The anti‐inflammatory and antiapoptotic effects of probiotic on induced neurotoxicity in juvenile hamsters

Abstract Brain inflammation and apoptosis play crucial roles in the pathogenesis of various neurodevelopmental disorders. Probiotics have been shown to confer protection against many stresses, including apoptosis and inflammation, by modulating the gut function. The short‐chain fatty acid, propionic acid (PPA), plays an important intermediate of cellular metabolism. Although PPA exhibits numerous beneficial biological effects, its accumulation is neurotoxic. This study focused on the therapeutic potency of probiotics against PPA‐induced apoptosis and neuroinflammation in hamsters. Five groups of male golden Syrian hamsters were treated as follows: Group I as control; Group II as PPA‐treated with three doses of 250 mg PPA/kg/day; Group III as clindamycin‐treated with a single dose of 30 mg clindamycin/kg; Group IV as PPA–probiotic; and Group V as clindamycin–probiotic were two therapeutic groups which were treated with the same doses of PPA and clindamycin, respectively, followed by treatment with 0.2 g kg‐1 d−1 of probiotic (ProtexinR, Probiotics International Limited) for three weeks. Proapoptotic markers, such as caspases 3 and 7; neuroinflammation markers, such as interleukins 1β and 8; and heat shock protein 70 were measured in the brain. Significant increase of all measured markers (p ˂ .001) was observed in PPA and clindamycin‐treated hamsters compared with controls. Probiotics significantly reduced the damages and ameliorated all the test markers in both therapeutic groups compared with the control. Our results confirmed that probiotics can be utilized as a feasible strategy for managing apoptotic and inflammation‐related stresses in brain disorders by retaining the gut function.

with the development of neurodevelopmental disorders such as autism (El-Ansary et al., 2012). Propionic acid and other short-chain fatty acid (SCFAs) are synthetized through the degradation of dietary amino acids and carbohydrates by many intestinal bacteria, including those involved in antibiotic-associated diarrhea (Kuijper et al., 2006) such as Clostridia. Generally, SCFAs are metabolized in the liver (Thomas et al., 2012); however, due to acquired and/or genetic metabolic abnormalities (MacFabe et al., 2007;Wajner et al., 2004), high blood levels of SCFAs can accumulate and gain access to the CNS after crossing the gut-blood and blood-brain barriers (BBB). Consequently, intracellular acidification may occur (Bonnet et al., 2000;Karuri et al., 1993), which affects cell signaling (Nakao et al., 1998), immune function (Le Poul et al., 2003), neurotransmitter release (DeCastro et al., 2005), lipid metabolism (Hara et al., 1999), mitochondrial function/CoA sequestration (Wajner et al., 2004), gene expression (Parab et al., 2007), and gap junction modulation (Rörig et al., 1996), thus causing neuropathologic and biochemical abnormalities. Numerous studies indicated correlation between behavioral abnormalities and neuroinflammation in rats exposed to PPA (El-Ansary et al., 2012;Shultz et al., 2008). Therefore, PPA seems to affect both the brain and behavior of laboratory animals in a way that is consistent with symptoms of neurological disorders. Intracerebroventricular (ICV) injection or oral administration of neurotoxic doses of PPA efficiently induced hyperactivity, repetitive behaviors, turning behavior, kindled seizures, retropulsion, neuroinflammation, and widespread oxidative stress in rats (MacFabe et al., 2007).
Moreover, animal models of neurotoxicity can be established through different methods, including the use of clindamycin, a broad-spectrum antibiotic prescribed for the treatment of bacterial infections (von Konow et al., 1992). Clindamycin has been employed to favor the overgrowth of propionic acid-producing bacteria such as Clostridium sp. and indirectly elevates PPA levels. Similarly to other antibiotics, clindamycin affects both pathogen and commensal host microbiota, thus inducing intestinal dysbiosis and stimulating the gut microbiota associated with immunologic, metabolic, and developmental disorders, besides increasing the susceptibility to infectious diseases (Cuthbertson et al., 2016;El-Ansary et al., ,2013Holmes et al., 2008). Buffie et al. (2012) and Al-Orf et al. (2018) revealed that a single dose of clindamycin could significantly decrease the diversity of intestinal microbiota, favor the growth of Clostridia, and promote the development of colitis and diarrhea.
Furthermore, the microbiota-gut-brain axis concept opens new horizons for developing novel therapies based on the modulation of gut microbiota to prevent or alleviate neurodevelopmental disorders. Therefore, consumption of fermented foods, which contain various probiotic strains, could play a major role in the therapy of several neurodevelopmental disorders, including anxiety disorder, autism, Parkinson's disease, and multiple sclerosis (Fung et al., 2017).
Microorganisms that act as probiotics have different properties; they persistently adhere to intestinal epithelial cells and mucus, reduce and exclude pathogenic adherence to healthy cells, propagate in a manner that allows fast multiplication and colonization, and generate reactive agents such as bacteriocins, hydrogen peroxide, and acids that can hamper pathogen multiplication. In addition, probiotics are safe, nonpathogenic, noncarcinogenic, and resist several microbicides, allowing them to form a balanced flora (Reid et al., 1990).

| Animals
All animal experiments were approved by the Ethics Committee of the College of Science at King Saud University and were carried out according to the national guidelines for use and care of animals.
Fifty young male golden Syrian hamsters (64.5 ± 3.9 g, 21 days old) were randomly assigned to five groups of ten hamsters each.
Animals in the control group were administered phosphate-buffered saline (PBS) via an orogastric tube daily. Hamsters in the PPA (Sigma-Aldrich)-treated group were orally administered 250 mg PPA/kg/ day for three consecutive days, followed by PBS till the end of experiment duration, whereas those in the clindamycin (Azupharma-Germany)-treated group received a single dose of clindamycin (30 mg/kg) through an orogastric tube followed by PBS, and were sacrificed the following day (El-Ansary et al., 2012). Animals in the two therapeutic groups were administered the same doses of PPA or clindamycin, followed by treatment with 0.2 g probiotic/kg/day dissolved in phosphate-buffered saline for three weeks. The probiotic used was Protexin R , (Probiotics International Limited, Somerset, UK), which consists of a combination of Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacusus bulgar, Lactobacillus casei, Lactobacillus rhamnosus, and Streptococcus thermophilus 1 109 CFU). Hamsters were maintained at 21 ± 1℃ on a standard diet and water ad libitum.
Carbon dioxide-anesthetized hamsters were decapitated at the end of feeding trials, and their brains were removed from the skull and dissected into small pieces. After homogenization in bidistilled water (1:10, w/v), brain samples were stored at −80°C. MBS035085) were investigated in the brain homogenates using ELISA kits (MyBioSource) following the manufacturer's instructions.

| Biochemical analyses
All measurements were performed in duplicate, and the mean of two different readings was calculated. Quality control assays were performed to evaluate experimental reproducibility through the interand intra-assay coefficients of variability (%CV).

| Statistical analysis
Data were analyzed using the statistical package for the social sciences (SPSS). Results are presented as mean and standard deviation (SD). Statistical comparisons and correlations between parameters were performed using the independent t test and Pearson's correlation coefficient (r), respectively. Furthermore, the receiver operating characteristics (ROC) curve and the area under the ROC curve (AUC) were used as a fundamental tool to evaluate brain neurotoxicity in animal modeling. Only p values ≤.005 were considered significant.

| RE SULTS
The data of the current study are expressed as mean ± SD of all investigated biochemical parameters in the different studied groups. Levels of CASP 3 and 7, HSP70, and IL-8 and IL-1 in the brain homogenates of the 5 groups as well as their percentage change in the treatment groups compared with the control group are reported in Table 1 and Figure 1.

TA B L E 1
Mean ± SD of all measured parameters in the brain homogenates of the 5 groups of hamsters studied (p < .05)  together with a remarkable increase in the production of proinflammatory cytokines IL-8 and IL-1β in the brain homogenates of PPAand clindamycin-treated hamsters compared with the control group.

| D ISCUSS I ON
Probiotics are live microorganisms that display health-promoting effects, prevent pathogen colonization of the gut, and ameliorate the symptoms of numerous diseases caused by deregulations of the immune system. Considering that the beneficial effects of probiotics can vary between strains, identifying the most efficient probiotic will be critical for their use in the prevention or treatment of various diseases. The present study explored the therapeutic effects of a combination of probiotics.  Heat shock proteins (HSPs) are a group of extremely conserved proteins that are usually identified as intracellular chaperones (Rudiger et al., 1997 reported that BBB disruption, an event preceding neurotoxicity, is often followed by increased HSP70 expression and apoptosis. This could also be related to our earlier study in which oral administration of PPA induced a significant increase in tail moment (comet DNA assay), tail length, and DNA fragmentation in PPA-treated rats compared with the control (El-Ansary et al., 2012).
Upon exposure to stress, such as infection and inflammation, HSP70 is released through unidentified mechanisms into the body fluids where it acts as an endogenous damage-associated molecular pattern to alter the secretion of proinflammatory cytokines (Ellis, 1990;Young, 1990

TA B L E 3 Multiple Regression using
Stepwise method between the measured markers, HSP70, CASP 7, CASP 3, IL-1β, and IL-8 by a remarkable increase in the production of proinflammatory cytokines IL-8 and IL-1β. It is well accepted that HSP70 plays a key role in inflammation by binding to toll-like receptor TLR4, thus activating nuclear factor kappa B (NF-κB) signaling to stimulate the production of inflammatory cytokines (Asea et al., 2000;Chalmin et al., 2010;Lee, Jeon, et al., 2019).

Numerous probiotics, including Bifidobacterium, Lactobacillus,
and Bacillus, are commonly used in therapy due to their beneficial effect on growth promotion and disease suppression (Král et al., 2012). to cell apoptosis and tissue damage (Salakou et al., 2007). More recently, Wu et al. (2019) demonstrated that probiotics downregulated Bax while upregulating Bcl-2 in the ileum mucosa.
Among the probiotics investigated in the present study, Bifidobacterium and Lactobacillus are the most commonly used to treat gut dysbiosis. They are supposed to restore the composition of gut microbiota by promoting healthy bacterial growth through immunomodulatory action in gut Lee, Jeon, et al., 2019;Schiffrin et al., 2009;Xia et al., 2011;Zhang et al., 2010).

Bacterial cells interact with a wide variety of cells that are present in
the intestine, such as epithelial cells, dendritic cells, and macrophages, which further stimulate the secretion of pro-and anti-inflammatory cytokines (Chon & Choi, 2010;Rodes et al., 2013;Roselli et al., 2006;Zhang et al., 2019). Certainly, these inflammation-related markers can affect the brain via the bidirectional gut-brain axis.
Although Much attention has been focused on clarifying the pathways through which inflammatory signals could diffuse from the periphery to the brain (D'Mello et al., 2009;Miller et al., 2009;Quan & Banks, 2007). Although these pathways still remain to be fully elucidated, some reports have hinted at the mechanism of direct diffusion of peripheral proinflammatory cytokines into the brain by binding to saturable transport proteins. Owing to their relatively large size, saturable transport receptors typically bind circulating proinflammatory cytokines and transport them into the brain across the BBB (Quan & Banks, 2007). However, direct entry of peripheral inflammatory cytokines into the brain through leaky regions of the BBB as a consequence to the neurotoxic effects of PPA has also been reported (El-Ansary et al., 2012;Quan & Banks, 2007). found to be affected with variable degrees by HSP70 and caspases 3 and 7 as proapoptotic-related parameters. Table 4 shows the ROC curves of the measured variables. It can be easily noticed that the 5 parameters recorded high AUCs, with satisfactory specificity and sensitivity.

| CON CLUS ION
This study ascertained the role of neuroinflammation and apoptosis as neurotoxic effects in the PPA-induced rodent model of autism.
Clindamycin showed relatively fewer neurotoxic effects compared with orally administered PPA. Moreover, our study showed that probiotics exert ameliorative effects and may be used as a novel noninvasive and safe treatment strategy.

ACK N OWLED G M ENTS
This work was financed by Researchers Supporting Project number (RSP-2020/237), King Saud University, Riyadh, Saudi Arabia.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest with respect to the authorship, and/or publication of this article.

E TH I C A L A PPROVA L
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at the Faculty of King Saud University (KSU-SE-17-10).

CO N S E NT TO PA RTI CI PATE
Not applicable.

CO N S E NT TO PU B LI S H
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.