Study On Hypoglycemic Effect Of Sha Qi Zhi Ji On KKAy Mice And Its Mechanism

Objective:Sha Qi preparation(SQ)is a new Mongolian drug preparation for exploratory research.This study observed the hypoglycemic effect of Sha Qi preparation on spontaneous type II diabetes KKAy mice and explored the mechanism of its effect,providing experimental basis for the treatment of type II diabetes.,Use as a basis for further development.Method:1.The KKAy mice were divided into model control group(MCG),low dose group(LSQG,0.8 g/kg),medium dose group(MSQG,1.6 g/kg),and high dose group(HSQG,3.2 g/kg)and Metformin hydrochloride(MEG,2.5 g/kg),and homologous C57BL/6mice as normal control groups(NCG).All mice were given gastric administration for 8weeks,once a day.Weekly detection of the change in body weight,24 h of eating,24 h of drinking water;Rats 0W,2W,4W,and 8W were used to determine random blood glucose and fasting 2h blood glucose in each group of mice,glucose tolerance was measured in the 6th week of administration,dynamic changes in fasting blood glucose were 24 h,and dynamic changes in random blood glucose were 24 h.After the end of 8weeks,various organ coefficients,serum blood glucose,insulin,blood lipids,and liver function indicators were determined.2.HE staining was performed on the liver and pancreas of the mice.The liver tissue was stained with glycogen(PAS)staining,Masson staining,and oil red staining.The effect of Sha Qi preparation on the liver Histopathology of KKAy mice was observed.3.Immunohistochemical staining to observe the effect of Sha Qi preparation on liver PTP1 B protein expression;The expression of PTP1 B protein in liver tissue was detected by Western Blotting method.PCR method for the detection of PTP1 B nucleic acid expression.4.Immunohistochemical staining to observe the effects of Sha Qi preparations on the expression of liver P-IRS2,P13 K,P-AKT,GSK3β and other proteins;The expression of P13 K,AKT,P-AKT,GLUT4,GSK3β in liver tissue was detected by Western Blotting;The PCR method detected the expression of nucleic acids such as IRS2,PI 3K,AKT,GLUT4,GSK3β.5.Because LD50 was not measured,the maximum tolerance and the effects on weight and viscera coefficients of mice were measured.Take 40 healthy mice,randomly divided into two groups,each group of 20,half male and half female.The first group was a high dose group of Sha Qi preparations,with a maximum volume(0.2 m L/10g)and a maximum administration concentration(1g/m L)of Sha Qi preparations 3times(3hours apart)within 12 hours.The total dose was 60g/kg.Equivalent to 380 times the amount of raw drugs used by clinical people.The second group was a blank control group,which gave the same volume of distilled water at one time.Results:1.Over time,the weight of each group of mice gradually increased,and compared with the normal group,the weight of the model group increased significantly(P< 0.01);There was a gradual decrease in weight in each group of Sha Qi preparations,but there was no statistical difference with the model group(P>0.05);The positive group had no significant effect on the weight of mice.Compared with the normal group,the consumption of 24 h and drinking water increased significantly in the model group(P<0.01);Compared with the model group,there was a decreasing trend in the consumption of mice,but no statistical significance in each group(P>0.05);After 4weeks of administration,the amount of drinking water in each group and the positive group of mice began to decrease,and the amount of high-dose drinking water from the7 th week was significantly lower than that of the model group,similar to that of the positive group(P<0.01).Compared with the normal group,the random blood glucose and fasting 2h blood glucose in the model group increased significantly(P<0.01);Compared with the model group,the randomized blood glucose and fasting 2h blood glucose were significantly reduced in each group and the positive group(P< 0.01);When the glucose load was 30 min,compared with the model group,the positive group had the largest degree of inhibition of blood glucose increase(P<0.01),followed by the high dose group of Sha Qi preparations,the medium dose group of Sha Qi preparations,and the low dose group of Sha Qi preparations(P< 0.01);In 60 min of glucose load,blood glucose levels were significantly lower in each group than in the model group(P<0.01)The most significant reduction was in the positive group,which was close to the normal group;When the glucose load was 120 min,the most significant decrease was in the positive group(P<0.01)The blood glucose level of each group of Shaqi preparations was lower than that of the positive group;AUC order is normal group>Positive group>Sha Qi preparation in high dose group>Middle dose group>Low dose group of Sha Qi preparation>Model Group(P<0.01).Compared with the normal group,24 h fasting blood glucose increased significantly in the model group(P<0.01);Compared with the model group,the groups of Sha Qi preparations had a declining trend when fasting 2h,6h,and 12 h of 24 h fasting glucose in mice(P>0.05),fasting 18 h,24h fasting fasting blood glucose is higher than the model group.Compared with the normal group,24 h random blood glucose increased significantly in the model group(P<0.01);Compared with the model group,the blood glucose of mice at 10:00 was significantly reduced in high dose group(P<0.01)There was no significant change in the rest of the time.Compared with the normal group,the liver coefficient,spleen coefficient and kidney coefficient increased significantly in the model group(P<0.01);Compared with the model group,there was no significant change in liver,spleen and kidney coefficients in each group and the positive group.2.Blood glucose indicator: Compared with the normal control group,serum GHb,GSP,and AGEs levels in the model control group increased significantly(P<0.01);Compared with the model control group,the serum GHb,GSP,and AGEs in each group and positive group decreased significantly(P<0.01).Insulin index: Compared with the normal control group,the serum levels of INS and C peptide in the model control group increased significantly(P<0.01);Compared with the model control group,the levels of serum INS and C peptides in each group and positive group decreased significantly,and the levels of HDL-C decreased significantly(P<0.01).Lipid index: Compared with the normal control group,serum levels of TC,TG and LDL-C increased significantly in the model control group.Compared with the model control group,the levels of serum TC,TG and LDL-C in each group and positive group were significantly reduced,and HDL-C levels increased.Liver function index: Compared with the normal control group,the serum AST,ALT,and direct bilirubin levels in the model control group increasedsignificantly(P<0.01);Compared with the model control group,the serum AST and ALT in each group and positive group decreased significantly and the direct bilirubin level decreased significantly(P<0.01).Compared to the normal control group,serum albumin levels in the model control group decreased significantly(P<0.01)The total protein has a decreasing trend;Compared with the model control group,the serum total protein and albumin levels of each group and positive group of Sha Qi preparations increased to different degrees(P<0.05 or P< 0.01).3.HE staining pathology: Compared with the normal control group,the pathological changes of pancreas and liver tissues in the model control group were significantly aggravated.Compared with the control group,the pathological changes of pancreas and liver in each group and the positive group were significantly reduced.Pathologic section of glycogen(PAS)staining: Compared with the normal control group,the content of liver glycogen decreased significantly in the model control group.Compared with the model control group,the content of liver glycogen in each drug group increased significantly.Masson staining: Fibrosis increased significantly in the model control group compared to the normal control group.Compared with the model control group,liver tissue fibrosis decreased significantly in each group.Carmine stain: Compared with the normal control group,the liver tissue fat content of the model control group increased significantly.Compared with the model control group,the liver tissue fat content of each group decreased significantly.4.The results of immunohistochemical observation of PTP1 B protein showed that the expression of PTP1 B protein in liver tissue of mice in the model group was significantly higher than that of normal groups(P<0.01);Compared with the model group,the expression of PTP1 B protein in liver tissues of each group and positive group of Sha Qi preparations decreased significantly(P<0.01).The results of Western Blotting method for the detection of PTP1 B protein in liver tissues of mice showed that the expression of PTP1 B protein in liver tissues of mice in the model group was significantly increased compared with the normal group(P<0.01);Compared with the model group,the expression of PTP1 B protein in liver tissues of each group and positive group of Sha Qi preparations was significantly reduced(P<0.01);The m RNA expression of PTP1 B inliver tissue of mice detected by PCR method showed that the m RNA expression of PTP1 B in liver tissue of mice in the model group was significantly increased compared with the normal group(P<0.01);Compared with the model group,the m RNA expression of PTP1 B in liver tissue of each group and positive group of Sha Qi preparations was significantly reduced(P<0.01).5.The immunohistochemical observations of the key factors of insulin signaling pathways P-IRS 2,PI3 K,P-AKT,and GSK3β showed that: Compared with the normal group,The expression of P-IRS 2,PI3 K,P-AKT protein and the expression of GSK3βprotein were significantly decreased in the liver tissue of mice in the model group(P<0.01);Compared with the model group,the expression of P-IRS2,PI3 K,P-AKT protein and GSK3β protein in liver tissues of each group and positive group were significantly increased and the expression of GSK3β protein was significantly decreased(P<0.01).The results of Western Blotting detection of key proteins in the insulin pathway PI3 K,AKT,P-AKT,GLUT4,GSK3β in mice showed that: Compared with the normal group,The expression of PI3 K,P-AKT,GLUT4 protein in liver tissue of mice in the model group was significantly reduced,and the expression of GSK3β White was significantly increased(P<0.01)There was no significant difference in AKT protein expression;Compared with the model group,the expression of PI3 K,P-AKT,GLUT4 protein in liver tissues of each group and positive group of Sha Qi preparations was significantly increased,and the expression of GSK3β protein was significantly reduced(P<0.01)There was no significant difference in AKT protein expression.The m RNA expression of IRS2,PI3 K,AKT,GLUT4,GSK3β in liver tissue of mice was detected by PCR method.The expression of m RNA in the liver tissues of mice in the model group was significantly reduced,and that of GSK3β was significantly increased(P<0.01);Compared with the model group,the m RNA expression of IRS2,PI3 K,AKT,GLUT4 was significantly increased in each group and positive group liver tissue,and the m RNA expression of GSK3β was significantly reduced(P<0.01).6.The acute toxicity test for mice with Sha Qi preparations was taken orally for 14 days with a maximum tolerance(dose of 60 g/kg,equivalent to 380 times the clinical dose).No toxicity was observed in the high dose group and no death occurred in allmice.And I didn’t find anything else related to the administration.Conclusion:1.Sha Qi preparation can reduce the blood glucose of type II diabetes KKAy mice,promote glucose metabolism,improve glucose tolerance,and stabilize blood glucose.2.Sha Qi preparation can regulate the blood lipid level of type II diabetes KKAy mice and improve lipid metabolism.3.Sha Qi preparation can reduce the damage of pancreatic islet beta cells in type II diabetic KKAy mice,reduce the lesions of liver and pancreatic tissue,and play a certain protective role in pancreas and liver tissue;Sha Qi preparation can increase the content of liver glycogen in type II diabetes KKAy mice;Sha Qi preparation can reduce liver fibrosis of type II diabetes KKAy mice;Sha Qi preparation can reduce the liver fat content of KKAy mice with type II diabetes.4.Sha Qi preparations can increase the expression of key factors IRS2,P13 K,AKT,GLUT4 proteins and genes in insulin signaling pathways,and lower PTP1 B,GSK3βproteins and gene expression,which may be the internal mechanism for its role in reducing glucose.5.No acute toxicity was found in Sha Qi preparations.