| Objective:In the present study,rats were exposed to simulated weightlessness with different tail lengths,and the effects of rat intestinal flora and liver injury by simulated weighlessness were studied by examining the changes of rat intestinal microorganisms.Specifically,we examined liver oxidative stress,inflammation,and liver damage to explore the possible routes by which liver damage could be affected by simulated weightlessness and to provide a scientific basis for these findings.Methods:Male SPF-grade Wistar rats with body weight of 200±20g were selected,and a total of 20 animals were used.They were fed for 1 week in an animal feeding center,and after they were acclimated,they were placed on the experimental condition.Each observation endpoint ended tail-suspension and fasting for 24 hours.The rats were weighed,anesthetized,whole blood was collected,and the rats were dissected and taken.Histopathological changes in liver were observed using hematoxylin-eosin(HE)staining;interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)levels in rat plasma and liver were observed using ELISA;liver function indicators alanine aminotransferase(ALT)and aspartate aminotransferase(AST)level,oxidative stress indicators malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)levels were detected using colorimetric assay;reactive oxygen species(ROS)levels in rat liver were detected using fluorescent colorimetric assay;Real time q PCR was used to measure liver inflammation-related genes(TLR4,NF-p65,NOD2);The expression of liver inflammation-related proteins(TLR4,NF-κB-p65,NOD1)was taken using immunohistochemical assays.IBM SPSS 24.0 software was utilized for statistical analysis.For normally distributed measures,the data were expressed as ?±s,Multiple group comparisons were done using one-way ANOVA,while two-way comparisons were done using Tukey’s method.Spearman rank correlation test was used to analyze the correlation between inflammation-related genes and inflammatory factors and oxidative stress indicators,and the correlation between intestinal flora and inflammation-related and oxidative stress indicators;Alpha diversity and Beta diversity were used to study the diversity of the intestinal microbial community.The clustering of the samples was visualized using principal coordinate analysis(PCo A).Lef Se analysis was used to compare four groups of samples to derive biometric markers that were significantly different between groups;Results of the analysis were visualized using R software(4.2.2);statistical significance was determined by selecting the P value at least 0.05,the test level was set at α=0.05.Results:1.General damage to rats,results showed: both tail-suspension groups had significantly lower weights than the control group(P<0.05);the liver coefficient was found to be lower in rats at 6 weeks than in other groups(P<0.05).2.HE staining results showed,the hepatocytes in the 4 weeks group showed swelling,the 5 weeks group showed disorganization of hepatocyte cords,and the 6 weeks group showed increased granular degeneration and cell necrosis.3.Effects on liver function in rats,results showed: rats in the control group had significantly lower plasma ALT activity than 6 weeks,and there was a significant difference in liver tissue ALT activity between tail-hanging groups(P<0.05);6 weeks rats showed significantly higher plasma AST activity than the control group and 4weeks(P<0.05).4.Effects on oxidative stress in rats,results showed the ROS,MDA,SOD and GSH were found to be higher in the plasma and liver of rats at 6 weeks group than the control group.(P<0.05).5.Effect on inflammatory factors in rats,results showed rats in the 6 weeks had significantly higher plasma and liver IL-1β levels than rats in the other groups(P<0.05);plasma IL-6 levels were shown to be higher in the 6 weeks group than in the 5 weeks group,moreover,liver IL-6 levels were seen to be significantly different between the 5and 4 weeks groups(P<0.05);plasma TNF-α levels were detected to be higher in rats in the 4 and 6 weeks groups than in the control group(P<0.05),in addition,TNF-α was observed to be significantly different in liver tissue between all tail suspension groups and the control group(P<0.05).6.Effects on inflammatory genes in rats,results showed with increasing tail suspension time,liver TLR4 m RNA expression in rats in the tail-hanging group gradually decreased,compared to the control group,it was significantly lower after 6weeks(P<0.05);when tail-suspension time was extended,liver NF-κB-p65 m RNA expression level gradually increased in the rats in the tail-suspension group,a significant elevation was observed in the six-week group compared to the control group(P<0.05).A significant difference was found between the m RNA levels of NOD2 in the livers of rats after 4 and 5 weeks compared to the control rats,and there was a significant difference between control group and 6 weeks group(P<0.05).7.Correlation analysis of inflammation-related genes and oxidative stress indicators showed that NF-κB-p65 was significantly and positively correlated with ROS,MDA,and GSH,TLR4 was significantly and negatively correlated with ROS,MDA,SOD,and GSH(P<0.05),and NOD2 was significantly and positively correlated with ROS,SOD,and GSH(P<0.05).8.Effects on inflammation-related proteins in rats,the results showed that hepatic TLR4,NF-κB-p65 and NOD1 protein were markedly enhanced in the tail suspension group of rats(P<0.05).9.The intestinal flora composition of rats in the tail-suspension group group was significantly different from the control group(P<0.05),Peptostreptococcaceae and Romboutsia were significantly lower(P<0.05),and Rikenellaceae,Alistipes and Bifidobacterium catenulatum were significantly higher(P<0.05).10.Rat liver ROS,MDA,SOD and GSH were negatively correlated(P<0.05);rat plasma TNF-α was significantly negatively correlated with Peptostreptococcaceae,Romboutsia(P<0.05)and positively correlated with Bifidobacterium catenulatum(P<0.05);TLR4 was positively correlated with Peptostreptococcaceae,Romboutsia(P<0.05).Conclusions:1.The simulated weightlessness can induce a decrease in body weight as well as alterations in liver coefficients,and liver pathological damage and liver function changes in rats.2.The simulated weightlessness induced oxidative stress and inflammation in the liver of rats,and damaged the liver.3.The simulated weightlessness can cause a decrease in the number of beneficial intestinal bacteria and an increase in the number of pathogenic bacteria in rats,and a dysregulation of intestinal flora homeostasis.Dysbiosis of intestinal flora homeostasis may damage rat liver by triggering inflammatory response and oxidative stress in rat liver. |
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