Effect of probiotics on olanzapine-induced metabolic syndrome in Wistar albino rats

Purpose: Olanzapine is the most viable second-generation antipsychotic (SGA) used in the treatment of schizophrenia and at the same time, it is the most notorious SGA to cause metabolic syndrome (MS). The target of this study is to assess the adequacy of probiotics in ghting the unfriendly impacts of olanzapine treatment such as weight gain, hyperlipidemia, and hyperglycemia in the olanzapine-induced MS model in rodents. Methods: Thirty-six Wistar rodents were haphazardly separated into six groups (n=6). The groups were treated for a month as follows: Group-I: Normal saline 1 ml/kg/day orally, Group-II: olanzapine 2 mg/kg/day i.p., Group-III: probiotic-VSL#3: 0.6 g/kg/day orally, Group-IV: VSL#3: 1.2 g/kg/day orally, Group-V: olanzapine 2 mg/kg/day i.p. + VSL#3: 0.6 g/kg/day orally, and Group-VI: olanzapine 2 mg/kg/day i.p. + VSL#3: 1.2 g/kg/day orally. Bodyweight, fasting blood glucose (FBG), and lipid prole was assessed at baseline and consequently at the end of each week. Data were analyzed by applying repeated measures ANOVA, followed by post-hoc Bonferroni test. P-value <0.05 was considered statistically signicant. Results: There was a noteworthy increment in the total cholesterol and triglycerides level after olanzapine treatment (P<0.01), and similarly a decline in the total cholesterol and triglycerides level in the probiotic treated groups (p < 0.05). There was a decrease in weight increase and FBG levels instigated by olanzapine in the probiotic-treated groups. Conclusion: Probiotics forestalled the advancement of hyperlipidemia and decreased the weight addition and increment in FBG levels initiated by olanzapine. A long-haul evaluation should be directed to additionally assess the impact of probiotics on MS and their plausible mechanism.


Introduction
Atypical/Second-Generation Antipsychotics (SGAs) are the mainstay of treatment in most psychotic disorders. [1] Among the SGAs, olanzapine (OLZ) is more e cacious than other SGAs in terms of improving the general mental state and reducing associated depressive and anxiety symptoms. [1][2][3][4] SGA's have been associated with initiating and aggravating metabolic syndromes such as obesity (increased BMI), diabetes, and hyperlipidemia. [4] Among the SGAs, even though OLZ is more e cacious than other SGAs, it also has the maximum potential to cause MS. [5][6][7][8] Obesity is observed to be a precursor for MS, and treating it with physical activities, counseling for behavioral reforms, calorie-restricted diets, administration of drugs for weight loss, as well as surgery for weight loss can be the key aspects in the management and control of MS. However, strategies like physical exercise and behavior reforms require strong mind control and are di cult to adopt by patients who are mentally ill. Diet modi cation strategies are also found to be less effective. On the contrary, the existing pharmacological management has its drawback of adverse side-effects and high cost of treatment. [9] Probiotics are live microorganisms, generally regarded as safe, which when administered in adequate amounts confer health bene ts on the host. [10] It is the probiotic component of the gut microbiota that plays a crucial role in the maintenance of general homeostasis and health. [11] The two key members of this group include Lactobacilli and Bi dobacteria. [11][12] With oral supplementation of probiotics manipulating the gut, the microbiota has proved to be a crucial strategy against high-fat-diet (HFD) induced MS. [12] Various strains of bene cial microorganisms have been used alone or a lot more as cocktails, and have shown promising results to combat metabolic complications like obesity, insulin resistance, and/or hepatic steatosis in HFD-fed rodents. These strains include Bi dobacterium spp., Lactobacillus spp., Streptococcus thermophilus, Bacteroides uniformis, and Akkermansia muciniphila. [13][14][15][16][17][18][19][20][21] VSL#3 probiotics, a multistrain cocktail containing Streptococcus thermophilus and a few types of lactobacilli and bi dobacteria, are found to advance the arrival of the hormone GLP-1, bringing about decreased food consumption and improved glucose resistance. The VSL#3-initiated changes were related to an expansion in the levels of SCFAs, which leads to the release of GLP-1 from intestinal L-cells, proposing a potential mechanism for the VSL#3-related effects. [22] Notwithstanding, little is known about the impact of probiotics, on antipsychotic-induced MS. 23 Thus, in the current situation, the advancement of dietary methodologies, i.e., planning natural food items with probiotics and prebiotics that regulate MS will be a nancially savvy approach without the dread of unfavorable effects on health.

Materials And Methods
The study was done for over 28 days, after getting the permission of the Institutional Animal Ethics Committee, MAHE, Manipal.

Animal selection
A sum of 36 adult male Wistar albino rats, each weighing 150-250 grams were acquired. Male rodents were picked over female rodents as the latter's estrous cycles meddle in initiating the MS. All rodents were housed in the central creature research o ce, Manipal, at room temperature (23 ± 2°C) with 12 hours of light: dark environment. They were given standard research centre feed and water. The study was carried out as per the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines.
Drugs, reagents, and other materials: A ne powder of olanzapine was blended in with a little volume (0.1 ml) of glacial acetic acid, and later made to volume utilizing normal saline. [24] Probiotic VSL#3 (manufactured in India by Sun Pharmaceutical Ind. Ltd.) is a freeze-dried pharmaceutical probiotic preparation containing 112.5 billion CFU/capsule (a mix of Streptococcus thermophiles, Bi dobacterium longum/lactis, Bi dobacterium breve, Bi dobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii subsp. bulgaricus) was bought from the drug store and made to volume with an ample volume of normal saline. [22][23] Reagents for the biochemical test (serum lipid pro le) were purchased from Aspen Laboratories Pvt Ltd. Computerized glucometer and testing strips (from AccuChek), centrifuge, and semiautomatic analyzer from the Department of Pharmacology, Kasturba Medical College, Manipal Academy of Higher Education, Manipal were used.
Selection of dose of medications: The dose (2 mg/kg/day), term (a month), and route (intraperitoneali.p.) of olanzapine were picked depending on past investigations that indicated positive induction of MS in female Wistar rodents with this regimen. [24] VSL#3 dose by chose by converting the adult human dose of a mean 70 kg man into rat dose using the standard dose conversion formula. [25] The VSL#3 capsules were stored at 2 to 8 ∘ C before administration and were dissolved in normal saline just before administering to the rats. [22][23][24] Experimental groups: Thirty-six rats were randomly divided into six groups (n=6), as shown in gure 1.
Group I -Rats received normal saline, 1 ml/kg/day orally for four weeks as a single daily dose.
Group II -Rats received olanzapine, 2 mg/kg/day i.p. for four weeks as a single daily dose.
Group III -Rats received VSL#3 (Dose I), 0.6 gm/kg/day for four weeks orally as a single daily dose.
Group IV -Rats received VSL#3(Dose II), 1.2 gm/kg/day for four weeks orally as a single daily dose.
Group V -Rats received olanzapine, 2 mg/kg/day i.p. + VSL#3 (Dose I), 0.6 g/kg/day orally for four weeks as a single daily dose.

Parameters assessed
Total body weight: Baseline weight was measured before the commencement of the doses, and at the end of each week for four weeks, and values were noted in grams. [24] Fasting blood glucose: Rats were fasted overnight (for 12 h) and blood glucose levels were measured by taking blood from tail pricks using a digital glucometer before the dosing started (as baseline) and once a week thereafter for four weeks. All values were noted down in mg/dL. [28] Serum lipid pro le: Venous blood was acquired from all rats from the retro-orbital venous plexus for assessment of lipid parameters -total cholesterol, HDL, and triglycerides. The blood was centrifuged, and the serum was exposed to a semiautomatic analyzer (utilizing the reagent kits standard procedure). All the values were noted down in mg/dl. This assessment was done at baseline and the nish of every week from that point for four weeks. [24] Estimation of total cholesterol: 10 μl of test serum sample was added to 1000 μl of the testing reagent, blended well, and incubated for ten minutes at 37°C. The absorbance was compared against the reagent blank. To get the estimation of total cholesterol, the absorbance of the test was divided by absorbance of standard and multiplied by the calibrator concentration factor. Estimation of HDL: 50 μl of test serum sample was added to 1 ml of the testing reagent, blended well, and incubated for ten minutes at 37°C. The absorbance was perused at 505 nm. To get the estimation of HDL, the absorbance of the test sample was divided by absorbance of standard and multiplied by the calibrator concentration factor, and two.
Estimation of triglycerides: 50 μl of test serum test was added to 1 ml of the testing reagent, blended well, and incubated for ten minutes at 37°C. The absorbance was perused at 505 nm. To get the estimation of HDL, the absorbance of the sample was divided by absorbance of standard and multiplied by the calibrator concentration factor, and two.
Statistical analysis: The data obtained was analyzed using IBM Statistical Package for Social Sciences (SPSS) v. 20.0. The signi cance of differences within the groups at a various time focuses was evaluated by applying repeated measures of One-way Analysis of Variance (ANOVA), trailed by the post-hoc Bonferroni test. All experimental groups were compared against the control and standard groups to nish up the outcomes. The p-value of less than 0.05 was considered statistically signi cant.

Results
The present study demonstrated the effects of various groups on body weight, total cholesterol, triglycerides, HDL cholesterol, and blood glucose levels as shown below.

Effect on body weight
The body weight was measured at baseline and the end of every week for four weeks. At baseline, the bodyweight of all the groups was comparable. After four weeks, there was a signi cant increase in the body weight in the OLZ treated group (250 ± 7.82), when compared to the baseline (211.67 ± 12.24) (p<0.001). There were no signi cant changes in body weight at four weeks in the groups treated with probiotics when compared to baseline. Intergroup comparison showed a signi cant increase (p<0.001) in the bodyweight of the group treated with olanzapine when compared to the control group at the end of week four. There was a decrease in the weight of the animals treated with probiotics (237.6 ± 1.5, 195.2 ± 9.38, 177.75 ± 7.02, 227.33 ± 11.56) when compared to the olanzapine treated group (250 ± 10, 211.67 ± 12.24, 230 ± 0, 200 ± 0, 180 ± 4.89, 232 ± 11.13), though not statistically signi cant. The mean ±SEM of the animals treated with probiotic (dose I) was 237.6 ± 1.5 (baseline value 230), probiotic (dose II) 195.2 ± 9.38 (baseline value 200), and that of olanzapine was 250 ± 7.82, though there was a decrease in weight, it was not statistically signi cant.  There was a statistically signi cant increase in total cholesterol levels at the end of the rst, third, fourth week (p<0.001), and second week (p<0.05) when compared with the control group as shown in gure 2. There were statistically signi cant differences (p<0.001) of decrease in total cholesterol levels at the end of four weeks when probiotics were given alone (both low and high dose) and along with olanzapine therapy when compared with the olanzapine group (p<0.001). There was a statistically signi cant decrease (p<0.001) in total cholesterol levels at the end of the rst week when probiotics were given alone at a low dose. Olanzapine showed a statistically signi cant increase in triglyceride levels at the end of the fourth week (p<0.05) as compared with the control group as shown in gure 3. There was a statistically signi cant decrease in triglyceride levels in group 3 (p<0.001) at the end of second and third weeks, and also at the end of the rst week (p<0.001) and second week (p<0.05) in group 4 when compared with the olanzapine standard group. Probiotics when given alone (groups 3 and 4) and along with olanzapine therapy (groups 5 and 6) decreased triglycerides levels at the end of the fourth week when compared with olanzapine standard group (p<0.001). Probiotics administered along with the olanzapine therapy showed a decrease in the triglycerides levels at the end of the second and third weeks in group 5 (p<0.001), and also at the end of the second, third, and fourth weeks in group 6 (p<0.05). There was an increase in the HDL levels of probiotics (groups 3 and 4) at the end of the rst week when compared with the olanzapine group (p<0.001) as shown in gure 4. There was an increase in HDL levels in group 3 (p<0.001) and group 4 (p<0.05) when compared with the control group. There was a statistical increase in the HDL levels in group 5 (p<0.001) and group 6 (p<0.05) when compared with the olanzapine group.

Effect on fasting blood glucose levels
In gure 5, FBG levels of the baseline and at the end of the fourth-week treatment of rats of all the different experimental groups are shown. The values are noted down in mg/dl. There was an increase in the FBG level observed after treatment with olanzapine and probiotics at the end of the fourth week when compared with the baseline FBG level, but it was not statistically signi cant.

Discussion
Metabolic syndrome is a cluster of cardiovascular diseases, and various risk factors such as an increase in blood pressure, insulin resistance, and blood coagulability cause an increase in obesity and neutral fat, and LDL-C, decreases in HDL-C, and the effect of dyslipidemia. [29] Olanzapine is an SGA; it is used in the present study to induce metabolic syndrome at the dose of 2mg/kg. [27] The study showed that with olanzapine treatment, there was a statistically signi cant (p<0.001) increase in body weight, total cholesterol, and triglycerides levels which is suggestive of metabolic syndrome.
Mechanism of OLZ induced MS-The histamine H 1 receptor antagonism promotes feeding in rodents. The metabolic complications with olanzapine therapy begin with increased appetite and weight gain, and progress to obesity, insulin resistance, and dyslipidemia with an increase in fasting triglyceride levels.
Ultimately, hyperinsulinemia advances to pancreatic ß-cell failure, prediabetes, and then diabetes. Once diabetes is established, the risk for cardiovascular events is further increased, as is the risk of premature death. [30] In the present study, probiotics when given alone in low and high doses and along with olanzapine therapy, decreased bodyweight, total cholesterol, and triglycerides levels at the end of four weeks.
Probiotics alone in low dose and along with olanzapine therapy, decreased the lipid pro le at the end of every week, with p<0.001 at the end of the fourth week. There was also an increase in the HDL cholesterol level even with the rst and fourth weeks of administration of probiotics.
Past investigations indicated that probiotics directly introduce beneficial bacteria, for example, Lactobacilli and Bifidobacteria into the gut. [31] Supplementation of probiotics was found to improve increased lipid pro le levels in HFD fed rodents and humans. [32][33] The outcomes from the past studies have demonstrated a reduction in plasma cholesterol and triglycerides levels. Bile acid deconjugation by probiotics and cholesterol bonding has additionally been proposed as likely components of bringing down cholesterol by probiotics. [34] Probiotics may change cholesterol into coprostanol, which is straightforwardly excreted through feces. [35] They have viably restored gut barrier function, decreased intestinal in ammation, and saved the intestinal microstructure in HFD fed rodents. [36] Probiotics likewise re-established gut barrier dysfunction and diminished concentrations of circulating glucocorticoids and pro-inflammatory cytokines. These cytokines weaken the integrity of the blood-brain barrier and allow conceivably pathogenic or inflammatory components. Probiotics additionally increment antiinflammatory cytokines, which upgrade the integrity of the blood-brain barrier, the gut barrier, and decrease inflammation. [31] Another investigation demonstrated that Lactobacilli species are t for strengthening the epithelial boundary and may forestall lipopolysaccharide-interceded in ammation and hyperglycemia. [37] Dysbiosis is a state portrayed by alteration in microbiota composition, a change in bacterial metabolic action, or potentially a more local distribution of microorganism communities. Intestinal dysbiosis is vital in the comprehension of the pathophysiology of a few metabolic diseases, including obesity and T2D. [34] The gut microbiota is overwhelmed by two phyla, Firmicutes and Bacteroidetes, which characterize about 90% of the apparent multitude of bacterial species in the gut. [39][40] Many vital studies have exhibited that the control of the gut microbiota and its metabolic pathways can in uence the host's adiposity and metabolism. [37] Gordon and associates utilizing germ-free (which lack microbiota) mice showed that they have 40% less fat versus fat in normally raised mice, and are impervious to diet-induced obesity. They additionally found that obese mice have an 'obesity-related microbiome' comprising of corresponding movements in the two most bountiful phyla of microorganisms-rmicutes (expanded) and bacteriodetes (diminished), comparative outcomes were shown by another investigation where rodents were given HFD, it diminished Bacteroidetes. They also gave produced mounting proof suggestive of alterations in the gut microbiome as a vital factor in the prevalence of obesity. [39,[41][42][43][44] Supplementation of probiotics prevented the development of hyperlipidemia and hyperglycemia induced by olanzapine in the Wistar albino rats. There was a clinical difference observed in the gain of body weight of the rats treated with probiotics and olanzapine treated groups for four weeks, which was statistically not signi cant, which could be attributed to their dietary pattern.

Limitations
Long-term studies to evaluate the effects of probiotics on olanzapine-induced metabolic syndrome should have been done.
The effect of different SGAs in inducing metabolic syndrome needs to be assessed.
Long-term studies need to be done to check for a change in the weight gain and lipid pro le by both olanzapine and probiotics on rats. Further evaluation of the gut hormones level, short-chain fatty acids, and enteroendocrine neurotransmitters could give an insight into the pathophysiology leading to change in the lipid pro le and weight gain in rats treated with olanzapine and probiotics administered to counteract the olanzapine-induced MS.
Declarations Figure 1 Experimental groups Figure 2 Total cholesterol levels in the different experimental groups at the end of 4th week. Inter-group comparison of total serum cholesterol levels. * p < 0.001, olanzapine vs control; # p < 0.001, probiotic (low and high dose)/probiotic (low and high dose)+olanzapine vs olanzapine; p-value calculated by Oneway ANOVA followed by post-hoc Bonferroni test).

Figure 3
Triglyceride levels in the different experimental groups at the end of 4th week. Inter-group comparison of triglyceride levels. * p < 0.05, olanzapine vs control group; # p < 0.001 probiotics (low and high dose) alone and along with olanzapine vs olanzapine group; p-value calculated by One-way ANOVA followed by post-hoc Bonferroni test).
Page 18/19 Figure 4 HDL-cholesterol levels in the different experimental groups at the end of 4th week. Inter-group comparison of HDL cholesterol levels. * p< 0.001, probiotic low dose + olanzapine vs olanzapine; # p < 0.05, probiotic high dose + olanzapine vs olanzapine; p-value calculated by One-way ANOVA followed by post-hoc Bonferroni test).