and a necrotizing enterocolitis model. It was found that enteritis could lead to the occurrence and aggravation of brain injury, which might be associated with the increased secretion of inflammatory factor interleukin-17 caused by the down-regulation of suppressor of cytokine signaling 1 (SOCS1). Further studies showed that sodium butyrate can up-regulate the expression of SOCS1, and increase the secretion of anti-inflammatory interleukin-10, which may alleviate the neurological dysfunction caused by brain injury. This study provides a new theoretical basis for further exploration of the mechanism of brain injury repair in premature infants.

Biological sciences/Developmental biology

Biological sciences/Immunology

Biological sciences/Neuroscience

Health sciences/Diseases

Health sciences/Neurology

Sodium butyrate

Gut-immune-brain axis

Neonatal hypoxia-ischemia

Necrotizing enterocolitis

Suppressor of cytokine signaling-1

Brain injury in the preterm infants is a severe complication, and it is one of the important causes of life-long neurological disorders, such as cerebral palsy, major cognitive deficits and motor disability, visual and auditory deficits^{[1, 2]}. There are currently very limited treatment options. In clinical practice, only symptomatic treatment can be used, but there is no effect on the outcome of death or disability, and the life-long neurological outcomes do not improve^[3]. The etiology of preterm brain injury is complex and multifactorial and includes antenatal infection, hypoxia-ischemia, nutrition, etc. Factors such as mechanical ventilation, hyperoxia and neonatal infection, may further exacerbate brain injury in premature infants after birth^[4].

At present, research shows that necrotizing enterocolitis (NEC) is associated with the neurological outcomes in preterm infants through mechanisms that remains unclear^[5]. By analyzing 4,239 infants (427 with NEC) from 11 studies, Schulzke et al. found that the odds ratio (OR) of 1.82 (95% CI 1.46–2.27) for long-term neurodevelopmental impairment in infants with NEC ^[6]. Rees et al. analyzed the data from 7,843 preterm infants (821 with NEC) and found infants with NEC were more likely to have neurodevelopmental impairment than the controls (OR 1.58; 95% CI 1.25–1.99)^[7]. The effect of NEC on neurocognitive outcome may partly be mediated by comorbidity such as sepsis^[5]. T lymphocytes are the key mediators of brain injury induced by sepsis, which may be related to bad neurocognitive results^{[8, 9]}. Meanwhile, current animal experiments show that NEC induces T lymphocyte–mediated injury in neonatal mouse brain^[10]. Adult animal models further reveal that enteritis and hypoxic-ischemic (HI) brain injury are associated with the imbalance between regulatory T (Treg) and T helper cell 17 (Th17)^[11–13].The mechanism of these findings may be associated with the production of anti-inflammatory factor IL-10 by Treg cells, inhibiting the inflammatory immune response in the gut and brain, and the production of inflammatory factor IL-17 by Th17 cells, aggravating the gut and brain injury^{[9, 13–15]}. Suppressor of cytokine signaling 1 (SOCS1), a negative regulator of cytokine signaling, plays an important role in the integrity and function of Treg cells by protecting the cells from excessive inflammatory cytokines being converted into Th 17 or Th 1 cells and maintaining IL-10/IL-17 balance ^{[16, 17]}.

By analyzing the stool microbiome profiles in preterm infants, Mohan et al. found NEC may be related to the decrease in the abundance of Firmicutes in intestinal flora ^[19]. Butyrate is a short-chain fatty acid produced by Gram-positive Firmicutes fermentation in the colon^[20], it up-regulates SOCS1,which improves Foxp3 expression in colon Treg cells, and can prevent Treg cells from converting into Th1 or Th17 cells, maintaining the homeostasis of intestinal Treg cells^{[16, 21, 22]}. Although, the potential neuroprotective of sodium butyrate (SB) after neonatal hypoxia-ischemia has been shown^[23], the specific mechanism of SB in alleviating brain injury in premature infants caused by enteritis remains unclear. Therefore, we established hypoxic-ischemic brain injury model and enteritis model in neonatal rats to determine the potential mechanisms by which enteritis could lead to injury in the brain and how SB alleviates brain injury.

Animals

Newborn male or female Sprague-Dawley (SD) rats weighing 6–7 g were purchased from Experimental Animal Center of North Sichuan Medical College. All operations performed on the animals were approved by the Animal Ethics Committee at North Sichuan Medical College, and all experiments were performed in accordance with Committee guidelines and regulations. The animal experiments complied with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines, and all efforts were made to minimize animal suffering and the number of animals used^[24].

Rats were fed by dams for 21 days after birth and had free access to food and water after 21 days under controlled conditions (12-h/12-h light/dark cycle at 40–70% relative humidity and 23 ± 2°C).

Experimental design

One hundred and fifty-two rats were randomized into five groups regardless of sex: Control, HI, NEC, NEC + HI, and SB-treated groups (16 rats in control, 17 rats in HI, 21 rats in NEC, 34 rats in NEC + HI, and 64 rats in SB-treated groups). The main purpose of this study to determine the potential mechanisms by which enteritis could lead to injury in the brain and how SB alleviates brain injury. In fact, our main experimental group was NEC + HI, while the NEC and HI groups existed as a control group. The control group was used as a reference without any interference.

Establishment of the experimental model in rat

HI: HI brain injury in premature infant model was produced in 3-day-old rats of either sex by a permanent unilateral common carotid artery ligation, followed by systemic hypoxia. Rats were anesthetized by isoflurane inhalation (4% induction, 2% maintenance). When the rat lost consciousness completely, the left common carotid artery was separated out through incision of the neck skin to be fully exposed, where both sides were ligated with a surgical wire, and a cut made between the two ligatures. The incision was then sutured with monofilament nylon. Anesthesia lasted on average 5 min. After surgery, the rat pups were returned to their home cage for 1 h to recover. Later, the animals were placed for 2.5 h in a hypoxic chamber containing 7.8% oxygen balanced with nitrogen with controlled humidity and temperature maintained at 35°C^[25].

NEC: Premature infant enteritis model was started 2 h after birth, pups were given 3% dextran sodium sulfate 0.01 ml/g (Sigma Aldrich, St. Louis, MO, USA) by intragastric administration every 8 h for three days. After irrigation of the stomach, pups were sent back to their mother^[26].

NEC + HI: Enteritis + HI brain injury in premature infant model was established by given 3% DSS by gavage 3 times a day for 3 consecutive days from birth, and hypoxia–ischemia treatment (treatment method is the same as HI model) was administered on the third day^[27].

SB-treated: Except the control all groups received intragastric administration of SB 300 mg/kg (Sigma Aldrich, St. Louis, MO, USA) once a day for 5 days after the model was established^{[28, 29]}.

Control: Normal rats without any intervention.

During the treatment, 1 rat in HI, 1 rat in HI + SB, 5 rats in NEC, 3 rats in NEC + SB, 18 rats in NEC + HI, and 12 rats in NEC + HI + SB groups died. Finally, a total of 98 rats (14 rats in each group) were included in the subsequent analysis. Forty-two rats (3 rats in each group) were anesthetized and sacrificed by decapitation at 24 h and 48 h after treatment, and the plasma, CSF, intestinal and brain tissues were harvested for WB and Elisa experiments. Thirty-five rats (5 rats in each group) were used for evaluating P28 spatial memory performance and P8 examination of neurobehavioral development. Twenty-one rats (3 rats in each group) were anesthetized and sacrificed by decapitation at P8, and used for pathological examination.

Morris water maze

At 28 days after birth, spatial memory performance was evaluated by using a Morris water maze (180 cm in diameter, 45 cm high) virtually divided into four equal imaginary quadrants by Top Scan software (CleverSys Inc., Reston, VA, USA). The water was darkened with ink. The platform (10 cm in diameter) was 1cm above the water surface. Training consisted of a swim followed by a 30 s platform sit. The escape latency to find the platform was measured for individual animals on each day. The experimenter guided rats to the platform if they did not find it within 120 s ^[30].

Short-term neurofunctional outcome

The examination of neurobehavioral development was performed for all rat pups from P8 to P21 and was carried out daily between 10 a.m. and 12 a.m. The day of eye opening of the rat pups was recorded. Pups were tested for the following neurological reflexes: (1) Negative geotaxis: this test examines the sensorimotor function of neonatal rats. Rat pups were placed upside down in the middle of a slope (45°) of 30 cm. The latency to turn 180° to an upward direction was recorded. When the animal turned to go up, the time (seconds) it took to reach the top was recorded. If the maximum duration of the recording was less than 30s, the test was considered positive. (2) Sensory reflex: the pups' ears and eyelids were touched with a cotton swab, and the first day of the ear twitch reflex and eyelid contraction was recorded. (3) Auditory startle: the first day of the startle response to a clapping sound was noted. (4) Limb grasp: the forelimbs were touched with a thin rod, and the first day of grasping onto the rod was recorded. (5) Gait: the animals were placed at the center of a silver metal circle (Ø = 13 cm). The day they started to move out of the circle with their two front paws was recorded, and the time (in seconds) it took the animals to leave the circle was estimated. If the animal leaves the circle within 30 seconds, the test is considered positive. To evaluate the development of neurological reflexes in rats, the earlier the positive reflex appears, the greater the capacity is for the development of neurological reflexes^[30].

Cerebrospinal fluid and plasma biomarker analysis

The rat pups were anesthetized by isoflurane inhalation (4% induction, 2% maintenance) and fixed by one hand with the head positioned at a 90° angle. A sagittal incision of the skin was made below the occiput. The dura mater of the cisterna magna was then penetrated with an 8-cm-long glass capillary, which had a narrowed tip with an inner diameter of about 0.3 mm so that the cerebrospinal fluid (CSF) flowed into the capillary. Blood was collected from the carotid artery in EDTA-K2 Vacutainer tubes. After collection, each sample was centrifuged at 3000 rpm for 10 min at 4°C, and the supernatant was aliquoted and stored at − 80°C for biochemical assays^[31]. Approximately 15 ul and 0.1 ml of CSF and plasma were collected each time. ELISA kits (IL-10 2R-KMLJr30194, and IL-17 2R-KMLJr30201 Camilo, Nanjing, Jiangsu, China) were used to detect IL-10/IL-17 in plasma and CSF.

Western blot analysis

Rats were anesthetized with isoflurane and sacrificed by cervical dislocation, and the intestine and brain were collected on dry ice. The scalp was cut in the middle along the top of the head to the nose, and the skull was pushed aside, leaving the brain exposed. The whole brain tissue was separated from the optic nerve and brainstem and then placed in a specimen box. For intestinal tissue collection, we first opened the abdominal cavity from the ventral midline and rapidly separated the mesentery and blood vessels. Then, the antral and duodenal junction, mesenteric reticulum, and rectum near anus were identified and quickly cut, followed by the removal of the entire intestinal tissue. The intestine tissue was washed with sterile saline at 4°C and also placed in a specimen box. All tissue samples were stored at − 80°C until used. Brain and intestinal tissues were homogenized in RIPA lysis buffer. Lysates were clarified by centrifugation at 12,000 rpm for 10 min at 4°C. The supernatant was collected, and protein concentration was determined using a BCA protein assay kit (Beyotime, Hangzhou, Zhejiang, China). Samples (30 µg protein) were run on 10–15% SDS-PAGE gels and transferred onto a polyvinylidene fluoride membrane, which was subsequently blocked with 5% skimmed milk for 2 h. After blocking, the membranes were incubated with Rabbit monoclonal anti-Occludin(A12621, 1:2000 dilution; ABclonal, Wuhan, Hubei, China, RRID: AB_2759466), rabbit polyclonal anti-SOCS1 (A7754, 1:2000 dilution; ABclonal, Wuhan, Hubei, China, RRID: AB_2772339), and mouse monoclonal anti-β-Tubulin (AC021,1:5000 dilution; ABclonal, Wuhan, Hubei, China, RRID: AB_2773004) antibodies and then incubated with biotin-labeled goat anti-mouse IgG(H + L) (Ab6789, 1:5000 dilution; Abcam, Cambridge, UK, RRID: AB_955439) and biotin-labeled goat anti-rabbit IgG(H + L) (S0001, 1:5000 dilution; Affinity Biosciences Cincinnati, OH, USA,RRID: AB_2839429). Immunoblot signals were visualized using the ECL chemiluminescence kit (Zen-bio, Shanghai, China). To verify an equal loading of protein per lane, the β-Tubulin antibody was used as an internal control for each immunoblotting. After the images had been captured, further analysis was conducted using Tanon GIS chassis control software V2.0 (Tanon, Shanghai, China). The relative expression of the proteins was presented as the gray value ratio between the band of protein to be assessed relative to that of β-Tubulin.

Hematoxylin and eosin (H&E) staining

Rats were anesthetized with isoflurane and sacrificed by cervical dislocation. The collected brain and intestinal tissues were histopathologically observed using H&E staining. The preserved tissue samples were prepared as paraffin sections after fixation, dehydration, embedding, and sectioning. Dewaxed paraffin sections received the hematoxylin solution and were incubated for 5 min at room temperature. After washing and differentiation for 30 s, paraffin sections were treated with eosin for 2 min, followed by xylene decolorization and neutral gum sealing. Finally, a light microscope (800 ×) was used to observe the pathological features of the periventricular tissues. Pathological changes in brain tissue were identified according to the grading method by Uehara et al^[32] .

Statistical analysis

SPSS 26.0 software (SPSS Inc., Chicago, IL, USA, RRID: SCR_019096) was used for the statistical analysis of the data obtained. Comparisons between animal groups were performed using one-way analysis of variance (ANOVA) or repeated measures ANOVA (RM-ANOVA) followed by the LSD test for multiple comparisons. The data were considered significant at P < 0.05.

SB alleviates brain injury

Compared to brain tissue of the control group, we found that the white matter fibers around the right ventricle were loose and disordered in the HI and NEC group. Brain tissues in the NEC + HI group showed loose and irregularly arranged fibrous structures and formed mesh-like softening foci (Fig. 1).

To be quantified (Table 1), compared with the control group the brain injury score increased in NEC (1 ± 0.58, P < 0.05), HI (1.17 ± 0.29, P < 0.01), and NEC + HI (1.83 ± 0.29, P < 0.001) groups. NEC + HI group has the highest score in the model group (P < 0.05). After 5 days of intragastric administration of sodium butyrate, the pathological manifestations of these experimental groups were alleviated, the brain injury score decreased in NEC + SB (0.67 ± 0.47, P = 0.291), HI + SB (0.5 ± 0.5, P < 0.05), NEC + HI + SB (1.17 ± 0.69, P < 0.05), suggesting that sodium butyrate may promote the repair of brain injury.

Table 1

Comparisons of brain injury scores between normal, HIBI, NEC + HI, SB-treated groups
	Group	HI	NEC	NEC + HI	Control	F	P
Brain injury score	Model	1.17 ± 0.37^{** &}	1 ± 0.58^{* &}	1.83 ± 0.37^***	0.17 ± 0.37	7.56	<0.001
	SB-treated	0.5 ± 0.5^#	0.67 ± 0.47	1.17 ± 0.69^#

HI hypoxic-ischemic brain injury, NEC necrotizing enterocolitis, SB-treated sodium butyrate-treated

*Compared with control; #compared with model; & compared with NEC + HI

*P < 0.05, **P < 0.01, ***P < 0.001, &P < 0.05, #P < 0.05 indicate statistical significances

SB reduced the inflammatory status in the central and periphery.

We analyzed the IL-10 and IL-17 levels in plasma and CSF of each group at 24 h and 48 h after model establishment, and took generating an IL-17 to IL-10 level ratio as an inflammatory status marker. As shown in Fig. 2, the higher the ratio of IL-17/IL-10, the higher inflammatory status. At 24 h and 48 h in groups NEC (Fig. 2C), HI (Fig. 2B) and NEC + HI (Fig. 2D), IL-17/IL-10 level in plasma and CSF increased significantly (P < 0.001, P < 0.01, P < 0.001, respectively). Specifically in NEC + HI, IL-17/IL-10 level increased more than in HI and NEC (P < 0.001, P < 0.05, respectively) in plasma and CSF at 24 h and 48 h (Fig. 2A). In NEC the IL-17/IL-10 level increased more than in HI (P < 0.01) in plasma and CSF at 24 h and 48 h. Further experiments revealed that SB significantly decrease IL-17 level in the plasma and CSF of groups HI, NEC and NEC + HI (P < 0.01, P < 0.001, P < 0.001, respectively), except for the 24 h CSF of HI group (P = 0.056).

SB up-regulates the expression of SOCS1 in the gut and brain

We determined the expression of SOCS1 protein in every group. We found that protein expression of intestinal SOCS1 in NEC (24 h, 0.743 ± 0.051, P = 0.111 ; 48 h, 0.463 ± 0.107, P < 0.001) (Fig. 3C) and NEC + HI (24 h, 0.368 ± 0.049, P<0.001; 48 h, 0.463 ± 0.107, P < 0.001) (Fig. 3G) was decreased after model establishment, protein expression of brain SOCS1 in HI (24 h, 0.501 ± 0.12, P < 0.001 ; 48 h, 0.447 ± 0.104, P < 0.001) (Fig. 3F) and NEC + HI (24 h, 0.193 ± 0.071, P < 0.001 ; 48 h, 0.128 ± 0.049, P < 0.001)(Fig. 3H) was decreased, and protein expression of intestinal SOCS1 in HI (24 h, 1.368 ± 0.132, P = 0.022 ; 48 h, 1.24 ± 0.195, P < 0.203) (Fig. 3D) and protein expression of brain SOCS1 in NEC (24 h, 1.333 ± 0.12, P = 0.107 ; 48 h, 1.276 ± 0.237, P = 0.12) (Fig. 3E) was increased, whereas protein expression of intestinal SOCS1 in HI increased and was the highest at 24 h (1.368 ± 0.132, P < 0.001) (Fig. 3A). Compared with HI and NEC (Fig. 3A, 3B), intestinal and brain SOCS1 protein expression was lowest in NEC + HI at 24 h and 48 h (P < 0.05). It is suggested that the aggravation of brain injury by enteritis may be associated with the downregulation of SOCS1. Further experiments revealed that SB intervention significantly increased SOCS1 protein expression in the brain in HI and NEC + HI (P < 0.01) (Fig. 3F, 3H) as well as in the intestine in NEC and NEC + HI (Fig. 3C, 3G) (P < 0.05).

SB affects the blood-brain barrier and intestinal barrier

By measuring the tight junction protein Occludin associated with barrier function^[33], we indirectly speculated the changes in gut - brain barrier function. As shown in Fig. 4,we found that Occludin protein expression in HI, NEC and NEC + HI decreased significantly in intestine (Fig. 4C, 4E, 4G) and brain (Fig. 4D, 4F, 4H) at 24 h and 48 h (p < 0.01). At the same time, expression of intestinal Occludin protein in NEC (0.467 ± 0.005) at 24 h was lower than that in HI (0.597 ± 0.047, P < 0.05) and NEC + HI (0.734 ± 0.103, P < 0.001), while the expression of brain Occludin protein in HI (24 h, 0.698 ± 0.039, P < 0.05; 48 h, 0.357 ± 0.052, P < 0.001) was lower than in NEC (24 h, 0.829 ± 0.022; 48 h, 0.663 ± 0.031) at 24 h and 48 h (Fig. 4A, 4B). Compared with NEC (0.816 ± 0.081, P < 0.001) and NEC + HI (0.279 ± 0.022, P < 0.05), expression of Occludin protein in intestine of HI (0.164 ± 0.007) was lower at 48h (Fig. 4A). Paradoxically, after administration of SB, we found that SB not only improved barrier function, but also exacerbated the barrier damage. After administration of SB, at 24 h, expression of Occludin protein in intestine and brain in NEC increased (NEC + SB: intestine, 0.580 ± 0.050, P < 0.001; brain, 1.097 ± 0.106, P < 0.01) (Fig. 4C, 4D), but decreased in HI (HI + SB: intestine, 0.225 ± 0.023, P < 0.05; brain, 0.531 ± 0.027, P < 0.05) (Fig. 4E, 4F). At 48 h, expression of Occludin protein in intestine and brain in NEC + HI (NEC + HI + SB: intestine, 0.529 ± 0.038, P < 0.001; brain, 0.925 ± 0.091, P < 0.001) (Fig. 4G, 4H)and HI (HI + SB: intestine, 0.410 ± 0.076 P < 0.001; brain, 0.543 ± 0.053, P < 0.01) (Fig. 4E, 4F)increased, but decreased in NEC (NEC + SB: intestine, 0.066 ± 0.013, P < 0.001; brain, 0.276 ± 0.011, P < 0.001) (Fig. 4C, 4D).

SB partially improves neurological maturation of reflexes in neonatal rats.

Brain injury may lead to the delay of neurological reflexes. As shown in Fig. 5, compared with the control, several neurological reflexes, such as negative geotaxis, ear twitch reflex, auditory startle, hindlimb grasp, day eyes open, and gait reflex appeared significantly later in HI and NEC + HI (p < 0.01) (Fig. 5A, 5B). Besides, the outcomes we found did not show delayed maturation of neurological reflexes in NEC, and the neurological reflexes appearing in NEC + HI were not significantly later than in HI. Therefore, we speculate that enteritis may not cause delayed neurological reflexes. SB improved several neurological reflexes: negative geotaxis, sensory reflex, auditory startle, day eyes open and gait reflex appeared significantly earlier in HI (p < 0.05), and sensory reflex, auditory startle, left eye opening day and gait reflex appeared significantly earlier in NEC + HI (p < 0.05).

Sodium butyrate improves long-term spatial learning ability

To determine the effect of enteritis on cognitive capacities, an RM ANOVA test revealed significant differences in the escape latency of the second between the experimental groups in the Morris water maze. As shown in Fig. 6, the results showed that the platform-finding latency of rats in NEC + HI was the most significantly increased (P < 0.01) (Fig. 6a). SB effectively improved the performance in HI, NEC and NEC + HI in the Morris water maze. (P < 0.001, P < 0.01, P < 0.001) (Fig. 6b, 6c, 6d)。

The occurrence of brain injury in premature infants is influenced by many factors inside and outside the uterus, especially hypoxia-ischemia and inflammation in vivo. However, mild hypothermia therapy, which can improve the long-term neurological prognosis of full-term infants, has limited application in premature infants, and has poor therapeutic effect on brain injury caused by inflammation^[3]. Therefore, it is very important to study the pathogenesis of brain injury in premature infants and explore potential therapeutic targets for improving the long-term prognosis of premature infants.

In this study, we established the NEC and HI brain injury model in the neonatal rat, and SB was used for intervention. We explored whether intestinal damage of premature infants participated in or exacerbated brain injury via the gut-microbiota-immune-brain axis, and how SB reduced brain injury by affecting the gut-brain axis. We previously showed that brain injury was associated with the mediation of inflammatory chemokines, and that NEC + HI shows more severe pathological damage than the NEC or the HI^[27]. In this study, we found that SB can not only alleviate the pathological damage of NEC + HI, but also alleviate the pathological damage of NEC and HI. While the inflammatory chemokine IL-17 was elevated within one day and peaked on the third day after ischemia injury ^[34]. On this basis, we further explored the pathogenesis of enteritis participating in or aggravating brain injury in acute stage. We found that the when enteritis and HI brain injury occurred simultaneously, the expression of SOCS1 protein was lower than in HI brain injury or intestinal injury. SB could increase the expression of SOCS1 protein in both intestine and brain. It was partly similar to the finding by Sitolo et al.^[35] that SB up-regulated the colon SOCS1 protein. Therefore, we speculate that the decrease in SOCS1 protein expression may be the key to the aggravation of brain injury caused by enteritis, while SB may alleviate the aggravation of brain injury caused by enteritis by up-regulating the expression of SOCS1 protein.

SOCS1, a negative regulator of cytokine signaling, is associated with Treg/Th17 balance^{[16, 17]}.Its expression may indirectly lead to the change of the balance between IL-10 and IL-17, and then affect the inflammatory reaction in the brain. We found that whether it was HI, NEC or NEC + HI, the inflammatory status in plasma and CSF increased obviously, especially NEC + HI. This indicates that the decrease in SOCS1 protein expression may lead to the increase of inflammation status in plasma and CSF, further leading to the increase of inflammation status in the central and periphery, finally leading to the aggravation of brain injury. Further studies showed that SB decreased the inflammatory status in plasma and CSF, which also verified the results of the previous studies. Up-regulation of SOCS1 by SB increased IL-10 ^{[16, 21, 22]}.

An adult rat stroke model found that the activated intestinal T cells in the acute stage of stroke exacerbated brain injury by releasing inflammatory mediators into the circulation, instead of directly entering the brain parenchyma^[36]. Therefore, the same mechanism may exist in neonatal rats. The inflammatory factors produced by the abundant T cells in the intestine may enter the brain through the blood-brain barrier and intestinal barrier. We measured Occludin, a tight junction protein associated with brain-gut barriers permeability. The results showed that NEC and HI can increase the permeability of the brain-gut barriers, which makes the brain more vulnerable to inflammatory factors from the circulation. Although both NEC and HI can compromise the function of brain-gut barriers, we also found that the expression level of Occludin protein was not positively correlated with the severity of brain injury. Even in the group of NEC + HI, brain-gut barrier function was not significantly lower than that with HI alone. At present, there is no experiment that can completely explain this phenomenon, but according to the existing findings, we speculate that it may be related to the trafficking of intestinal γδT cells to the meninges. Trafficking of intestinal γδ T cells to the meninges after stroke^[9], which makes γδT cells disappear from intestinal tissue and abandon their intraepithelial support function, further increase intestinal permeability and disrupts the gut barrier^[37]. Besides, γδ T cells secrete IL-17 and exacerbate the damage to brain^[9]. Recent experimental results showed that SB improved the expression of IL-10 in Treg cells and contributed to the recovery of barrier function^[38]. Our study also partially agreed with this. The expression of Occludin protein increased in HI and NEC + HI at 48 h after administration of SB, indicating that the repair of brain-gut barriers by SB was time-dependent. However, Occludin protein decreased significantly after 48h in NEC group and 24h in HI group, which may be related to the immature barrier function of neonatal rats ^[39]. Excessive butyrate accumulation in the intestine will lead to further damage of barrier function^[40], and the early deterioration of barrier function caused by SB needs further study. Although SB can not repair the gut-brain barrier well, it can induce the secretion of IL-10 by Treg cells^[41], and IL-10 further enters the brain through the blood-brain barrier with increased permeability to alleviate the brain injury.

Enteritis in preterm infants is closely associated with the outcome of neurological dysplasia^{[5, 39]}. The destruction of the brain-gut barrier directly causes the intestinal inflammatory factor IL-17 to enter the brain, affecting the development of the nervous system and impairing long-term spatial learning ability. Our study found that NEC and HI can lead to the impairment of long-term spatial learning ability in neonatal rats, especially NEC + HI being more serious. SB can not only partially improve the neonatal rats’ delayed maturation of neurological reflexes caused by HI, but also promote the long-term spatial learning ability in neonatal rats with NEC + HI.

In conclusion, our study found that the reason why enteritis exacerbates brain injury in neonatal rats may be the change in IL-10/IL-17 balance caused by the decreased expression of SOCS1 protein and the impairment of brain-gut barriers function, which in turn leads to the inflammatory factor IL-17 entering the central nervous system through the disrupted brain-gut barrier to participate in brain injury. This indicates that the gut-microbiota-immune-brain axis plays an important role in brain injury in neonatal rats. SB can reduce brain injury caused by enteritis by intervening this pathway, increase SOCS1 protein expression, restore IL-10/IL-17 balance, and benefit long-term neural development. This provides a new theoretical basis for further exploring the mechanism of brain injury repair in preterm infants.

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

Funding

This work was supported by Sichuan Provincial Medical Scientific Research Project (No.S2300), School Cooperation Research Fund of Nanchong City (No. 20SXZRKX003), Research and development program of Affiliated Hospital of North Sichuan Medical College (No. 2021ZK004 and No. 2021LC010).

Author Contribution

Jing Zhao and Zhiyuan Feng wrote the main manuscript text , Can Yang amd Jun Zhang prepared figures 1-3, Lin Jiang and Li Hou prepared figures 4-6 . All authors reviewed the manuscript.

Acknowledgement

Not applicable.

Data Availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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Effect of butyrate, a short-chain fatty acid, in repairing brain injury in neonatal rats

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Version 1

Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS

Animals

Experimental design

Establishment of the experimental model in rat

Morris water maze

Short-term neurofunctional outcome

Cerebrospinal fluid and plasma biomarker analysis

Western blot analysis

Hematoxylin and eosin (H&E) staining

Statistical analysis

RESULTS

SB alleviates brain injury

SB up-regulates the expression of SOCS1 in the gut and brain

SB affects the blood-brain barrier and intestinal barrier

Sodium butyrate improves long-term spatial learning ability

DISCUSSION

Declarations

Conflicts of Interest

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

Version 1