Study On The Correlation Between Muscle Function Of Gene Variant Mice On Mitochondrial Serine Protease Omi/HTRA2 And Intestinal Flora

BackgroundIn recent years,more and more studies have revealed the important biological connection between skeletal muscle and intestinal flora.It is believed that muscle function depends to a large extent on the number and structure of intestinal flora.In modern life,people pay more attention to the impact of the living environment on human health.In particular,the environment may directly or indirectly affect the basic functions of human life such as sports and cognition by affecting the intestinal flora.Therefore,the intestinal flora and skeletal muscle Function has important research value.Mitochondria regulate cell apoptosis,redox reaction and cell signal transduction,as well as the intersection of sugar,amino acid and fat metabolism.The role of mitochondria in the regulation of muscle function is currently a hot and difficult research topic.There are also significant differences in muscle fat content between the mitochondrial serine protease Omi/HTRA2 gene variant mice and the wild type.Our previous work found that compared with wild-type mice,mitochondrial serine protease Omi/HTRA2 mutant mice had significantly reduced skeletal muscle function and muscle atrophy.The research of the intestinal flora polymorphism and fat muscle metabolism gene expression level of mice with different stocking densities based on Omi/HTRA2 gene variant mice will help to reveal the relationship between the intestinal flora composition and muscle function.By establishing different living environments with different densities,changing the range of motion of mice,we detected muscle mitochondrial function,the composition of microbes in the intestines,and genes related to fat muscle metabolism,and the expression of genes related to skeletal muscle myosin heavy chain(My HC)subtypes,etc.It further reveals the influence of the living environment on the composition of intestinal flora and muscle function,and provides some experimental and theoretical basis for guiding exercise,diet and other health management and disease prevention.Methods1.Through 16 S r RNA metagenomic sequencing,OTU cluster analysis,Alpha diversity analysis and other methods,the intestinal flora polymorphism of Omi/HTRA2 gene mutant mice was studied.2.Using q PCR,western blot and immunotissue staining techniques,the muscle fat content and fat muscle metabolism genes,including lipoprotein esterase(LPL),fatty acid transfer protein(CD36),carnitine palmitoyl transferase 1(CPT1).The expression levels of sterol regulatory element binding protein 1(SREBF1),triglyceride hydrolase(PNPLA2),and skeletal muscle myosin heavy chain(My HC)subtypes were studied.3.By setting different rearing densities-low-density rearing group(3per cage),medium-density rearing group(5 per cage),high-density rearing group(10 per cage),the effects of rearing density on the weight and activity status of mice were observed.4.The muscle fat content of mice under different stocking densities,the expression levels of the aforementioned fat muscle metabolism genes and their intestinal flora polymorphism were studied.Results1.The intestinal flora of Omi/HTRA2 mutant mice was significantly different from that of wild-type mice.Compared with the intestinal flora of wild-type mice,the intestinal flora of Omi/HTRA2 mutant mice had Actinobacteria(Adlercrertzia and Odoribacter).Bacteroidetes(Parabacteriodes,Alitipes,and Rikenella).Firmicutes(Lactobacillus,Turicibacter,Clostridium,Coporococcus,Ruminododdus)flora content was significantly reduced.In-depth analysis of the above-mentioned bacterial genera revealed that the altered bacterial genera were closely related to the occurrence of fat metabolism,obesity and diabetes.2.The expression of MHY1 in the gastrocnemius muscle of the Omi/HTRA2 gene variant mice did not change significantly,while the transcription of MYH4 and MYH7 were significantly decreased,indicating that the dysfunction of the Omi/HTRA2 gene leaded to abnormal expression of myosin.In the gastrocnemius muscle of the Omi/HTRA2 gene variant mice,the expression of gene carnitine palmitoyltransferase 1(CPT1),the key to the breakdown of fatty acids,significantly decreased;while the expression of fatty acid synthase(FASN),the key rate-limiting enzyme for fatty acid synthesis,significantly increased.The above results indicated that the partial loss of Omi/HTRA2 function leaded to the disorder of fatty acid metabolism in the gastrocnemius muscle.3.The experimental observation of different stocking densities showed that different stocking densities had significant effects on the body weight of KM mice.The weight of the animals in the high-density feeding group was significantly lower than that in the low-density feeding group and the medium-density feeding group.The feeding density of mice was negatively correlated with the animal body weight.4.SPF mice in different stocking density groups were kept for different times,and the medium density stocking group was used as a benchmark to observe the expression of genes related to skeletal muscle fat synthesis and muscle function.(1)Lipoprotein esterase(LPL),fatty acid transporter(CD36),fatty acid synthase(FASN)and acetyl-coenzyme A carboxylase(ACACA)are all key molecules in the regulation of fat synthesis.With the extension of the feeding time,the expression levels of the above 4 genes in the lowdensity feeding group and the high-density feeding group were significantly reduced after 12 weeks of feeding.This analysis suggests that the stocking density affects the fat synthesis capacity in skeletal muscle.(2)Carnitine palmitoyl transferase 1(CPT1)and triglyceride hydrolase(PNPLA2)are the key genes for fatty acid decomposition.At12 weeks of rearing,the expression level of CPT1 in the low-density rearing group and the high-density rearing group decreased significantly.The expression level of PNPLA2 increased,and the increase was more significant in the high-density feeding group.Comprehensive comparison,analysis may be that PNPLA2 plays a role in the density-influence of skeletal muscle lipolysis.(3)The expression levels of some skeletal muscle myosin heavy chain(My HC)subtypes MYH1,MYH2,MYH4,MYH7,MYH1,MYH2,MYH4 encode fast muscle fibers,and MYH7 encode slow muscle fibers.The expression levels of MYH1,MYH2,MYH4 genes encoding fasttwitch fibers in the low-density feeding group were significantly reduced,but there was no statistical difference in the high-density feeding group;the expression of MYH7 genes encoding slow-twitch fibers was significantly increased in the low-density feeding group.It is suggested that the low-density feeding group and the high-density feeding group may cause changes in skeletal muscle function according to changes in the surrounding environment and the amount of exercise,and the changes in skeletal muscle function may be caused by lipid metabolism disorders.(4)The expression level of mitochondrial interstitial protease Omi/HTRA2 in skeletal muscle tissue.The expression level of Omi/HTRA2 in the low-density and high-density feeding groups was significantly reduced,suggesting that different feeding densities may affect the fat metabolism of skeletal muscle through Omi/HTRA2.5.SPF mice in different stocking density groups were reared for 1week and 12 weeks.The medium-density rearing group was used as a benchmark,and the abundance of intestinal flora was observed through16 S r RNA sequencing analysis,OTU cluster analysis and other methods.The results showed different results.There are differences in the abundance statistics of intestinal flora in the same feeding density group during the feeding time;there are also differences in the abundance of intestinal flora in the same feeding time and different feeding density groups,which are all statistically significant.The above results were analyzed for multi-level species composition of a single sample,and the results showed that an unnamed Porphyromonadaceae,unclassified_Porphyromonadaceae is a dominant bacterial group in each group,and this bacteria is closely related to the regulation of skeletal muscle quality.In the 1-week and 12-week high-density feeding groups,although the bacteria had the highest abundance,the flora accounted for close to 50%,suggesting that high-density feeding caused disorders of the intestinal flora of mice.Conclusion1.Compared with the wild type,the muscle function of mitochondrial Omi/HTRA2 gene variant mice is reduced exercise endurance and exercise adaptability,and changes in skeletal muscle function may be closely related to the remodeling of intestinal flora;2.The mitochondrial Omi/HTRA2 gene may be reduced by the range of motion(degree)which rebuilds the intestinal flora.3.Lactobacillus and unclassified_Porphyromonadaceae which in the intestinal flora may be involved in the regulation of muscle function.4.Mitochondrial Omi/HTRA2 gene may indirectly regulate skeletal muscle fat metabolism through intestinal flora,and affect muscle function.