| ObjectiveUnder the guidance of TCM theory,to explore the regulatory effect and mechanism of Huoxue-Jiangtang decoction on glucose and lipid metabolism,insulin resistance and intestinal flora homeostasis in type 2 diabetic rats;to detect and analyze the main active components of Huoxue-Jiangtang decoction and further explore there's effects on glucose and lipid metabolism and insulin resistance in type 2 diabetic rats;In addition,to ensure the repeatability of the studies,we established the quality control standard for the study of Huoxue-Jiangtang decoction.Materials and methodsThis study mainly includes the establishment of the quality control standard for the study of Huoxue-Jiangtang decoction and the effect and mechanism of Huoxue-Jiangtang decoction on glucose and lipid metabolism,insulin resistance and intestinal flora homeostasis in type 2 diabetic rats.1.52 male Wistar rats of SPF grade,7 weeks old,weighing 150±20g.After 7 days of adaptive feeding,all rats were randomly divided into normal group and model group according to their weight.10 rats in the normal group were fed with normal standard feed,the rest rats were fed with high-fat feed(D12492,60%fat)in the model group.At the end of the 4th week after feeding,all rats were fasted for 12 hours,and then the model rats were injected 2%streptozocin(STZ)(35mg/kg)intraperitoneally in the next day.Before injection,STZ was fully dissolved in the sterile citric acid buffer with pH=4.4.STZ solution was prepared on ice,and the injection was completed within 30 minutes.Rats in the normal group were given 0.1mol/1 citric acid buffer of equal volume.The fasting blood glucose(fasting for 12 hours)of rats were measured by blood glucose meter on the 3rd,7th and 14th day of STZ injection.Model rats with fasting blood glucose between 11.1-33.3 mmol/L and stable for 14 days were included in the study.The model rats were randomly divided into 5 groups(n=10/group)according to their body weight and fasting blood glucose:model control group,metformin group,HXJTL group and HXJTH group.Another normal control group(normal n=10).The rats in the normal and model control groups were given dual steaming water,the dose was 5ml/kg b.w;the rats in the metformin group were given metformin,the dose was 90 mg/kg;the rats in the low-dose group were given Huoxue-Jiangtang freeze-dried powder 1.25g/kg,and the rats in the high-dose group were given Huoxue-Jiangtang freeze-dried powder 5g/kg.The drug was given by gavage once a day for 8 weeks.2.Observe and record the state of rats and the bedding change every day,all rats were weighed once a week,the water intake and the average food intake were recorded weekly.The fasting blood glucose(fasting for 12 hours)was measured weekly by Roche blood glucose meter by the tail vein blood of rats.The oral glucose tolerance test(OGTT)was carried out at the end of 8 weeks after administration.After 12 hours of fasting,fasting blood glucose was measured with a blood glucose meter once,and then 2g/kg glucose solution was given to gavage.Blood glucose in tail vein was measured with a blood glucose meter at 0,30,60 and 120 minutes respectively.3.In the three time points of animal experiment,before the administration of STZ(-4w),after the establishment of STZ model and before the intervention treatment(-2w),and before the end of 8 weeks of treatment,the intestinal feces of rats were collected,and the genomic DNA of intestinal microflora was extracted by tianamp stool DNA kit.The gene sequencing platform sequenced the V4 region of 16S rRNA of all microorganisms in the feces,and analyzed the species richness and diversity of intestinal flora.4.After the intervention,the blood of rats and tissues of pancreas,liver,skeletal muscle and kidney were collected;the blood lipid profile(TG,TC,LDL-C,HDL-C)level,serum creatinine,urea nitrogen,SOD,MDA,glycogen synthetase,hexokinase and other active enzymes were detected by enzyme labeling;the fasting insulin,serum leptin,adiponectin,TNF-? and so on were detected by ELISA The expression of P-acc,p-Akt protein,PI3K,Akt,P-acc and GLUT4 protein in liver and skeletal muscle were detected by anthrone method,he staining and immunohistochemistry staining,TUNEL method,Western blot.Detection of apoptosis related proteins in pancreas by Western blot.5.The two active components(catalpol and hydroxysafflor yellow a)with the highest content in Huoxue Jiangtang Decoction were also tested in type 2 diabetic rats.The specific method is the same as before.6.In addition,the 2015 edition of Pharmacopoeia of traditional Chinese medicine was used as the criterion of detection of the pharmaceutical ingredients in the compound solution.The components of the Huoxue-Jiangtang decoction prepared by different processing and extraction processes were detected by HPLC,and the extraction process of Huoxue-Jiangtang decoction was optimized.Based on the optimized processing technology of Huoxue-Jiangtang decoction,the known components of eight kinds of traditional Chinese medicine were mined out through the database of traditional Chinese medicine components,and then combined with the existing database,the most likely known main active components with metabolic regulation were searched,and the prepared Huoxue-Jiangtang decoction(freeze-dried powder)was used in type 2 diabetes.At last,the preparation standard of the traditional Chinese medicine compound was used as the quality control standard of Huoxue-Jiangtang decoction.Results1.The type 2 diabetic rats were established by high-fat feed and following by intraperitoneal injection of a low-dose(35mg/kg)for one-time,which were similar to human type 2 diabetic.The T2DM rats have obvious insulin resistance,polydipsia and dyslipidemia,etc.;40 rats of type 2 diabetic have been successfully established,with the success rate of 95.24%,and the fasting blood glucose in the model group tends to be stable throughout the whole process;the Huoxue-Jiangtang decoction could significantly reduce the fasting blood glucose,the intake of energy and water in T2DM rats,although there have not a significant effect on body weight;after 8 weeks of intervention,the fasting blood glucose in the low-dose and high-dose Huoxue-Jiangtang decoction groups decreased by 54.83%and 56.16%,respectively,significantly higher than that in the model group(P<0.05);the area under the OGTT curve in the drug administration group was also significantly lower than that in the model group(P<0.01),but the high-dose group was not significantly better than that in the low-dose group(P<0.01)Quantity group.2.After 8 weeks of treatment,the insulin resistance index and ?-cell function of rats in the Huoxue jiangtangyin group were significantly improved(P<0.05 or P<0.01);compared with the model group,the blood lipid level(TG,TC,LDL-C)in the Huoxue jiangtangyin group was significantly decreased(P<0.05),the serum creatinine level was also significantly decreased(P<0.01),but there was no significant difference in HDL-C,AST,alt,bun,etc Influence.3.Compared with the model group,the level of SOD and MDA,TNF-? and free fatty acid in the serum of the group were significantly increased(P<0.05).Compared with the model group,the content of liver glycogen,muscle glycogen,glycogen synthetase and hexokinase were also increased(P<0.05).4.HE staining,immunohistochemical staining and TUNEL analysis showed that Huoxue-Jiangtang decoction had a better protective effect on pancreas.It can maintain proud ?-cell morphology,increase the number of islet cells,up regulate the expression of insulin,inhibit ?-cell apoptosis,etc.;Western blot results showed that Huoxue Jiangtang decoction can up regulate the expression of P-acc,Akt(p-Akt)protein in liver and skeletal muscle(P<0.05),up regulate the expression of PI3K and GLUT4 protein in skeletal muscle(P<0.05).5.The results of genome sequencing of intestinal flora showed that,compared with the normal control group,the abundance and diversity of intestinal flora in the model group decreased,and the species and number of pathogenic bacteria increased,showing a significant imbalance of intestinal homeostasis;Huoxue Jiangtang decoction could significantly increase the species and number of beneficial bacteria such as Lactobacillaceae,lachnospiraceae,and reduce such beneficial bacteria as prevotellacae and porphyromonaceae,etc The correlation analysis showed that the intestinal flora regulated by Huoxue-Jiangtang decoction was significantly related to fasting blood glucose,blood lipid and insulin resistance,and KEGG analysis also showed that these regulated intestinal flora mainly involved in the pathway of glucose and lipid metabolism.6.The two main active components of Huoxue-Jiangtang decoction compound also significantly improved the glycolipid metabolism of type 2 diabetic rats.Compared with the model group,catalpol and hydroxysafflor yellow a could significantly reduce the fasting blood glucose,blood lipid level and insulin resistance index of T2DM rats(P<0.05).7.In addition,the extraction and preparation process of Huoxue-Jiangtang decoction was optimized.22 main active ingredients,which may have metabolic regulation were screened out through database and literature search.The freeze-dried powder of Huoxue-Jiangtang decoction has the regulation effect on the metabolism of glycolipid in type 2 diabetic rats.The preparation and processing process and quality control standard can be used as the formulation standard for the study of Huoxue-Jiangtang decoction accurate,to ensure the consistency of each compound study.Conclusion1.Through HFD feeding and STZ injection,a stable type 2 diabetes model that similar to human disease can be established;Huoxue-Jiangtang decoction can significantly improve the symptoms of water intake and polydipsia,reduce fasting blood glucose and lipid levels,improve insulin and leptin resistance;at the same time,Huoxue-Jiangtang2.decoction can also improve the inflammation and oxidative stress caused by endotoxemia It can maintain the shape of islet cells,inhibit the apoptosis of islet ?-cells,and promote the expression and secretion of insulin.3.The potential mechanism of Huoxue-Jiangtang decoction in the treatment of type 2 diabetes may be that it can increase the content of liver glycogen,muscle glycogen,glycogen synthetase and hexokinase,improve the state of inflammation and oxidative stress,and improve insulin resistance,which is related to PI3K/Akt/GLUT4 signaling pathway.4.The homeostasis of intestinal flora in the model of type 2 diabetic rats established by HFD feeding and STZ injection was obviously out of balance.Huoxue Jiangtang decoction could significantly improve the state of intestinal flora disorder in type 2 diabetic rats,which was mainly manifested in reducing the number of pathogenic Gram-negative bacteria such as prevotellace and increasing the number of beneficial bacteria such as Lactobacillus These beneficial bacilli are closely related to butyrate production and negatively related to the biochemical indicators of glycolipid metabolism.They may be involved in the regulation of glycolipid metabolism and insulin resistance in type 2 diabetes through the increase of SCFA metabolites.5.Catalpol and hydroxysafflor yellow A,the main active components of Huoxue-Jiangtang decoction compound,were also proved to have obvious effects on the regulation of glycolipid metabolism and the improvement of insulin resistance through the intervention experiments on type 2 diabetic rats.But whether the effect of active monomers of traditional Chinese medicine is similar to that of the compound in human body The effect needs further study.At the same time,it also provides an idea for the study of the whole and the split prescription of traditional Chinese medicine.6.In addition,it is feasible to establish the quality control standard of Huoxue-Jiangtang decoction through the main active components of Huoxue-Jiangtang decoction,and it almost has the same effect through different experiments.It also provides a good reference for the idea of repeatable research of traditional Chinese medicine. |
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