The potential effects of the combination (Cytidine, Uridine and Zinc) on gut microbiota and clinical parameters of healthy rats

The potential effects of the combination (Cytidine, Uridine and Zinc) on gut microbiota and clinical parameters of healthy rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The potential effects of the combination (Cytidine, Uridine and Zinc) on gut microbiota and clinical parameters of healthy rats Maram A. Othman, Muna Barakat, Amin Omar, Tamara Athamneh, Rafat Zrieq, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4667549/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background/Aims As healthy gut flora contributes to mammalian health, the focus of this study is to investigate the effect of the combination of (Uridine, Cytidine, and Zinc) compared to individual supplements on the diversity of gut microbiota in healthy rats, and on other clinical parameters. Methods The 16S rRNA gene sequencing was used to study the diversity and abundance of gut microbiota. Liver enzymes were monitored by measuring serum level of (ALP and ALT), serum fasting blood glucose level, latency to fall, index of nociception and body weight were monitored. The previously mentioned analysis was done to study the effect of orally taken combination of (Uridine, Cytidine and Zinc) and the separate intake of these supplements on gut microbiota and other parameters in 35 healthy white Wistar rats for six weeks. Results The study findings showed that both the combination and the individual supplements enhanced the abundance of Clostridia and decreased Bacteroidia. Clostridia: Bacteroidia ratio increased from 1:2.5 to 1:1.2 by the last day of treatment in the combination group. Moreover, combination supplement reduced fasting blood glucose level ( P < 0.0001), improved rats’ motor activity ( P < 0.05), reduced nociception Index ( P < 0.0001) and decreased the ALP liver enzyme level in healthy rats ( P < 0.05). Conclusions The studied combination supplement was found to be safe and no harmful change on gut microbiota was noticed. This was clear from the normal level of the tested clinical parameters such as fasting blood glucose level and motor activity. Biological sciences/Microbiology Biological sciences/Microbiology/Applied microbiology Biological sciences/Microbiology/Clinical microbiology Microbiota uridine cytidine zinc nucleosides clinical parameters Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The adult human gastrointestinal (GI) tract harbors more than 10 14 diverse microbial populations 1 . The healthy human gut microbiota is mainly predominated by two important phyla, Bacteroidetes and Firmicutes 2 . The composition of this normal gut microbiota can be influenced by several factors since infancy, such as mode of delivery, the type of feeding during infancy, diet, antibiotics, and other factors 3 . Gut microbiota contributes to the disease or health state of humans and animals. For instance, it can act as an immunomodulator, and play a remarkable role in nutrients metabolism and vitamins K and B synthesis 4 – 6 . Besides, it plays a role in the therapeutic efficiency of various medications and in preserving the structure and function of a healthy GI tract 7 – 10 . Meanwhile, dysregulated gut flora contributed to different pathological conditions such as cardiovascular, CNS, inflammatory bowel diseases, and even depression 11 – 13 . Nucleotides are involved in almost all biological processes, like the synthesis of nucleic acids, energy metabolism, signal transduction, and participate in the process of enzymatic regulation 14 . Endogenous synthetic processes are the primary source of nucleosides, besides other exogenous natural sources such as plant- and animal-based food 15 . The demand for nucleotides during growth and other stressful conditions, such as infections, had provoked the need for nucleotide dietary supplements to become intriguingly semi-essential nutrients 16 . Uridine and cytidine as nucleosides showed therapeutic potential in different diseases. In recent studies, uridine supplementation was correlated to increased diversity of gut microbiota in High Fat Diet (HFD)-fed mice 17 , and significantly enhanced the growth and development of animals during weaning 18 . Cytidine monophosphate (CMP) can modify gut microbiota 19 , improve growth 20 , and enhance memory and learning 21 . Zinc is fundamental element for enzymes function and protein’s structure. Diverse physiological processes including cell replication are affected by zinc deficiency. It is also necessary for growth, immune systems, intestinal function, and brain development 22 . Zinc deficiency or over supplementation played a role in the pathogenesis of multiple diseases. Substantially, the balanced levels of zinc and other transition metals in the body contributed to the diversity and abundance of gut microbiota 23 , 24 . Zinc deficiency has been associated with depression, reduced cognitive function, chronic inflammation, growth retardation, delayed wound healing, and reduced diversity and richness in gut microbiota 25 , 26 . Furthermore, other gastrointestinal conditions such as animal dysbiosis, irritable bowel syndrome, and Crohn's disease were related to zinc deficiency 27 – 29 . Thus, zinc supplementation is vital to maintain balanced endogenous levels of zinc to maintain health. Recently, the use of nucleosides as supportive regimens for multiple neurodegenerative diseases has increased. Accordingly, as the reliance on these supplements is on the rise, having the opportunity to study the effect of zinc, uridine and cytidine, alone and in combination, on the diversity and abundance of normal gut microbiota while knowing the importance and role of balanced gut microbial colonization and its contribution to the health of humans and animals will allow for further functional and biological deep understanding while evaluating other clinical parameters that may indicate the safety and effectiveness of these supplements. Basically, scientific research has discussed the potential effects of zinc, uridine, and cytidine in a separate manner 17 , 18 , 23 , 30 . However, the effect of their combination on gut microbiota has not been reported yet, especially in healthy rats. Therefore, this study was designed to study the effect of the combination of uridine, cytidine, and zinc on the gut microbiota, besides other important clinical parameters in healthy rats. Results Impact of combined and individual supplements on the diversity and abundance of gut microbiota in healthy rats Alpha microbial diversity 16S rRNA sequencing analysis was carried out in all animal groups in replicates, relative abundances at baseline and the last day of treatment were calculated accordingly for each group. Shannon and Simpson diversity Indices were calculated to reflect microbial diversity (Table 1 and Fig. 1 ). The Uridine group had the most apparent drop in bacterial diversity among groups throughout the treatment period as the Shannon diversity index increased from 3.7 to 4.2 on day 42. The cytidine group had a similar trend to the uridine group, as the Shannon diversity index increased from 4.2 to 4.4 throughout the treatment period. Meanwhile, no changes in the Shannon diversity index were noticed in the zinc group. On the other hand, the combination group showed a slight reduction in the Shannon diversity index, indicating a slight rise in the biodiversity. Furthermore, the difference in Shannon or Simpson diversity indices, whether increase or decrease at day 42 for all animal groups, was insignificant compared to control at baseline upon performing One-Way ANOVA statistical analysis (Fig. 1 ). Table 1 Mean Shannon Index, mean Simpson Index, mean bacterial count, evenness and average population size calculated at day 0 (baseline) and day 42 (the last day of treatment) for the combination, uridine, cytidine, and zinc treated groups. Treatment group Combination Uridine Cytidine Zinc Days d 0 d 42 d 0 d 42 d 0 d 42 d 0 d 42 Mean Shannon Index # 4.5±0.06 4.4±0.16 3.7±0.03 4.2±0.07 4.2±0.06 4.4±0.02 4.3±0.0 4.3±0.21 Mean Simpson Index # 0.08±0.01 0.09±0.02 0.16±0.0 0.09±0.01 0.07±0.002 0.08±0.002 0.08±0.0 0.09±0.02 Mean bacterial count 40788 48841 51786 36773 48842 46481 49798 44219 Evenness 0.78 0.76 0.65 0.80 0.77 0.82 0.79 0.79 Average population size 832 997.0 1328 943 1191 1134 1186 1028 # Results are presented as mean ± SD, knowing that the lower the Simpson index value, the higher the diversity, while higher value of Shannon index means more biodiversity in the sample. Changes at the class level Bacteroidia and Clostridia were the most dominant bacterial classes in all animal groups before starting the treatment at day 0 (Fig. 2 ). The relative abundance of Clostridia class increased in all treatment groups after 42 days of treatment to reach its highest levels 41.6% in zinc group. Meanwhile, Bacteroidia class decreased at the end of the treatment period to reach 34.3% in the control group, which was the lowest among others. Besides, the Clostridia to Bacteroidia ratio was calculated during day 42 of treatment. Combination group showed a 1:1.2 ratio and cytidine group showed a 1:1.3 ratio, meanwhile; zinc treated group showed a 1:0.9 ratio. However, uridine treated group showed a 1:2.5 ratio. Generally, the ratio increased by the end of the treatment period when compared to baseline in all treatment groups, (Table 2 ). Table 2 The table summarizes calculated Clostridia: Bacteroidia ratio for the combination, uridine, cytidine, and zinc treated groups, at baseline (day 0) and at the last day of treatment (day 42). Clostridia: Bacteroidia Combination Uridine Cytidine Zinc Day 0 (baseline) 1:2.5 1:3 1:4 1:1.2 Day 42 (the last day of treatment) 1:1.2 1:2.5 1:1.3 1:0.9 Changes at the family level The mean relative abundance of bacterial families showed three dominant families in all animal groups, Prevotellaceae , Lachnospiraceae and Bacteroidaceae . The most abundant family was Prevotellaceae among others; however, it displayed inconsistent decrease and increase trends between the groups. The Lachnospiraceae family; on the other hand, increased in all animal groups except for the zinc-treated group, which remained almost the same by day 42, while cytidine treated group showed the highest abundance of Lachnospiraceae among others. Moreover, all groups exhibited a decline in the relative abundance of Bacteroidaceae from day 0 up to day 42 (Table 3 ). No significant difference between the relative abundances of different families in different treatment groups at day 42 when compared to baseline control at day 0 ( p- value > 0.05 ). In addition, several families were shared between the different groups, while the combination and the uridine treatments showed the highest number of unique families (Fig. 3 ). Table 3 relative abundances of Prevotellaceae, Lachnospiraceae and Bacteroidaceae, the most abundant bacterial families, in the combination, uridine, cytidine and zinc treatment groups at baseline before the start of treatments (day 0) and at the last day of treatment (day 42). Data is presented as mean ± SD. Family Control Combination Uridine Cytidine Zinc d 0 d 42 d 0 d 42 d 0 d 42 d 0 d 42 d 0 d 42 Prevotellaceae% 26.3±4.4 19.6±4.4 26.7±5.3 29.1±9.8 43.2±2.5 31.1±12.5 26.9±3.1 23.7±10.9 17.6 26.9±12.2 Lachnospiraceae% 10.9±6.5 13.8±1.9 7.4±1 17.3±11 4.7±0.46 12.7±5.6 5.0±0.03 16.4±8.7 20.6 20.3±7.9 Bacteroidaceae% 16.6±0.41 8.4±1.8 23.4±9.2 6.2±0.13 14.6±2.2 7.8±4.1 21.4±0.47 14.7±0.9 10.3 4.8±0.43 Changes at the species level At the species level, the mean relative abundances showed different patterns among the studied groups, and some common and unique trends. The species Allobaculum stercoricanis increased uniquely in the combination group, while decreased in other groups. Moreover, the relative abundance of Bacteroides acidifaciens and Bacteroides uniformis species had uniquely increased in the cytidine group by day 42, in contrast to the combination and uridine groups at which it had declined. On the other hand, the relative abundance of Bacteroides plebeius had a remarkable increase in the uridine group by day 42, while decreased in the other three treatment groups. Statistical analysis showed that the differences in species relative abundances for different treatment groups at day 42 and compared to baseline control were insignificant (Fig. 4 ). Clinical parameters Effect on body weight in healthy rats At days 0, 21, 42 and after the discontinuation of treatments; at day 49, there were no significant differences in the mean body weight among different treatment groups, compared to control ( p -value > 0.05). For instance, the increase of weight for the rats in all groups at baseline and day 42 was similar to that of the control group, suggesting no effect of the different treatments on gaining weight (Fig. 5 A). Effect on Fasting blood glucose level in healthy rats A significant reduction in the FBG level for the animal group which received the combination supplement was noticed directly after three weeks of treatment (day 21) and it was kept even after the discontinuation of therapy at week seven (day 49) (****p- value < 0.0001). Moreover, a significant reduction in the FBG level was noticed at days 21, 42 and 49 when compared to baseline in other animal groups which received individual supplements, uridine, cytidine (** p -value < 0.01) and zinc alone (* p -value < = 0.05). Accordingly, the combination therapy showed a more significant reduction in glucose levels after three weeks of therapy in comparison to other individual treatments (Fig. 5 B). Effect on motor activity in healthy rats Statistical analysis showed no significant differences in the mean latency to fall (sec) time among different animal groups or even when compared to control at each time interval in the study. However, the combination group showed significant improved motor activity at day 21 (* p -value < 0.05 ), compared to cytidine and uridine groups at baseline (Fig. 5 C). Effect on the nociceptive response in healthy rats A significant reduction in the mean index of nociception was obvious in the combination group at day 42 and day 49 in comparison to day 0 (**** p- value < 0.0001). Moreover, the same significant reduction was noticed in uridine group at days 21, 42 and 49 (**** p- value < 0.0001) (Fig. 5 D). Effect on liver enzymes level in healthy rats The baseline for normal ALP level was determined on day 0 for all animals (n = 35), which was 209.8±17.5 U/l, and the median was 202.2 U/l. The mean ALP level for the different treatment groups at baseline didn’t exceed the stated reference ALP level which was established by the kit manufacturer; (89–370 U/l). In general, the mean ALP level for the combination, uridine, cytidine, and zinc supplemented animal groups was decreasing throughout the study time starting from day 0 to day 49. Statistical analysis showed no significant differences between animal groups or in comparison to control group on days 0, 21, 42 and 49. Remarkably, the animal group that received the combination of (uridine, cytidine and zinc) resulted in a significant reduction in ALP level (M = 118.02 U/l) on day 42 (* p- value = 0.0148), and day 49 after treatment discontinuation (M = 122.5 U/l) (* p- value = 0.0257), compared to day 0 (M = 225 U/l) for this group. However, the cytidine group showed a more significant reduction in ALP level at day 49 (M = 148.6 U/l) compared to day 0 (M = 244.3 U/l) (* p- value = 0.0055) (Fig. 5 E). The baseline for normal ALT level was determined on day 0 for all animals (n = 35), which was 34.9±3.1 U/l, and the median was 36.5 U/l. The mean ALT level at baseline for different treatment groups didn’t exceed the stated reference ALT level which was established by the kit manufacturer (up to 42 U/l). In general, the mean ALT level in the different treatment groups has a common increase trend starting from baseline to the last day of treatment, followed by a decrease in ALT level after treatments discontinuation. The increase at day 42 compared to day 0 in the combination, cytidine and zinc groups was 2-fold, however, for the uridine group was 1.7-fold, but in all cases this increase was not significant ( p -value > 0.05), and it was followed by a subsequent decrease upon treatments discontinuation to reach the normal levels again at day 49 (Fig. 5 F). Discussion Dietary nucleotides are being widely used as supplemental therapy for a variety of neurodegenerative diseases. This study aimed to evaluate the effect of (zinc, uridine, and cytidine), both alone and in combination, on the diversity and abundance of normal gut microbiota while monitoring other clinical parameters as an indication for their safety and effectiveness. In agreement with previous studies, the results of this study showed an increase in Shannon and Simpson diversity indices in uridine and cytidine-supplemented groups, reflecting a decrease in microbial diversity, although it is insignificant. The richness and composition of gut microbiota were improved in formula-fed human infants containing nucleotides 19 , 31 . Furthermore, individually added nucleotides such as CMP and UMP and others changed the growth of certain bacterial strains in piglet’s small intestine 32 , and increased bacterial diversity in mice fed HFD 17 , 33 . However, according to the study findings no changes in the diversity indices of the combination or zinc supplemented groups were noticed. This is expected due to the relatively low zinc dose used in this study compared to the doses mentioned in literature. Basically, the role of low zinc concentrations in the combination supplement is to catalyze the cytidine deaminase enzyme, which converts cytidine into uridine 34 . Besides, different forms and concentrations of zinc supplements could change the gut microbiome 23 , 24 , 35 and in a dose-dependent manner 35 , 36 . In contrast to previous studies that were conducted under certain conditions, such as weaning or disease status, this study highlighted the changes of gut microbiota in healthy rats. The increased Clostridia: Bacteroidia ratio in all animal groups at the class level, may indicate positive outcomes on gut health. Increased relative abundance of Bacteroidia is normally correlated to inflammatory conditions due to increased cytokine production and endotoxins. However, increased levels of Clostridia are associated with decreased inflammation and improved gut function 37 – 39 . Members of Lachnospiraceae family are butyrate producing bacteria, which is considered as a source of energy for colonocytes and causes indirect activation for nervous and hormonal systems 40 . The anti-inflammatory and immunity inducing effects for butyrate were reported as well 41 . Importantly, Lachnospiraceae was correlated to improved glucose tolerance 42 , which may contribute to this study findings which showed significant reduction in the fasting blood glucose levels at week three in all animal groups in comparison to baseline. On the other hand, the relative abundance of the members belonging to Bacteroidaceae family, which showed a slight reduction in all animal groups, might be correlated to carbohydrates metabolism in humans and rodents 43 . The increased abundance of this family was correlated to type-2 diabetes 44 . Members of Prevotellaceae family, which showed insignificant raise in their relative abundance in the combination and zinc groups exclusively, is considered as commensal bacteria and infrequently involved in infections. Only a few members can cause endogenous infections such as chronic infections and anaerobic pneumonia 45 . Some previous studies supported that nucleotides supplementation lead to increased weight gain and head growth in formula-fed infants and enhanced growth in animals during weaning due to decreased intestinal inflammation 46 , 47 . On the other hand, neither UMP nor uridine had significant effects on the growth performance of weaned piglets 48 . CMP reduced weight gain in mice model which was fed high fat diet 33 . Furthermore, pharmacological zinc doses of 3000 ppm, which are much higher than the zinc dose used in this study, were able to boost growth in weaned piglets 49 . In other studies, zinc related growth enhancement has been observed at plasma zinc concentrations around 2.5 mg/L 50 , 51 . According to this study findings neither the individual supplements, nor their combination resulted in a significant increase in body weight compared to control at the last day of the study. According to previous studies, uridine in the form of UMP increased the plasma glucose levels significantly in weaned piglets 48 . Besides, uridine had increased gluconeogenesis, decreased insulin signaling activity and long term administration of uridine over 16 weeks had induced glucose intolerance and sever lipid accumulation in mice liver 52 . High doses of cytidine, on the other hand, improved glucose tolerance in HFD fed-mice 33 . it was concluded that zinc supplementation improved fasting insulin and glucose levels in obese mice 53 .It decreased the glycosylated hemoglobin HBA1C% after supplementing 30 mg of zinc over 3 months in type 2 diabetic patients, while other studies concluded that these effects will not be achieved over short time administration 54 , 55 . The changes in microbiome in the healthy rats in this study could be attributed to the reduced fasting blood glucose levels, as the abundance of certain species that is related to improved glucose tolerance and enhanced insulin activity. For example, the relative abundance of certain species increased such as Bacteroides acidifaciens and Bacteroides uniformis . Bacteroides acidifaciens can regulate intestinal dipeptidyl peptidase-4 (DPP-4) secretion and subsequently induce glucagon-like peptide 1 (GLP-1) production, which may contribute to glucose homeostasis and may have a role in the prevention of diabetes and obesity 56 . Furthermore, Bacteroides uniformis reduced body weight gain, increased oral glucose tolerance in HFD-fed mice, improved immunity and reduced inflammation 57 , 58 . Moreover, Lachnospiraceae family was strongly correlated to improved glucose tolerance, involved in the metabolism of carbohydrates and SCFAs production 59 , 60 . The abundance of Lachnospiracea family increased in all groups except the zinc group. This may explain the more significant reduction in the FBG level that was noticed in the combination supplemented group and could be attributed to the presence of cytidine, uridine, and zinc concomitantly, which may have resulted in a synergistic action to reduce the glucose levels. The catalytic zinc dose in the combination supplement may contribute to the activation of the cytidine deaminase enzyme that converts cytidine to uridine 34 . Furthermore, this study focused on evaluating the effect on healthy rats over a short time (six weeks), unlike previous studies that were implemented over a prolonged time on rats with pathological conditions, accordingly further studies in the future could answer this hypothesis. The significantly improved motor activity in the combination group could be attributed to the synergistic action between the three individual supplements. In agreement with this study result, it was concluded that uridine in combination with omega three fatty acids enhanced motor activity and increased latency to fall time in mice model with Parkinson disease via enhancing brain phospholipids levels and brain dopaminergic neurotransmission 61 . On the other hand, chronic treatment with uridine alone did not cause any changes in motor activity or rotation 62 . The improved motor activity in the cytidine-treated group could be attributed to CDP-choline metabolites such as choline, which were involved in the synthesis of acetylcholine that contributed directly to improved motor activity, and the protection of cholinergic neurons by sparing membrane phospholipids 63 . Moreover, CDP-choline administered intraperitoneally has improved motor activity in aged mice by acting on central neurotransmission and cognition 64 . Zinc deficiency has been associated with impaired motor activity in some studies 65 , while other studies in contrast, concluded that motor activity could not be affected by zinc deficiency 66 . The index of nociception which decreased significantly and only in the combination and uridine groups may indicate more pain/ nociception 67 . Nucleotides are involved in phospholipids and glycolipids synthesis and may promote nerve fiber regeneration. In a previous study, CMP and UMP didn’t improve nerve thermal conduction parameters in diabetic patients with neuropathy 68 . On the other hand, the combination of UMP with vitamin B12 and folic acid has significantly reduced neuropathic pain intensity in patients with peripheral neuropathy 69 . UTP administration has prolonged the thermal nociceptive latency in the tail-flick test 70 . Importantly, the hyper-polarization-activated, cyclic nucleotide-gated (HCN) channels in nociceptive neurones, were involved in peripheral nociception 71 , and some studies highlighted their direct contribution to neuropathic pain 72 , 73 . Most of the studies in literature discussed the contribution of cyclic nucleotides signalling pathway in chronic inflammatory pain induced by injury and stress; however, this study evaluated the antinociceptive effect of different supplements on induced acute non-inflammatory pain in healthy rats. ALT levels, according to our results have increased throughout the study time up to 2 folds the baseline level, but this increase in all cases was reversible upon treatment discontinuation and non-significant. ALP enzyme is found almost in all tissues, and on the surface of hepatocytes. Usually, elevated levels of ALP are associated with cholestatic disorders 74 , or could be referred to stress factors that the rats might have experienced during blood sampling and /or during dose administration of test substances 75 , 76 . On the other hand, the ALT enzyme is more indicative of hepatocytic necrosis, and its elevated levels reflect leakage from damaged tissues in the liver 77 . In a previous study, nucleotides at high doses were safe and decreased ALT levels but insignificantly, thus promoted liver function 20 . Nucleotides were able to reverse increased ALT and AST levels associated with alcohol induced liver injury in rats 78 , 79 . Unlike previous studies, this study examined the effect of various supplements on the levels of liver enzymes in healthy rats; additionally, the findings suggest that they are relatively safe, but if they were supplemented over a prolonged period, liver enzymes should be monitored. As the use of the combination of Uridine, Cytidine and Zinc by healthy people, as adjuvant treatment for many diseases grows, our findings showed that gut microbiota changes suggest beneficial clinical implications for gut health and could contribute to the revealed changes in FBG levels. Our results support the efficacy and safety of these supplements, with recommendation to monitor liver enzymes when used for prolonged time. Overall, further investigations in the future could provide further understanding of the promising clinical implications of metabolic and gut health, besides determining the best dosages and treatment regimens. Materials and methods All the experimental procedures in this study were ethically approved by the Institutional Review Board Committee for the field of Clinical Pharmacy and Pharmacy Practice at the Applied Science Private University (ASU), Faculty of Pharmacy (Approval No. 2022-PHA-33). Dietary supplements The supplements used in this experiment were purchased from community pharmacies in Amman-Jordan and USA. ISCHELIUM (150 mg CMP, 150 mg UMP and 10 mg zinc, Polifarma®, Roma-Italy) vials of 10 ml oral solution, CITICOLIN (250 mg Cytidine -5’-diphosphocholine capsules, Cognizin®, Kyowa Hakko Bio Co., Ltd.), URIDINE (300 mg Uridine -5’-monophosphate disodium salt capsules, Amazing Nutrition®, USA), ZINC (25 mg zinc gluconate tablets, Jamieson™). Experimental animals and experimental design A total of 35 male, healthy, eight-weeks old, wistar rats were utilized, and procured from the animal house laboratories, Pharmacy School, ASU- (Amman- Jordan), with a mean weight of 125 ± 5 g. All animals were housed and acclimatized in the animal room at the university, at a temperature of 21-23 ℃ and humidity of 35 % - 70 %, and under a 12-hour light/dark cycle. Rats were continuously fed with the standard diet, and a free access to water and food throughout the study period. Rats were randomly divided into 4 treatment groups and one control (n=7), and then each rat was identified in each group to monitor its clinical parameters. The doses were calculated by extrapolation based on human average body weight (70 Kg) while considering the average rat weight (130 g) at the age of eight weeks. Moreover, the specified concentrations of the solutions of the individual supplements were prepared by dissolving their contents in distilled water based on the solubility of the active ingredients and then were placed on the vortex for dissolution. Group I received 20 µl/day/rat of the commercially available combination of (Cytidine, Uridine and Zinc), ISCHELIUM® at a daily dose of 10 ml oral vial/day for humans. Group II received 8 µl/day/rat of Uridine alone at a daily dose of 50 mg/ml suspension. Group III received 4 µl/day/rat of Cytidine alone, at a daily dose of 100 mg/ml suspension. Group IV received 0.3 µl/day/rat of Zinc alone at an average daily dose of 0.25mg/ml solution. The last group was the control which received normal saline on daily basis. The study was conducted over six weeks of treatment during which, all previously mentioned doses have been administered by the orogastric gavage with another week (7 th week) of no treatments for all animal groups. Blood and fecal samples collection and analysis Blood and fecal samples were collected at the baseline (before the start of day one; day 0), and at the end of weeks three (day 21), six (day 42) and seven (day 49). All rats were fasted for 15 hours before the sampling time 80 . Blood samples were obtained from the rats’ orbital sinus veins using capillary tubes to assess blood glucose level directly. To prepare serum samples, part of the collected blood was set to clot directly at 4 ᵒC overnight, then the samples were centrifuged at 1000 rpm for 5 min. Sterile Eppendorf tubes were used to collect serum and finally the samples were stored at -80 ᵒC until further analysis to assess liver enzymes. Moreover, fresh fecal samples were collected in a previously autoclaved Eppendorf tubes to investigate the changes in gut microbiota and were frozen immediately at -80 ℃ for further analysis. Barcoded sequencing of the 16S rRNA gene and data analysis Bacterial DNA was extracted from fecal samples using G-spin™ Total DNA Extraction Mini Kit, purchased from QIAGEN, Germany, following the manufacturer's protocol. The extracted DNA was sent to the Molecular Research Lab (www.mrdnalab.com, MR DNA, Shallowater, TX) for 16S rRNA sequencing and data analysis. The 16S rRNA gene V4 variable region was amplified with PCR primers 27F-519R. PCR products were then pooled, purified to be sequenced using Illumina on a MiSeq following the manufacturer's guidelines. Sequencing data were processed using MR DNA analysis pipeline (MR DNA, Shallowater, TX, S.A.). Sequences were denoised, chimeras were removed, and operational taxonomic units (OUT) were generated. Final OTUs were taxonomically classified using BLASTN against a curated database derived from NCBI (National Center for Biotechnology Information, USA). Assessment of clinical parameters All clinical parameters were assessed at the baseline of the study (day 0), and at the end of week three (days 21), week six (day 42), and after the discontinuation of treatments at week seven (day 49) except for the body weight which was monitored on weekly basis. Body weight Fasting Blood Glucose Test (FBG) Blood glucose concentrations were determined immediately after collecting blood samples using an Accu-Chek® Performa glucose meter, (Roche, Germany). Motor activity The rota-rod test was used to assess the motor activity behaviour of each rat 81 . The rota-rod treadmill (Muromatchi kikai Co., Ltd., Mk-630B, Tokyo, Japan) that was used in the study is a single-lane treadmill, thus enabling one rat to walk . Initially, rats had multiple practice sessions on the device before they were placed individually on the accelerating rod with different speeds, ranging from 4 rpm to 40 rpm and facing away from the experimenter. The latency to fall was reported for a maximum time of 300 sec. Antinociceptive effect (The hot plate test) Paw-licking and paw-lifting behaviours were monitored as acute, non-inflammatory pain indicators 82,83 . The rats were placed individually on a hot-plate analgesia meter (Muromatchi Kikai Co., Ltd, Mk 350D, Tokyo, Japan), heated at 55℃, which was optimal to induce pain, not burn, then the timer was started immediately until the rat had shown a nociceptive response such as paw-licking, paw-lifting, or jumping within a maximum latency time of 60 sec. Liver Function Thawed serum samples were used to test the levels of ALT and ALP using liver enzymes spectroscopic diagnostic kits (BioSystems S.A, Barcelona, Spain), by following the accompanying kits protocol and the manufacturer’s instructions. Statistical analysis The significant difference among the different groups means was assessed by one-way and two-way analysis of variance (ANOVA) and Post-hoc analysis using Tukey’s test, by GraphPad Prism 9 (USA), and the results were presented as mean ± standard error of the mean (SEM). The Shannon and Simpson diversity indices were used to assess the effects of the different treatments on the composition and abundance of gut microbiota. In data presentation, p -value ≤ 0.05 was considered a significance cut-off point and tagged by one asterisk (*), p -value < 0.01 was tagged by two asterisks (**), p -value <0.001 was tagged by three asterisks (***) and p -value <0.0001 was tagged by four asterisks (****). Declarations Authors contribution Conceptualization: Maram A. Othman, Muna Barakat, Amin Omar, Mohammad AA Al-Najjar. Data curation: Maram A. Othman, Formal analysis: Maram A. Othman. Investigation: Maram A. Othman, Muna Barakat, Ruaa R. Al-Alwany, , Mohammad AA Al-Najjar. Methodology: Maram A. Othman, Muna Barakat, Tamara Athamneh, Ruaa R. Al-Alwany, , Mohammad AA Al-Najjar. Project administration: Muna Barakat, Ruaa R. Al-Alwany, , Mohammad AA Al-Najjar. Resources: Maram A. Othman, Tamara Athamneh, Ruaa R. Al-Alwany. Software: Maram A. Othman. Supervision: Muna Barakat, Amin Omar, Rafat Zrieq, , Mohammad AA Al-Najjar. Validation: Muna Barakat, Amin Omar, Tamara Athamneh, Rafat Zrieq, Mohammad AA Al-Najjar. Visualization: Muna Barakat, Amin Omar, Tamara Athamneh, Rafat Zrieq, Mohammad AA Al-Najjar. Writing – original draft: Maram A. Othman, Amin Omar, Rafat Zrieq. Writing – review & editing: Maram A. Othman, Muna Barakat, Amin Omar, Tamara Athamneh, Rafat Zrieq, Mohammad AA Al-Najjar. Acknowledgment The authors acknowledge the support from the Applied Science Private University in using the lab facilities to conduct this research. They also acknowledge First Care medical company for providing the Ischelium dietary supplement used in this research study. Conflict of interest No potential conflict of interest relevant to this article was reported. Data Availability Statement Data is available within the article or its supplementary materials. Funding Statement This study was not supported by any national or international institution. Ethical Approval Statement This study is reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All animal experiments were conducted following the ethical standards and guidelines set forth by our institution and approved by the Animal Research Ethics Committee at the Applied Science Private University (ASU), Faculty of Pharmacy (Approval No. 2022-PHA-33)., and all procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. References Backhed, F., Ley, R. 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Cunningham, J. J., Fu, A., Mearkle, P. L. & Brown, R. G. Hyperzincuria in individuals with insulin-dependent diabetes mellitus: concurrent zinc status and the effect of high-dose zinc supplementation. Metabolism 43 , 1558-1562 (1994). Gunasekara, P., Hettiarachchi, M., Liyanage, C. & Lekamwasam, S. J. J. D. Blood Sugar lowering effect of zinc and multi vitamin/mineral supplementation is dependent on initial fasting blood glucose. 1 (2011). Yang, J. et al. Gut commensal Bacteroides acidifaciens prevents obesity and improves insulin sensitivity in mice. 10 , 104-116 (2017). Gauffin Cano, P., Santacruz, A., Moya, Á. & Sanz, Y. Bacteroides uniformis CECT 7771 ameliorates metabolic and immunological dysfunction in mice with high-fat-diet induced obesity. (2012). Cani, P. D. et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 56 , 1761-1772 (2007). Devillard, E., McIntosh, F. M., Duncan, S. H. & Wallace, R. J. J. J. o. b. Metabolism of linoleic acid by human gut bacteria: different routes for biosynthesis of conjugated linoleic acid. 189 , 2566-2570 (2007). Sheridan, P. O. et al. Polysaccharide utilization loci and nutritional specialization in a dominant group of butyrate-producing human colonic Firmicutes. 2 (2016). Perez-Pardo, P. et al. Gut–brain and brain–gut axis in Parkinson's disease models: effects of a uridine and fish oil diet. 21 , 391-402 (2018). Myers, C., Napolitano, M., Fisher, H., Wagner, G. J. P. o. t. S. f. E. B. & Medicine. Uridine and stimulant-induced motor activity. 204 , 49-53 (1993). Conant, R. & Schauss, A. G. Therapeutic applications of citicoline for stroke and cognitive dysfunction in the elderly: a review of the literature. Alternative medicine review 9 , 17-31 (2004). Drago, F. et al. Effects of cytidine-diphosphocholine on acetylcholine-mediated behaviors in the rat. 31 , 485-489 (1993). Golub, M. S. et al. Modulation of behavioral performance of prepubertal monkeys by moderate dietary zinc deprivation. 60 , 238-243 (1994). Cole, T. B., Martyanova, A. & Palmiter, R. D. Removing zinc from synaptic vesicles does not impair spatial learning, memory, or sensorimotor functions in the mouse. Brain research 891 , 253-265 (2001). Ledowski, T. J. B. j. o. a. Objective monitoring of nociception: a review of current commercial solutions. 123 , e312-e321 (2019). Verdugo, R. J. et al. PS-51-7 Cytidine monophosphate (CMP), uridine triphosphate (UTP) and hydroxycobalamin in the treatment of diabetic polyneuropathy: preliminary study. 97 , S219-S220 (1995). Negrão, L. et al. Effect of the combination of uridine nucleotides, folic acid and vitamin B12 on the clinical expression of peripheral neuropathies. 4 , 191-196 (2014). Okada, M., Nakagawa, T., Minami, M., Satoh, M. J. J. o. P. & Therapeutics, E. Analgesic effects of intrathecal administration of P2Y nucleotide receptor agonists UTP and UDP in normal and neuropathic pain model rats. 303 , 66-73 (2002). Sartiani, L., Mannaioni, G., Masi, A., Romanelli, M. N. & Cerbai, E. J. P. r. The hyperpolarization-activated cyclic nucleotide–gated channels: from biophysics to pharmacology of a unique family of ion channels. 69 , 354-395 (2017). Jiang, Y.-Q. et al. Axonal accumulation of hyperpolarization-activated cyclic nucleotide-gated cation channels contributes to mechanical allodynia after peripheral nerve injury in rat. 137 , 495-506 (2008). Chaplan, S. R. et al. Neuronal hyperpolarization-activated pacemaker channels drive neuropathic pain. Journal of Neuroscience 23 , 1169-1178 (2003). Rosalki, S. & Mcintyre, N. i. J. O. t. o. c. h., Ed. Biochemical investigations in the management of liver disease. 2 , 506-507 (1999). Gao, X., Zeng, Y., Liu, S., Wang, S. J. L. i. H. & disease. Acute stress show great influences on liver function and the expression of hepatic genes associated with lipid metabolism in rats. 12 , 1-6 (2013). Al-Najjar, M. A. et al. Evaluation of the orally administered calcium alginate aerogel on the changes of gut microbiota and hepatic and renal function of Wistar rats. PLoS One 16 , e0247633 (2021). Rosen, H., Keeffe, E. J. L. d. d. & management. Evaluation of abnormal liver enzymes, use of liver test, and the serology of viral hepatitis. 24-35 (2000). Cai, X. et al. Dietary nucleotides supplementation and liver injury in alcohol-treated rats: a metabolomics investigation. Molecules 21 , 435 (2016). Osama I. Ramadan, M. A. A. Hepatoprotective effects of silymarin on cyclophosphamide-induced hepatotoxicity in male rats. Jordan Journal of Applied Science - Natural Science Series 17 , 24-27 (2023). Waner, T. & Nyska, A. J. V. C. P. The influence of fasting on blood glucose, triglycerides, cholesterol, and alkaline phosphatase in rats. 23 , 78-80 (1994). Shiotsuki, H. et al. A rotarod test for evaluation of motor skill learning. 189 , 180-185 (2010). WOOLFE, G., MacDonald, A. J. J. o. P. & Therapeutics, E. The evaluation of the analgesic action of pethidine hydrochloride (Demerol). 80 , 300-307 (1944). Barrot, M. Tests and models of nociception and pain in rodents. Neuroscience 211 , 39-50 (2012). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4667549","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":329682875,"identity":"061a7d63-8886-4393-af89-8049b5d03cd6","order_by":0,"name":"Maram A. 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Al-Alwany","email":"","orcid":"","institution":"Applied Science Private University","correspondingAuthor":false,"prefix":"","firstName":"Ruaa","middleName":"R.","lastName":"Al-Alwany","suffix":""},{"id":329682884,"identity":"64ff22a3-e7a3-4d50-a737-5001ebc1969c","order_by":6,"name":"Mohammad AA Al-Najjar","email":"","orcid":"","institution":"Applied Science Private University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"AA","lastName":"Al-Najjar","suffix":""}],"badges":[],"createdAt":"2024-07-01 11:09:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4667549/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4667549/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61197831,"identity":"75c2f82c-c509-4f21-a9d6-c0fa0f52b659","added_by":"auto","created_at":"2024-07-27 00:34:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16963,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plots show Shannon and Simpson diversity indices (amicrobial diversity) that were calculated on the last day of treatment (day 42) in replicates for the untreated, combination, uridine, cytidine and zinc animal groups, and in replicates at the baseline (Control group) before the start of treatments (day 0).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4667549/v1/10187c064ef508acbabb91f8.jpg"},{"id":61197832,"identity":"475137e3-4a62-4e4f-b751-88bae61bc5eb","added_by":"auto","created_at":"2024-07-27 00:34:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39560,"visible":true,"origin":"","legend":"\u003cp\u003eStacked columns show the relative abundances of 12 different bacterial classes in the combination, uridine, cytidine, and zinc group at baseline before the start of treatments (day 0), and at the last day of treatment (day 42). One-Way ANOVA analysis showed an insignificant difference in the relative abundance at day 42 for the different classes in different treatment groups when compared to baseline control (day 0), p-value \u0026gt; 0.05 significance level.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4667549/v1/1dac57ef1bba9a5d22abb6aa.jpg"},{"id":61197834,"identity":"5cdada61-26bb-42f2-ac81-5153907f88cc","added_by":"auto","created_at":"2024-07-27 00:34:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":33377,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram of common and unique families among the animal groups treated with the combination, uridine, cytidine, and zinc. The numbers in different colors in the diagram represent the common or unique bacterial families with the same relative abundance increase or decrease trend across groups.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4667549/v1/2223dbcbfa544c9552b0ab0c.jpg"},{"id":61197836,"identity":"d1b2a100-0a18-4952-b263-e80fbef56388","added_by":"auto","created_at":"2024-07-27 00:34:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":85799,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of the mean relative abundance of 47 common bacterial species between combination, uridine, cytidine, and zinc group at baseline before the start of treatments (day 0) and at the last day of treatment (day 42). The value of the mean relative abundance is displayed on the graph in different colours. One-way ANOVA indicated no significant statistical difference in the mean relative abundance of different species at day 42 and day 0 in the four different treatment groups, p-value \u0026gt;0.05.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4667549/v1/ffdff8bcce910bc3595cdf4a.jpg"},{"id":61197833,"identity":"118df103-b3f7-4bcb-b9c3-df0a51f0dff6","added_by":"auto","created_at":"2024-07-27 00:34:29","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37920,"visible":true,"origin":"","legend":"\u003cp\u003eColumn charts represents \u003cstrong\u003eA)\u003c/strong\u003e mean rats body weight (g) \u003cstrong\u003eB)\u003c/strong\u003e mean Fasting Blood Glucose level (FBG) (mg/dl) \u003cstrong\u003eC)\u003c/strong\u003emean latency to fall (sec) \u003cstrong\u003eD)\u003c/strong\u003e mean index of nociception (sec) \u003cstrong\u003eE)\u003c/strong\u003emean Alkaline Phosphatase enzyme level (ALP) (U/I) \u003cstrong\u003eF)\u003c/strong\u003e mean Alanine aminotransferase enzyme level (ALT) (U/I) of the five different animal groups, uridine, cytidine, zinc, combination, and control, at (day 0) before the start of treatments, after 3 weeks of treatment (day 21), after six weeks of treatment (day 42) and after the discontinuation of therapy (day 49) (Data presented as mean ±SEM, n=7). Statistical analysis was performed using Two-Way ANOVA and post hoc Tukey’s test, different groups were compared and to control, and were presented as means and standard error of the mean SEM.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4667549/v1/8b7c63fb9064cf473cca4fb6.jpg"},{"id":75687691,"identity":"dac96bd4-2ac7-4883-b97d-f7cba9e083c7","added_by":"auto","created_at":"2025-02-07 06:38:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1340221,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4667549/v1/420f949e-931a-4b77-b28a-000ebc6e85b2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The potential effects of the combination (Cytidine, Uridine and Zinc) on gut microbiota and clinical parameters of healthy rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe adult human gastrointestinal (GI) tract harbors more than 10\u003csup\u003e14\u003c/sup\u003e diverse microbial populations \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The healthy human gut microbiota is mainly predominated by two important phyla, \u003cem\u003eBacteroidetes\u003c/em\u003e and \u003cem\u003eFirmicutes\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The composition of this normal gut microbiota can be influenced by several factors since infancy, such as mode of delivery, the type of feeding during infancy, diet, antibiotics, and other factors \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Gut microbiota contributes to the disease or health state of humans and animals. For instance, it can act as an immunomodulator, and play a remarkable role in nutrients metabolism and vitamins K and B synthesis \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Besides, it plays a role in the therapeutic efficiency of various medications and in preserving the structure and function of a healthy GI tract \u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Meanwhile, dysregulated gut flora contributed to different pathological conditions such as cardiovascular, CNS, inflammatory bowel diseases, and even depression \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNucleotides are involved in almost all biological processes, like the synthesis of nucleic acids, energy metabolism, signal transduction, and participate in the process of enzymatic regulation \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Endogenous synthetic processes are the primary source of nucleosides, besides other exogenous natural sources such as plant- and animal-based food \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The demand for nucleotides during growth and other stressful conditions, such as infections, had provoked the need for nucleotide dietary supplements to become intriguingly semi-essential nutrients \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUridine and cytidine as nucleosides showed therapeutic potential in different diseases. In recent studies, uridine supplementation was correlated to increased diversity of gut microbiota in High Fat Diet (HFD)-fed mice \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and significantly enhanced the growth and development of animals during weaning \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Cytidine monophosphate (CMP) can modify gut microbiota \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, improve growth \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and enhance memory and learning \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eZinc is fundamental element for enzymes function and protein\u0026rsquo;s structure. Diverse physiological processes including cell replication are affected by zinc deficiency. It is also necessary for growth, immune systems, intestinal function, and brain development \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Zinc deficiency or over supplementation played a role in the pathogenesis of multiple diseases. Substantially, the balanced levels of zinc and other transition metals in the body contributed to the diversity and abundance of gut microbiota \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Zinc deficiency has been associated with depression, reduced cognitive function, chronic inflammation, growth retardation, delayed wound healing, and reduced diversity and richness in gut microbiota \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Furthermore, other gastrointestinal conditions such as animal dysbiosis, irritable bowel syndrome, and Crohn's disease were related to zinc deficiency \u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Thus, zinc supplementation is vital to maintain balanced endogenous levels of zinc to maintain health.\u003c/p\u003e \u003cp\u003eRecently, the use of nucleosides as supportive regimens for multiple neurodegenerative diseases has increased. Accordingly, as the reliance on these supplements is on the rise, having the opportunity to study the effect of zinc, uridine and cytidine, alone and in combination, on the diversity and abundance of normal gut microbiota while knowing the importance and role of balanced gut microbial colonization and its contribution to the health of humans and animals will allow for further functional and biological deep understanding while evaluating other clinical parameters that may indicate the safety and effectiveness of these supplements. Basically, scientific research has discussed the potential effects of zinc, uridine, and cytidine in a separate manner \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the effect of their combination on gut microbiota has not been reported yet, especially in healthy rats. Therefore, this study was designed to study the effect of the combination of uridine, cytidine, and zinc on the gut microbiota, besides other important clinical parameters in healthy rats.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eImpact of combined and individual supplements on the diversity and abundance of gut microbiota in healthy rats\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAlpha microbial diversity\u003c/h2\u003e \u003cp\u003e16S rRNA sequencing analysis was carried out in all animal groups in replicates, relative abundances at baseline and the last day of treatment were calculated accordingly for each group. Shannon and Simpson diversity Indices were calculated to reflect microbial diversity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The Uridine group had the most apparent drop in bacterial diversity among groups throughout the treatment period as the Shannon diversity index increased from 3.7 to 4.2 on day 42. The cytidine group had a similar trend to the uridine group, as the Shannon diversity index increased from 4.2 to 4.4 throughout the treatment period. Meanwhile, no changes in the Shannon diversity index were noticed in the zinc group. On the other hand, the combination group showed a slight reduction in the Shannon diversity index, indicating a slight rise in the biodiversity. Furthermore, the difference in Shannon or Simpson diversity indices, whether increase or decrease at day 42 for all animal groups, was insignificant compared to control at baseline upon performing One-Way ANOVA statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean Shannon Index, mean Simpson Index, mean bacterial count, evenness and average population size calculated at day 0 (baseline) and day 42 (the last day of treatment) for the combination, uridine, cytidine, and zinc treated groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCombination\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eUridine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eCytidine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eZinc\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDays\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean Shannon Index \u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.5\u0026plusmn;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.4\u0026plusmn;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7\u0026plusmn;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.2\u0026plusmn;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.2\u0026plusmn;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.4\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.3\u0026plusmn;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.3\u0026plusmn;0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean Simpson Index \u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026plusmn;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.09\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.16\u0026plusmn;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.09\u0026plusmn;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.07\u0026plusmn;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.08\u0026plusmn;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.08\u0026plusmn;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.09\u0026plusmn;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean bacterial count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40788\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e36773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48842\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46481\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e49798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e44219\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEvenness\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage population size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e997.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1328\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e943\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1191\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1134\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e# Results are presented as mean \u0026plusmn; SD, knowing that the lower the Simpson index value, the higher the diversity, while higher value of Shannon index means more biodiversity in the sample.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eChanges at the class level\u003c/h2\u003e \u003cp\u003eBacteroidia and Clostridia were the most dominant bacterial classes in all animal groups before starting the treatment at day 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The relative abundance of Clostridia class increased in all treatment groups after 42 days of treatment to reach its highest levels 41.6% in zinc group. Meanwhile, Bacteroidia class decreased at the end of the treatment period to reach 34.3% in the control group, which was the lowest among others. Besides, the Clostridia to Bacteroidia ratio was calculated during day 42 of treatment. Combination group showed a 1:1.2 ratio and cytidine group showed a 1:1.3 ratio, meanwhile; zinc treated group showed a 1:0.9 ratio. However, uridine treated group showed a 1:2.5 ratio. Generally, the ratio increased by the end of the treatment period when compared to baseline in all treatment groups, (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe table summarizes calculated Clostridia: Bacteroidia ratio for the combination, uridine, cytidine, and zinc treated groups, at baseline (day 0) and at the last day of treatment (day 42).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClostridia: Bacteroidia\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCombination\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUridine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCytidine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eZinc\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 0 (baseline)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1:2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1:1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDay 42 (the last day of treatment)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1:1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1:2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1:1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1:0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eChanges at the family level\u003c/h2\u003e \u003cp\u003eThe mean relative abundance of bacterial families showed three dominant families in all animal groups, \u003cem\u003ePrevotellaceae\u003c/em\u003e, \u003cem\u003eLachnospiraceae\u003c/em\u003e and \u003cem\u003eBacteroidaceae\u003c/em\u003e. The most abundant family was \u003cem\u003ePrevotellaceae\u003c/em\u003e among others; however, it displayed inconsistent decrease and increase trends between the groups. The \u003cem\u003eLachnospiraceae\u003c/em\u003e family; on the other hand, increased in all animal groups except for the zinc-treated group, which remained almost the same by day 42, while cytidine treated group showed the highest abundance of \u003cem\u003eLachnospiraceae\u003c/em\u003e among others. Moreover, all groups exhibited a decline in the relative abundance of \u003cem\u003eBacteroidaceae\u003c/em\u003e from day 0 up to day 42 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No significant difference between the relative abundances of different families in different treatment groups at day 42 when compared to baseline control at day 0 (\u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u003cem\u003e\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). In addition, several families were shared between the different groups, while the combination and the uridine treatments showed the highest number of unique families (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003erelative abundances of Prevotellaceae, Lachnospiraceae and Bacteroidaceae, the most abundant bacterial families, in the combination, uridine, cytidine and zinc treatment groups at baseline before the start of treatments (day 0) and at the last day of treatment (day 42). Data is presented as mean \u0026plusmn; SD.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFamily\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCombination\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eUridine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eCytidine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eZinc\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003ed 0\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003ed 42\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePrevotellaceae%\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.3\u0026plusmn;4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e19.6\u0026plusmn;4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.7\u0026plusmn;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e29.1\u0026plusmn;9.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e43.2\u0026plusmn;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e31.1\u0026plusmn;12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e26.9\u0026plusmn;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e23.7\u0026plusmn;10.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e17.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e26.9\u0026plusmn;12.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLachnospiraceae%\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e10.9\u0026plusmn;6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.8\u0026plusmn;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.4\u0026plusmn;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e17.3\u0026plusmn;11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4.7\u0026plusmn;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e12.7\u0026plusmn;5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e5.0\u0026plusmn;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e16.4\u0026plusmn;8.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e20.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e20.3\u0026plusmn;7.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacteroidaceae%\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.6\u0026plusmn;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.4\u0026plusmn;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.4\u0026plusmn;9.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e6.2\u0026plusmn;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e14.6\u0026plusmn;2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e7.8\u0026plusmn;4.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e21.4\u0026plusmn;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e14.7\u0026plusmn;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e4.8\u0026plusmn;0.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eChanges at the species level\u003c/h2\u003e \u003cp\u003eAt the species level, the mean relative abundances showed different patterns among the studied groups, and some common and unique trends. The species \u003cem\u003eAllobaculum stercoricanis\u003c/em\u003e increased uniquely in the combination group, while decreased in other groups. Moreover, the relative abundance of \u003cem\u003eBacteroides acidifaciens\u003c/em\u003e and \u003cem\u003eBacteroides uniformis\u003c/em\u003e species had uniquely increased in the cytidine group by day 42, in contrast to the combination and uridine groups at which it had declined. On the other hand, the relative abundance of \u003cem\u003eBacteroides plebeius\u003c/em\u003e had a remarkable increase in the uridine group by day 42, while decreased in the other three treatment groups. Statistical analysis showed that the differences in species relative abundances for different treatment groups at day 42 and compared to baseline control were insignificant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eClinical parameters\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffect on body weight in healthy rats\u003c/h2\u003e \u003cp\u003eAt days 0, 21, 42 and after the discontinuation of treatments; at day 49, there were no significant differences in the mean body weight among different treatment groups, compared to control (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026gt;\u0026thinsp;0.05). For instance, the increase of weight for the rats in all groups at baseline and day 42 was similar to that of the control group, suggesting no effect of the different treatments on gaining weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect on Fasting blood glucose level in healthy rats\u003c/h2\u003e \u003cp\u003eA significant reduction in the FBG level for the animal group which received the combination supplement was noticed directly after three weeks of treatment (day 21) and it was kept even after the discontinuation of therapy at week seven (day 49) \u003cem\u003e(****p-\u003c/em\u003evalue\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.0001).\u003c/em\u003e Moreover, a significant reduction in the FBG level was noticed at days 21, 42 and 49 when compared to baseline in other animal groups which received individual supplements, uridine, cytidine (**\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and zinc alone (*\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05). Accordingly, the combination therapy showed a more significant reduction in glucose levels after three weeks of therapy in comparison to other individual treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffect on motor activity in healthy rats\u003c/h2\u003e \u003cp\u003eStatistical analysis showed no significant differences in the mean latency to fall (sec) time among different animal groups or even when compared to control at each time interval in the study. However, the combination group showed significant improved motor activity at day 21 (*\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u003cem\u003e\u0026lt;\u0026thinsp;0.05\u003c/em\u003e), compared to cytidine and uridine groups at baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEffect on the nociceptive response in healthy rats\u003c/h2\u003e \u003cp\u003eA significant reduction in the mean index of nociception was obvious in the combination group at day 42 and day 49 in comparison to day 0 (****\u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Moreover, the same significant reduction was noticed in uridine group at days 21, 42 and 49 (****\u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect on liver enzymes level in healthy rats\u003c/h2\u003e \u003cp\u003eThe baseline for normal ALP level was determined on day 0 for all animals (n\u0026thinsp;=\u0026thinsp;35), which was 209.8\u0026plusmn;17.5 U/l, and the median was 202.2 U/l. The mean ALP level for the different treatment groups at baseline didn\u0026rsquo;t exceed the stated reference ALP level which was established by the kit manufacturer; (89\u0026ndash;370 U/l). In general, the mean ALP level for the combination, uridine, cytidine, and zinc supplemented animal groups was decreasing throughout the study time starting from day 0 to day 49. Statistical analysis showed no significant differences between animal groups or in comparison to control group on days 0, 21, 42 and 49. Remarkably, the animal group that received the combination of (uridine, cytidine and zinc) resulted in a significant reduction in ALP level (M\u0026thinsp;=\u0026thinsp;118.02 U/l) on day 42 (*\u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;=\u0026thinsp;0.0148), and day 49 after treatment discontinuation (M\u0026thinsp;=\u0026thinsp;122.5 U/l) (*\u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;=\u0026thinsp;0.0257), compared to day 0 (M\u0026thinsp;=\u0026thinsp;225 U/l) for this group. However, the cytidine group showed a more significant reduction in ALP level at day 49 (M\u0026thinsp;=\u0026thinsp;148.6 U/l) compared to day 0 (M\u0026thinsp;=\u0026thinsp;244.3 U/l) (*\u003cem\u003ep-\u003c/em\u003evalue\u0026thinsp;=\u0026thinsp;0.0055) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The baseline for normal ALT level was determined on day 0 for all animals (n\u0026thinsp;=\u0026thinsp;35), which was 34.9\u0026plusmn;3.1 U/l, and the median was 36.5 U/l. The mean ALT level at baseline for different treatment groups didn\u0026rsquo;t exceed the stated reference ALT level which was established by the kit manufacturer (up to 42 U/l). In general, the mean ALT level in the different treatment groups has a common increase trend starting from baseline to the last day of treatment, followed by a decrease in ALT level after treatments discontinuation. The increase at day 42 compared to day 0 in the combination, cytidine and zinc groups was 2-fold, however, for the uridine group was 1.7-fold, but in all cases this increase was not significant (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and it was followed by a subsequent decrease upon treatments discontinuation to reach the normal levels again at day 49 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDietary nucleotides are being widely used as supplemental therapy for a variety of neurodegenerative diseases. This study aimed to evaluate the effect of (zinc, uridine, and cytidine), both alone and in combination, on the diversity and abundance of normal gut microbiota while monitoring other clinical parameters as an indication for their safety and effectiveness.\u003c/p\u003e \u003cp\u003eIn agreement with previous studies, the results of this study showed an increase in Shannon and Simpson diversity indices in uridine and cytidine-supplemented groups, reflecting a decrease in microbial diversity, although it is insignificant. The richness and composition of gut microbiota were improved in formula-fed human infants containing nucleotides \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Furthermore, individually added nucleotides such as CMP and UMP and others changed the growth of certain bacterial strains in piglet\u0026rsquo;s small intestine \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, and increased bacterial diversity in mice fed HFD \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. However, according to the study findings no changes in the diversity indices of the combination or zinc supplemented groups were noticed. This is expected due to the relatively low zinc dose used in this study compared to the doses mentioned in literature. Basically, the role of low zinc concentrations in the combination supplement is to catalyze the cytidine deaminase enzyme, which converts cytidine into uridine \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Besides, different forms and concentrations of zinc supplements could change the gut microbiome \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and in a dose-dependent manner \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In contrast to previous studies that were conducted under certain conditions, such as weaning or disease status, this study highlighted the changes of gut microbiota in healthy rats.\u003c/p\u003e \u003cp\u003eThe increased Clostridia: Bacteroidia ratio in all animal groups at the class level, may indicate positive outcomes on gut health. Increased relative abundance of Bacteroidia is normally correlated to inflammatory conditions due to increased cytokine production and endotoxins. However, increased levels of Clostridia are associated with decreased inflammation and improved gut function \u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMembers of Lachnospiraceae family are butyrate producing bacteria, which is considered as a source of energy for colonocytes and causes indirect activation for nervous and hormonal systems \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The anti-inflammatory and immunity inducing effects for butyrate were reported as well \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Importantly, Lachnospiraceae was correlated to improved glucose tolerance \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, which may contribute to this study findings which showed significant reduction in the fasting blood glucose levels at week three in all animal groups in comparison to baseline. On the other hand, the relative abundance of the members belonging to Bacteroidaceae family, which showed a slight reduction in all animal groups, might be correlated to carbohydrates metabolism in humans and rodents \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The increased abundance of this family was correlated to type-2 diabetes \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Members of Prevotellaceae family, which showed insignificant raise in their relative abundance in the combination and zinc groups exclusively, is considered as commensal bacteria and infrequently involved in infections. Only a few members can cause endogenous infections such as chronic infections and anaerobic pneumonia \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSome previous studies supported that nucleotides supplementation lead to increased weight gain and head growth in formula-fed infants and enhanced growth in animals during weaning due to decreased intestinal inflammation \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. On the other hand, neither UMP nor uridine had significant effects on the growth performance of weaned piglets \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. CMP reduced weight gain in mice model which was fed high fat diet \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Furthermore, pharmacological zinc doses of 3000 ppm, which are much higher than the zinc dose used in this study, were able to boost growth in weaned piglets \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In other studies, zinc related growth enhancement has been observed at plasma zinc concentrations around 2.5 mg/L \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. According to this study findings neither the individual supplements, nor their combination resulted in a significant increase in body weight compared to control at the last day of the study.\u003c/p\u003e \u003cp\u003eAccording to previous studies, uridine in the form of UMP increased the plasma glucose levels significantly in weaned piglets \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Besides, uridine had increased gluconeogenesis, decreased insulin signaling activity and long term administration of uridine over 16 weeks had induced glucose intolerance and sever lipid accumulation in mice liver \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. High doses of cytidine, on the other hand, improved glucose tolerance in HFD fed-mice \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. it was concluded that zinc supplementation improved fasting insulin and glucose levels in obese mice \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.It decreased the glycosylated hemoglobin HBA1C% after supplementing 30 mg of zinc over 3 months in type 2 diabetic patients, while other studies concluded that these effects will not be achieved over short time administration \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. The changes in microbiome in the healthy rats in this study could be attributed to the reduced fasting blood glucose levels, as the abundance of certain species that is related to improved glucose tolerance and enhanced insulin activity. For example, the relative abundance of certain species increased such as \u003cem\u003eBacteroides acidifaciens\u003c/em\u003e and \u003cem\u003eBacteroides uniformis\u003c/em\u003e. \u003cem\u003eBacteroides acidifaciens\u003c/em\u003e can regulate intestinal dipeptidyl peptidase-4 (DPP-4) secretion and subsequently induce glucagon-like peptide 1 (GLP-1) production, which may contribute to glucose homeostasis and may have a role in the prevention of diabetes and obesity \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Furthermore, \u003cem\u003eBacteroides uniformis\u003c/em\u003e reduced body weight gain, increased oral glucose tolerance in HFD-fed mice, improved immunity and reduced inflammation \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Moreover, \u003cem\u003eLachnospiraceae\u003c/em\u003e family was strongly correlated to improved glucose tolerance, involved in the metabolism of carbohydrates and SCFAs production \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The abundance of \u003cem\u003eLachnospiracea\u003c/em\u003e family increased in all groups except the zinc group. This may explain the more significant reduction in the FBG level that was noticed in the combination supplemented group and could be attributed to the presence of cytidine, uridine, and zinc concomitantly, which may have resulted in a synergistic action to reduce the glucose levels. The catalytic zinc dose in the combination supplement may contribute to the activation of the cytidine deaminase enzyme that converts cytidine to uridine \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Furthermore, this study focused on evaluating the effect on healthy rats over a short time (six weeks), unlike previous studies that were implemented over a prolonged time on rats with pathological conditions, accordingly further studies in the future could answer this hypothesis.\u003c/p\u003e \u003cp\u003eThe significantly improved motor activity in the combination group could be attributed to the synergistic action between the three individual supplements. In agreement with this study result, it was concluded that uridine in combination with omega three fatty acids enhanced motor activity and increased latency to fall time in mice model with Parkinson disease via enhancing brain phospholipids levels and brain dopaminergic neurotransmission \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. On the other hand, chronic treatment with uridine alone did not cause any changes in motor activity or rotation \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The improved motor activity in the cytidine-treated group could be attributed to CDP-choline metabolites such as choline, which were involved in the synthesis of acetylcholine that contributed directly to improved motor activity, and the protection of cholinergic neurons by sparing membrane phospholipids \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Moreover, CDP-choline administered intraperitoneally has improved motor activity in aged mice by acting on central neurotransmission and cognition \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Zinc deficiency has been associated with impaired motor activity in some studies \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, while other studies in contrast, concluded that motor activity could not be affected by zinc deficiency \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe index of nociception which decreased significantly and only in the combination and uridine groups may indicate more pain/ nociception \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Nucleotides are involved in phospholipids and glycolipids synthesis and may promote nerve fiber regeneration. In a previous study, CMP and UMP didn\u0026rsquo;t improve nerve thermal conduction parameters in diabetic patients with neuropathy \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. On the other hand, the combination of UMP with vitamin B12 and folic acid has significantly reduced neuropathic pain intensity in patients with peripheral neuropathy \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. UTP administration has prolonged the thermal nociceptive latency in the tail-flick test \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Importantly, the hyper-polarization-activated, cyclic nucleotide-gated (HCN) channels in nociceptive neurones, were involved in peripheral nociception \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, and some studies highlighted their direct contribution to neuropathic pain \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e,\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. Most of the studies in literature discussed the contribution of cyclic nucleotides signalling pathway in chronic inflammatory pain induced by injury and stress; however, this study evaluated the antinociceptive effect of different supplements on induced acute non-inflammatory pain in healthy rats.\u003c/p\u003e \u003cp\u003eALT levels, according to our results have increased throughout the study time up to 2 folds the baseline level, but this increase in all cases was reversible upon treatment discontinuation and non-significant. ALP enzyme is found almost in all tissues, and on the surface of hepatocytes. Usually, elevated levels of ALP are associated with cholestatic disorders \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e, or could be referred to stress factors that the rats might have experienced during blood sampling and /or during dose administration of test substances \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. On the other hand, the ALT enzyme is more indicative of hepatocytic necrosis, and its elevated levels reflect leakage from damaged tissues in the liver \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. In a previous study, nucleotides at high doses were safe and decreased ALT levels but insignificantly, thus promoted liver function \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Nucleotides were able to reverse increased ALT and AST levels associated with alcohol induced liver injury in rats \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Unlike previous studies, this study examined the effect of various supplements on the levels of liver enzymes in healthy rats; additionally, the findings suggest that they are relatively safe, but if they were supplemented over a prolonged period, liver enzymes should be monitored.\u003c/p\u003e \u003cp\u003eAs the use of the combination of Uridine, Cytidine and Zinc by healthy people, as adjuvant treatment for many diseases grows, our findings showed that gut microbiota changes suggest beneficial clinical implications for gut health and could contribute to the revealed changes in FBG levels. Our results support the efficacy and safety of these supplements, with recommendation to monitor liver enzymes when used for prolonged time. Overall, further investigations in the future could provide further understanding of the promising clinical implications of metabolic and gut health, besides determining the best dosages and treatment regimens.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eAll the experimental procedures in this study were ethically approved by the Institutional Review Board Committee for the field of Clinical Pharmacy and Pharmacy Practice at the Applied Science Private University (ASU), Faculty of Pharmacy (Approval No. 2022-PHA-33).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDietary supplements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe supplements used in this experiment were purchased from community pharmacies in Amman-Jordan and USA.\u0026nbsp;ISCHELIUM (150 mg CMP, 150 mg UMP and 10 mg zinc, Polifarma\u0026reg;, Roma-Italy) vials of 10 ml oral solution, CITICOLIN (250 mg Cytidine -5\u0026rsquo;-diphosphocholine capsules, Cognizin\u0026reg;, Kyowa Hakko Bio Co., Ltd.), URIDINE (300 mg Uridine -5\u0026rsquo;-monophosphate disodium salt capsules, Amazing Nutrition\u0026reg;, USA), ZINC (25 mg zinc gluconate tablets, Jamieson\u0026trade;).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental animals and experimental design \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 35 male, healthy, eight-weeks old, wistar rats were utilized, and procured from the animal house laboratories, Pharmacy School, ASU- (Amman- Jordan), with a mean weight of 125\u0026nbsp;\u0026plusmn;\u0026nbsp;5 g. All animals were housed and acclimatized in the animal room at the university, at a temperature of 21-23 ℃ and humidity of 35 % - 70 %, and under a 12-hour light/dark cycle. Rats were continuously fed with the standard diet, and a free access to water and food throughout the study period. Rats were randomly divided into 4 treatment groups and one control (n=7), and then each rat was identified in each group to monitor its clinical parameters. The doses were calculated by extrapolation based on human average body weight (70 Kg) while considering the average rat weight (130 g) at the age of eight weeks. Moreover, the specified concentrations of the solutions of the individual supplements were prepared by dissolving their contents in distilled water based on the solubility of the active ingredients and then were placed on the vortex for dissolution. Group I\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ereceived 20 \u0026micro;l/day/rat of the commercially available combination of (Cytidine, Uridine and Zinc), ISCHELIUM\u0026reg; at a daily dose of 10 ml oral vial/day for humans.\u0026nbsp;Group II\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ereceived 8\u0026nbsp;\u0026micro;l/day/rat of Uridine alone at a daily dose of 50 mg/ml suspension. Group III received 4 \u0026micro;l/day/rat of Cytidine alone, at a daily dose of 100 mg/ml suspension.\u0026nbsp;Group IV\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ereceived 0.3 \u0026micro;l/day/rat of Zinc alone at an average daily dose of 0.25mg/ml solution. The last group was the control which received normal saline on daily basis. The study was conducted over six weeks of treatment during which, all previously mentioned doses have been administered by the orogastric gavage with another week (7\u003csup\u003eth\u003c/sup\u003e week) of no treatments for all animal groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood and fecal samples collection and analysis \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood and fecal samples were collected at the baseline (before the start of day one; day 0), and at the end of weeks three (day 21), six (day 42) and seven (day 49). All rats were fasted for 15 hours before the sampling time\u0026nbsp;\u003csup\u003e80\u003c/sup\u003e. Blood samples were obtained from the rats\u0026rsquo; orbital sinus veins using capillary tubes to assess blood glucose level directly. To prepare serum samples, part of the collected blood was set to clot directly at 4 ᵒC overnight, then the samples were centrifuged at 1000 rpm for 5 min. Sterile Eppendorf tubes were used to collect serum and finally the samples were stored at -80 ᵒC until further analysis to assess liver enzymes. Moreover, fresh fecal samples were collected in a previously autoclaved Eppendorf tubes to investigate the changes in gut microbiota and were frozen immediately at -80 ℃ for further analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBarcoded sequencing of the 16S rRNA gene and data analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial DNA was extracted from fecal samples using G-spin\u0026trade; Total DNA Extraction Mini Kit, purchased from QIAGEN, Germany, following the manufacturer\u0026apos;s protocol. The extracted DNA was sent to the Molecular Research Lab (www.mrdnalab.com, MR DNA, Shallowater, TX) for 16S rRNA sequencing and data analysis. The 16S rRNA gene V4 variable region was amplified with PCR primers 27F-519R. PCR products were then pooled, purified to be sequenced using Illumina on a MiSeq following the manufacturer\u0026apos;s guidelines. Sequencing data were processed using MR DNA analysis pipeline (MR DNA, Shallowater, TX, S.A.). Sequences were denoised, chimeras were removed, and operational taxonomic units (OUT) were generated. Final OTUs were taxonomically classified using BLASTN against a curated database derived from NCBI (National Center for Biotechnology Information, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of clinical parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll clinical parameters were assessed at the baseline of the study (day 0), and at the end of week three (days 21), week six (day 42), and after the discontinuation of treatments at week seven (day 49) except for the body weight which was monitored on weekly basis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBody weight \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFasting Blood Glucose Test (FBG)\u003c/p\u003e\n\u003cp\u003eBlood glucose concentrations were determined immediately after collecting blood samples using an Accu-Chek\u0026reg; Performa glucose meter, (Roche, Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMotor activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rota-rod test was used to assess the motor activity behaviour of each rat\u0026nbsp;\u003csup\u003e81\u003c/sup\u003e. The rota-rod treadmill (Muromatchi kikai Co., Ltd., Mk-630B, Tokyo, Japan) that was used in the study is a single-lane treadmill, thus enabling one rat to walk\u003cstrong\u003e.\u003c/strong\u003e Initially, rats had multiple practice sessions on the device before they were placed individually on the accelerating rod with different speeds, ranging from 4 rpm to 40 rpm and facing away from the experimenter. The latency to fall was reported for a maximum time of 300 sec. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntinociceptive effect (The hot plate test)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePaw-licking and paw-lifting behaviours were monitored as acute, non-inflammatory pain indicators\u0026nbsp;\u003csup\u003e82,83\u003c/sup\u003e. The rats were placed individually on a hot-plate analgesia meter (Muromatchi Kikai Co., Ltd, Mk 350D, Tokyo, Japan), heated at 55℃, which was optimal to induce pain, not burn, then the timer was started immediately until the rat had shown a nociceptive response such as paw-licking, paw-lifting, or jumping within a maximum latency time of 60 sec.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiver Function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThawed serum samples were used to test the levels of ALT and ALP using liver enzymes spectroscopic diagnostic kits (BioSystems S.A, Barcelona, Spain), by following the accompanying kits protocol and the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe significant difference among the different groups means was assessed by one-way and two-way analysis of variance (ANOVA) and\u0026nbsp;Post-hoc analysis using Tukey\u0026rsquo;s test, by GraphPad Prism 9 (USA), and the results were presented as mean\u0026nbsp;\u0026plusmn;\u0026nbsp;standard error of the mean (SEM). The Shannon and Simpson diversity indices were used to assess the effects of the different treatments on the composition and abundance of gut microbiota.\u0026nbsp;In data presentation,\u003cem\u003e\u0026nbsp;p\u003c/em\u003e-value \u0026le; 0.05 was considered a significance cut-off point and tagged by one asterisk (*), \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0.01 was tagged by two asterisks (**),\u003cem\u003e\u0026nbsp;p\u003c/em\u003e-value \u0026lt;0.001 was tagged by three asterisks (***) and \u003cem\u003ep\u003c/em\u003e-value \u0026lt;0.0001 was tagged by four asterisks (****).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch1\u003eAuthors contribution\u003c/h1\u003e\n\u003cp\u003eConceptualization: Maram A. Othman, Muna Barakat, Amin Omar, Mohammad AA Al-Najjar. Data curation: Maram A. Othman, Formal analysis: Maram A. Othman. Investigation: Maram A. Othman, Muna Barakat, Ruaa R. Al-Alwany, , Mohammad AA Al-Najjar. Methodology: Maram A. Othman, Muna Barakat, Tamara Athamneh, Ruaa R. Al-Alwany, , Mohammad AA Al-Najjar. Project administration: Muna Barakat, Ruaa R. Al-Alwany, , Mohammad AA Al-Najjar. Resources: Maram A. Othman, Tamara Athamneh, Ruaa R. Al-Alwany. Software: Maram A. Othman. Supervision: Muna Barakat, Amin Omar, Rafat Zrieq, , Mohammad AA Al-Najjar. Validation: Muna Barakat, Amin Omar, Tamara Athamneh, Rafat Zrieq, Mohammad AA Al-Najjar. Visualization: Muna Barakat, Amin Omar, Tamara Athamneh, Rafat Zrieq, Mohammad AA Al-Najjar. Writing \u0026ndash; original draft: Maram A. Othman, Amin Omar, Rafat Zrieq. Writing \u0026ndash; review \u0026amp; editing: Maram A. Othman, Muna Barakat, Amin Omar, Tamara Athamneh, Rafat Zrieq, Mohammad AA Al-Najjar.\u003c/p\u003e\n\u003ch1\u003eAcknowledgment\u003c/h1\u003e\n\u003cp\u003eThe authors acknowledge the support from the Applied Science Private University in using the lab facilities to conduct this research.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThey also acknowledge First Care medical company for providing the Ischelium dietary supplement used in this research study.\u0026nbsp;\u003c/p\u003e\n\u003ch1\u003eConflict of interest\u0026nbsp;\u003c/h1\u003e\n\u003cp\u003eNo potential conflict of interest relevant to this article was reported.\u003c/p\u003e\n\u003ch1\u003eData Availability Statement\u003c/h1\u003e\n\u003cp\u003eData is available within the article or its supplementary materials.\u003c/p\u003e\n\u003ch1\u003eFunding Statement\u003c/h1\u003e\n\u003cp\u003eThis study was not supported by any national or international institution.\u0026nbsp;\u003c/p\u003e\n\u003ch1\u003eEthical Approval Statement\u003c/h1\u003e\n\u003cp\u003eThis study is reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All animal experiments were conducted following the ethical standards and guidelines set forth by our institution and approved by the Animal Research Ethics Committee at the Applied Science Private University (ASU), Faculty of Pharmacy (Approval No. 2022-PHA-33)., and all procedures were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBackhed, F., Ley, R. E., Sonnenburg, J. L., Peterson, D. A. \u0026amp; Gordon, J. I. Host-bacterial mutualism in the human intestine. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e307\u003c/strong\u003e, 1915-1920 (2005).\u003c/li\u003e\n\u003cli\u003eEckburg, P. 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Tests and models of nociception and pain in rodents. \u003cem\u003eNeuroscience\u003c/em\u003e \u003cstrong\u003e211\u003c/strong\u003e, 39-50 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microbiota, uridine, cytidine, zinc, nucleosides, clinical parameters","lastPublishedDoi":"10.21203/rs.3.rs-4667549/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4667549/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground/Aims\u003c/h2\u003e \u003cp\u003eAs healthy gut flora contributes to mammalian health, the focus of this study is to investigate the effect of the combination of (Uridine, Cytidine, and Zinc) compared to individual supplements on the diversity of gut microbiota in healthy rats, and on other clinical parameters.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe 16S rRNA gene sequencing was used to study the diversity and abundance of gut microbiota. Liver enzymes were monitored by measuring serum level of (ALP and ALT), serum fasting blood glucose level, latency to fall, index of nociception and body weight were monitored. The previously mentioned analysis was done to study the effect of orally taken combination of (Uridine, Cytidine and Zinc) and the separate intake of these supplements on gut microbiota and other parameters in 35 healthy white Wistar rats for six weeks.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe study findings showed that both the combination and the individual supplements enhanced the abundance of Clostridia and decreased Bacteroidia. Clostridia: Bacteroidia ratio increased from 1:2.5 to 1:1.2 by the last day of treatment in the combination group. Moreover, combination supplement reduced fasting blood glucose level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), improved rats\u0026rsquo; motor activity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reduced nociception Index (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and decreased the ALP liver enzyme level in healthy rats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe studied combination supplement was found to be safe and no harmful change on gut microbiota was noticed. This was clear from the normal level of the tested clinical parameters such as fasting blood glucose level and motor activity.\u003c/p\u003e","manuscriptTitle":"The potential effects of the combination (Cytidine, Uridine and Zinc) on gut microbiota and clinical parameters of healthy rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-27 00:34:24","doi":"10.21203/rs.3.rs-4667549/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d51d0668-2f48-4771-af3d-ed737833c2c1","owner":[],"postedDate":"July 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34898792,"name":"Biological sciences/Microbiology"},{"id":34898793,"name":"Biological sciences/Microbiology/Applied microbiology"},{"id":34898794,"name":"Biological sciences/Microbiology/Clinical microbiology"}],"tags":[],"updatedAt":"2025-02-07T06:38:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-27 00:34:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4667549","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4667549","identity":"rs-4667549","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}