Stress is a phenomenon that affects all humans for the duration of their lifespan. It has a variety of effects on the body's numerous biochemical regulatory systems and can ultimately contribute to a number of different disorders. (Reineke & Neilson,2019). The study of stress physiology has emerged as a crucial tool for scientists seeking a deeper comprehension of the pathophysiology of both physical and mental diseases. (Daryl B. O'Connor et al., 2019). Acute stress is characterized by the perception of a physical, emotional, or psychological peril that is imminent. Chronic stress is a prolonged exposure to stress that, for the majority of individuals, results in negative long-term health outcomes. (Nicholas Rohleder et al., 2019). The modern way of life is characterized by an increase in the consumption of high-sugar and high-fat foods, which is typically accompanied by an ongoing exposure to known stressful conditions (Tiago Batchauer et al., 2020). The alteration of commensal bacteria that reside in the digestive system, also known as the gut microbiota, is one of the numerous biological effects that can be caused by daily stress. (J. Philip Karl. et al., 2018). Several distinct disorders have been linked to dysbiosis of the gut microbiota (Amrita Vijay & Ana M. Valdes, 2022). This establishes the gut microbiota as a significant mediator of host health by modulating the host's stress response and stress-related sequelae. The interaction between the host and microbiota in the intestine influences a number of crucial processes, including the host's metabolism, immunology, and intestinal barrier function. (Stefani Lobionda et al., 2019). Practically all physiological systems are significantly regulated by the circadian schedule. Circadian rhythm is responsible for keeping internal time in harmony with external time throughout the course of a 24-hour period. A disruption in the circadian rhythm system may have negative effects on an individual's overall health. (Yanling Xie et al., 2019). Stress can influence the circadian rhythm via glucocorticoids, sympathetic nerves, oxidative stress, hypoxia, pH, cytokines, and temperature, among other mechanisms. (Johanneke E. Oosterman et al., 2020). The hypothalamic-pituitary-adrenal (HPA) axis necessitates the involvement of both the central nervous system and the body's tissues. These organs respond to stress by releasing glucocorticoids into the systemic circulation, which is primarily manifested by the adrenal glands' release of glucocorticoids (Haung, X et al., 2020). Even though glucocorticoid secretion contributes to the preservation and maintenance of homeostasis at normal operating levels, an excess of cortisol can cause physiological and psychological problems. Numerous health issues, including hypertension, obesity, metabolic syndrome, and insulin resistance, can be traced to the elevated levels of glucocorticoids that result from chronic stress. (Jerez-Cepa et al., 2019). Under stressful conditions, the primary target of glucocorticoids is in the liver, which produces glucose via glycolysis or gluconeogenesis (Douglas Carvalho Caixeta et al., 2018). Cortisol influences lipid catabolism by stimulating lipase activity and causing the conversion of triglycerides (TAG) to glycerol and free fatty acids (FFA). The liver secretes the hormone betatrophin, which regulates both the triglyceride (TG) and glucose metabolisms (Uzma khan, 2019). Betatrophin has a strong correlation with metabolic syndrome risk factors, for example dyslipidemia, hypertension, obesity and impaired glucose tolerance leading to the onset of type 2 diabetes (Hanako Niki et al., 2019).

Prebiotics are foods that promote the growth of beneficial bacteria in the gut. They are mainly fiber or complex carbs that human cells cannot digest. Probiotics are a mixture of live bacteria that lives in your body. Synbiotics are a combination of prebiotics and probiotics that are believed to have a synergistic effect by inhibiting the growth of pathogenic bacteria and enhancing the growth of beneficial organisms. Synbiotics are the end product of years of research into microorganisms. Their increase in the number of beneficial bacteria contribute significantly to the resolution of adverse effects of chronic stress (Hang-Yu Li et al., 2021).

Limited data is available on the effect of Synbiotic on the stress markers such as cortisol and betatrophin. Therefore, additional research on this is the need of hour. In the current study we determine and correlate serum levels of cortisol and betatrophin in rats consuming standard lab diet, probiotics and Synbiotic exposed to the stressors of immobilization and fasting. The results of this study will shed light on the effects of Synbiotic on stress markers

Animals A total of 50 male Sprague Dawley rats of 6 to 8-weeks with an approximate weight of 250 – 300 gm were selected, and they were acclimatized to the environmental conditions for at least seven days prior to experiment carried out in the NIH Animal House at a humidity of 50-70% and a room temperature of 24 + 2 oC, with a 12-hour light/dark cycle. The animals were then separated and placed in their respective enclosures. At the Animal House of NIH in Islamabad, standard diet was prepared in pellet form These rats were randomly allocated to two groups which are control and experimental. Group A: (Control Group): Ten rats given no stressor and given standard lab diet in the form of pellets. Group B: (Interventional Group): Group B was further divided into four groups. Group B1: Ten rats given prolonged overnight fasting (10pm-10am) with 2 hours of immobilization (08-10 am) and morning meal at 10am of standard lab diet in the form of pellets. Group B2: Ten rats given overnight fasting (10pm-08am) with 2 hours of immobilization (10 am) and morning meal (08am) of standard lab diet in the form of pellets. Group B3: Ten rats given overnight fasting (10pm-08am) with 2 hours of immobilization (10 am) and morning meal (08am) consisting of standard lab diet along with probiotics (Yogurt 8g/day) in the form of pellets. Group B4: Ten rats given overnight fasting (10pm-08am) with 2 hours of immobilization (10 am) and morning meal (08am) consisting of standard lab diet along with Synbiotic (Yogurt 8g/day and legumes 75g/day) in the form of pellets. Stressors Stressors used in this study were immobilization and fasting. In immobilization rats were confined into the containers where they were unable to move. In fating the rats were deprived of food Immobilization Each male Sprague Dawley rat was placed in a 57x19x6.5cm wooden container that was specially crafted by a carpenter. Each container contained 10 small, 17x5.5x5 cm compartments for individual rats. To prevent nibbling, these spaces were lined with metallic sheeting from the inside. Mesh was installed on the surface of the container for ventilation. On one side there was a hole for tails. The small size of the space precluded the animal from moving within it. The rodents were confined in these containers for two hours, five days per week. Fasting The experimental groups B2, B3, and B4 were subjected to the stressor of overnight fasting. They were fed their last meal at 10 p.m., after which the food was removed from their cages, preventing them from eating during fasting hours. Rats were given their morning meal at 8 a.m., ensuring 10 hours of fasting. Rats in group B1 had prolonged fasting for 12 hours, from 10 p.m. to 10 a.m., before receiving their morning meal at 10 a.m. Diet Preparation Standard diet was prepared in the form of pellets at Animal House of NIH, Islamabad according to the guiding principle given by the Universities federation for animal welfare and its composition is shown in Table: 1. Table1: Standard diet composition of 10 kg formulated according to the recommendation given by the Universities Federation for animal welfare Ingredients Weight (kg) Percentage (%) Wheat bran 2.85 28.5 Wheat flour 2.85 28.5 Dried skimmed milk powder 2.00 20.0 Fish meat 1.50 15.0 Common salt 0.05 0.5 Vitamins/minerals/amino acids 0.10 1.0 Mollasses 0.015 1.5 Soybean oil 0.51 ml 5L Total weight 10 kg 100% Synbiotic Synbiotic diet used in this study comprises of probiotic and prebiotic components. For probiotics yogurt was used and calculated as 8g/day, and for prebiotics legumes were used with daily consumption of 75g/day (1/2 cup). They were added in the pallets and given to the rats. Both yogurt and legumes were verified the National Agriculture Research Centre, Islamabad. Sampling Serum cortisol (ng/ml) and serum betatrophin (ng/ml) parameters were measured in blood samples. On the first day of the experiment blood was taken from rat tail vein. Intracardiac puncture was used to collect the sample after four and ten weeks of experimentation. Animals were euthanized after 10 weeks of experimentation. Data analysis Statistical analysis was done by ANOVA and Post hoc-Tukey tests to compare groups. Pearson’s correlation correlated between serum levels of cortisol and betatrophin. p-value < 0.05 was significant

Serum Cortisol (ng/ml)

A post hoc Tukey test was utilized to perform multiple comparisons of the mean ± SD serum cortisol levels among various groups of male Sprague Dawley rats. The results demonstrate a significant difference between group A and group B1 (p<0.001), B2 (p<0.001), B3 (p<0.001), and B4 (p<0.001), indicating that stress significantly elevated the mean ± SD serum cortisol levels in Group B1, B2, B3, and B4. Group B1 exhibited a significant difference compared to group B3 (p<0.002) and B4 (p<0.001), indicating the protective effects of probiotics and synbiotics in the diet during exposure to stressors. Similarly, B2 showed a significant difference compared to B3 (p<0.002) and B4 (p<0.001), suggesting the protective effects of probiotics and synbiotics in the diet during exposure to stressors. These findings indicate no significant difference between B3 and B4 (p<0.08), suggesting that probiotics and synbiotics have the same effects on cortisol.

The comparisons of mean ± SD among different groups (B1, B2, B3, B4) is shown in Figure 1.

Serum Betatrophin (ng/ml)

A post hoc Tukey test was employed to conduct multiple comparisons of the mean ± SD serum betatrophin levels among different groups of male Sprague Dawley rats. The results revealed a significant difference between group A and group B1 (p<0.001), B2 (p<0.001), B3 (p<0.04), and B4 (p<0.05), indicating that stress significantly elevated the mean ± SD serum betatrophin levels in Group B1, B2, B3, and B4. Group B1 exhibited a significant difference compared to group B2 (p<0.03), B3 (p<0.04), and B4 (p<0.05), suggesting that prolonged fasting led to a significant increase in the mean ± SD serum betatrophin levels compared to other groups. Similarly, B2 showed a significant difference compared to B3 (p<0.05) and B4 (p<0.04), indicating the protective effects of probiotics and synbiotics in the diet during exposure to stressors. There was a significant difference observed between B3 and B4 (p<=0.02), suggesting that synbiotics had more beneficial effects than probiotics.

Correlation between serum Cortisol and Betatrophin

Pearson correlation was used to determine the relationship between cortisol and betatrophin at 10 weeks. With a p value of 0.001, the results are extremely significant. Cortisol has a positive association with serum betatrophin, with a Pearson correlation coefficient of 0.621

Cortisol is the main stress hormone of the body which is released due to stimulation of HPA axis after exposure to stress. It ultimately effects all the systems of body. Current study shows that the exposure to chronic stress results in increase secretion of cortisol hormone in the body. These findings are in support of Lisa R. Starr et al, (2018) and Eleonora Iob et al, (2019) which states the increase in cortisol levels after the exposure to chronic stress. Another study Ekasit Lalitsuradej et al, (2022) states that the cortisol levels can be decreased by using synbiotics in diet with support the results of present study in which there is significant decrease in cortisol levels as seen after the use of synbiotic diet. However another study Asma Kazemi et al, (2019) results differ from our study in which there is no significant effect is noted on cortisol levels by the use of synbiotic diet. In present study the group of rats which skipped their morning meals and have prolonged fasting shows much increased in their cortisol levels as compared to other groups. These findings are not in support with the findings of Shreya Chawla et al, (2021) which stats that there is decrease in cortisol levels in persons who skipped their morning meals.

Betatrophin hormone is released from liver and plays important part in lipid and glucose metabolism. Limited data is available related to the effects of chronic stress, synbiotics and skipped morning meal on levels of Betatrophin. In present study there is significant increase in Serum betatrophin levels in the group of rats who skipped the morning meal and have prolonged fasting. These findings are different Mustafa Timurkaan et al, (2022) in this study fasting during the month of Ramazan did not affect the circulating levels of Betatrophin hormone. But this study cannot be correlated with present study as the timing of fasting and duration is different. Another study Purwo Sri Rejeki et al, (2022) stated that moderate exercise which also act as a stressor on body reduced the levels of Betatrophin. But in current study the stressors of fasting and immobilization caused significant increase in Betatrophin levels. These findings are also not correlated with present study as the type and duration of stressors are different. Present study also shows that there is significant decrease in the levels of betatrophin after the use of synbiotics diet. Betatrophin is also known as the indicator for cardiovascular diseases and metabolic disease so the decrease in its levels is beneficial for health,

Chronic stress disrupts the body's homeostasis and causes an increase in cortisol and betatrophin levels in the blood. Dietary modifications, such as the incorporation of synbiotics and the synchronisation of the circadian rhythm, can mitigate these negative effects of stress.

Limitations / Recommendation for Future Research

As the time of study falls in the duration of Covid-19 pandemic so it was difficult to perform this study on humans. It is recommended to perform this study in humans for better understanding. Also this study is of short duration of 10 weeks which also a limited time. A study of long duration is suggested for further evaluation of benefitial effects of synbiotics.

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