| Objective: Based on the theory of "spleen-stomach homologous",the spontaneously type 2 diabetes GK rats were used as research objects,and the gene chip and microbial 16 Sr DNA gene sequencing technology were applied to investigate the molecular mechanism of Shenqi compound in regulating islet cell function and intestinal flora,and provide theoretical basis for T2 DM prevention and treatment by traditional Chinese medicine.Methods: 60 rats of GK rats were fed adaptively for 1 week.Rats with two random blood glucose≥11.1mmol/L were screened and included in the experiment.They were randomly divided into 5 groups: model group,the high-,medium-,and low-dose of Shenqi compound group(the following are referred to as the Sh,Sm,and Sl respectively),and western medicine group.Wistar rats were also set as normal group.Wistar rats were fed with normal feed every day.GK rats were fed with high-fat diet daily for 4 weeks.Rats were randomly selected from each group of GK rats to detect random blood glucose.Verify that the diabetes model is successful.After successful modeling,high,medium and low doses of Shenqi compound were given high,medium and low doses of Shenqi compound extract,The western medicine group was given sitagliptin suspension,and the normal group and the model group were given physiological saline.After 8 weeks of continuous administration,the general condition of the rats was observed during the drug intervention,and the body weight of the mice was measured periodically every week,and the amount of gastric perfusion was adjusted according to the change of body weight.Blood glucose at five time points was measured once a week during the intervention period at 8:00,10:00,14:00,16:00,and 18:00.According to the five-point blood glucose level,the mean blood glucose(MBG),the standard deviation of blood glucose(SDBG),and the largest amplitude of glycemic excursions(LAGE)were used to reflect the fluctuation of blood glucose in the rats after 4 and 8 weeks of intervention.Rats in each group were sacrificed after 8 weeks of drug intervention.Blood samples were collected and the level of serum cholesterol(TC),triglyceride(TG),insulin(INS),glucagon(GC),glucagon-like peptide-1(GLP-)and high-sensitivity C-reactive protein(hs CRP)were measured by double antibody sandwich ABC-ELISA.The apoptosis of pancreatic islet βcells in each groupwas detected by Dead End TM TUNEL fluorescence method.The pathological morphology of pancreatic tissue in each group was observed by hematoxylin-eosin staining.50-100 mg of pancreatic tissue of 3 rats in each group of normal group,model group,Sm group and western medicine group were detected.Differential RNA detection was performed on all samples of RNA using the Agilent4*44k Whole Genome Expression Profile.Combined with GO and Pathway enrichment,PCR was performed on key differential genes GCG,KCNN4,PIK3R1,AKT1,CRY1,DBP.1-2 feces of 5 rats in each group of normal group,model group,Shenqi compound middle dose group and western medicine group were aseptically collected.The intestinal flora of rats was detected by microbial 16 Sr DNA gene sequencing technology.Results: After 8 weeks of intervention,Shenqi compound can improve the general condition of GK rats,and its hair color,color and lustre and mental state are better than the model group.With the prolonged drug intervention time,Shenqi compound can reduce the daily food intake and daily drinking water of rats,and has no significant effect on the body weight of rats.The high and medium doses of Shenqi compound can effectively reduce the TC level;the doses of Shenqi compound and western medicine can effectively reduce the TG level;the doses of Shenqi compound and western medicine can increase the level of GLP-1;The medium dose of Shenqi compound and western medicine can all reduce the hs CRP level;among them,the medium dose of Shenqi compound has the most significant effect.The doses of Shenqi compound and western medicine can improve the levels of INS and GC to a certain extent.Among them,the high dose of Shenqi compound and the western medicine have the most significant effect.With the prolonged drug intervention time,the doses of Shenqi compound and western medicine can reduce the levels of MBG,SDBG and LAGE to a certain extent and reduce the fluctuation of blood glucose.Among them,the effect of the medium dose of Shenqi compound and western medicine on blood sugar fluctuation was the most significant.After 8 weeks of intervention,both the Shenqi compound and the western medicine could reduce the islet βcell apoptosis.Among them,the medium dose of Shenqi compound has the most significant dose effect.The high and medium doses of Shenqi compound can improve the pathological morphology of pancreas.Gene chip differential gene screening found that there were 349 differential genes in the model group compared with the normal group,of which 128 genes were up-regulated and 211 genes were down-regulated.Compared with the model group,the Shenzhong group had 77 differential genes,of which 54 genes were up-regulated and 23 genes were down-regulated.Compared with the model group,Western medicine group shared 95 differential genes,of which 46 genes were up-regulated and49 genes were down-regulated.The GO function enrichment analysis of differential genes showed that the biological processes involved in the differential genes of the model group and the normal group include metabolic processes,cell secretion,apoptosis,circadian rhythm,and response to light stimulation.The biological processes involved in the differential genes of the Shenqi compound group and the model group include response to insulin stimulation,apoptosis,regulation of phosphatidylinositol 3-kinase,circadian rhythm,and response to light stimulation.Western medicine group compared with the model group,the biological process of differential genes including insulin secretion,apoptosis,phosphatidylinositol 3-kinase signaling pathway,circadian rhythm,regulation of steady state process,and regulation of inflammatory response and other biological processes.The differential genes were analyzed by pathway enrichment analysis.Compared with the normal group,the model group showed that the main signaling pathways were metabolic pathway,amebiasis,oocyte meiosis,progesterone-mediated oocyte maturation,arginine and proline metabolism,glycosphingolipid biosynthesis-lactose and new lactose series.Compared with the Sm group,the main pathways of the model group involved pancreatic cancer,regulation of actin cytoskeleton,m TOR signaling pathway,local adhesion,renal cell carcinoma,bacterial invasion of epithelial cells,VEGF signaling pathway,small cell lung cancer,Erb B signaling pathway,FcγR-mediated phagocytosis,Gn RH signaling pathway,T cell receptor signaling pathway,leukocyte transendothelial migration,axon guidance,cancer pathway,neurotrophin signaling pathway,toxoplasmosis.The main pathways involved in the differentially expressed genes in the western medicine group and the model group were arginine and proline metabolism,salivary secretion,and Gn RH signaling pathway.Rat pancreatic tissue gene chip differential gene binding GO function and pathway enrichment analysis found that: compared with the model group,Shenqi compound and western medicine can up-regulate the expression of insulin secretion related gene GCG,down-regulate the expression of insulin secretion related gene KCNN4 And the PCR verification results are consistent with the gene chip.Compared with the model group,Shenqi compound can up-regulate the expression of Islet cell apoptosis related genes PIK3R1 and AKT1,and the PCR results are basically consistent with the gene chip.Compared with the model group,Shenqi compound and western medicine can up-regulate the expression of biological clock rhythm related genes CRY1 and DBP,and the PCR verification results are consistent with the gene chip.The 16 Sr DNA gene sequencing results of rat feces showed that compared with the normal group,the bacterial population of GK rats was higher,and the richness of the western medicine group was higher than that of the model group and the Sm group.At the level of the phylum,the intestinal flora of each group had a high proportion of the Firmicutes and Bacteroidetes,which were the main dominant bacteria.Compared with the normal group,the ratio of Bacteroides/Firmicutes decreased in each GK rat group,and the ratio of Bacteroides/Firmicutes increased after the intervention of Shenqi compound.At the genus level,compared with the model group,the abundance of Bacteroides,Butyricimonas,Blautia,and Roseburia were relatively high in the Sm and western medicine groups.Conclusion: Shenqi compound can improve the general condition of GK rats,reduce the daily food intake and daily drinking water,reduce the levels of serum TC,TG,GC,hs CRP,increase the levels of INS and GLP-1,reduce the fluctuation of blood glucose,and inhibit the apoptosis of islet βcells and improve the pathological morphology of pancreas.It is speculated that the molecular mechanism of Shenqi compound in preventing and treating type 2 diabetes may be related to the regulation of insulin secretion related genes GCG,KCNN4,islet apoptosis related genes PIK3R1,AKT1,biological clock rhythm related genes CRY1,DBP expression;regulation of intestinal flora,improvement the ratio of Bacteroides/Firmicutes,increasing the relative abundance of butyric acid producing bacteria,improving islet cell function,regulating insulin secretion of islet cells and maintaining the body’s blood glucose homeostasis.It reflects the multi-target and multi-channel treatment characteristics of Shenqi compound,and its advantages may be related to the traditional Chinese medicine theory of "spleen-stomach homologous". |
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