Tangshen Recipe Based On The Mechanism Of FXR Pathway Regulating Lipid Metabolism "liver And Kidney Tongzhi"

Diabetes has become a chronic disease that seriously threatens global human health.The prevalence of diabetes in China has been increasing year by year,and it has reached 10.9%,among which 20-40%of diabetic patients will progress to diabetic kidney disease.The prevalence of NAFLD is 75%among patients with type 2 diabetes mellitus.DKD and NAFLD have become major disease burden of the type 2 diabetes patients.Despite strict glycemic control and blood pressure control are adopted for diabetic patients,more than 20%of type 2 diabetic patients in China still progress to DKD.The molecular mechanism underlying non-alcoholic fatty liver diasease(NAFLD)has not been fully understood,and no drugs are recommended for the therapy of non-alcoholic fatty liver diasease.Therefore,there is an urgent need to carry out research on the treatment of DKD and NAFLD to delay the progression of NAFLD and renal failure in diabetic patients.It is reported in the literature that abnormal lipid metabolism can accelerate the progress of DKD.Abnormal accumulation of lipids in the kidneys is called "lipid nephrotoxicity",which can cause oxidative stress through excessive lipid oxidation and mitochondrial production of reactive oxygen species,thereby impairing kidney function.Excessive lipid deposition in liver tissue is a typical pathological change of NAFLD.It is precisely because of this important feature that liver tissue may develop more severe fibrosis or cirrhosis.Recent studies indicate that nuclear hormone receptors play important role in regulating lipid and carbohydrate metabolism,fibrosis and inflammation.The farnesoid X receptor is a member of the nuclear receptor family.Farnesoid X receptor(FXR)is essential in regulating bile acid,lipid and carbohydrate metabolism,and is highly expressed in the liver and kidney.Chinese medicine has significant advantages in ameliorating glycolipid metabolism disorder and alleviating kidney and liver tissue injury.Exploring the mechanism of traditional Chinese medicine to delay liver and kidney damage is a realistic problem that needs to be solved urgently.Tangshen Formula is a traditional Chinese medicine compound for the effective prevention and treatment of DKD developed by Professor Li Ping on the basis of predecessors’ experience in the treatment of DKD and under the guidance of the theory of"simultaneous treatment of liver and kidney".Its achievements won the second prize of Chinese National Science and Technology Progress Award in 2016.The research group carried out a multi-center clinical trial of Tangshen Formula(TSF)in the treatment of DKD in the early stage.It is proved that Tangshen Formula can improve the glomerular filtration rate and protein excretion.In recent study,it has been found that Tangshen Formula alleviate kidney injury and steatosis of the liver by improving lipid deposits.Nevertheless,the mechanism by which Tangshen Formula improves lipid deposition in the kidney and liver of DKD is not fully understood.Further research is required to elucidate this issueThis study will use spontaneous type 2 diabetic db/db mice with DKD and NAFLD to give Tangshen Formula by gavage to evaluate the pharmacodynamic effects of Tangshen Formula on liver and kidney damage in db/db miceAt the same time,we will observe the effects of Tangshen Formula on kidney cortex and liver lipid metabolism in db/db mice,and explore the molecular pathways of Tangshen Formula to improve liver and kidney lipid metabolism.Combining with in vitro sodium palmitate(PA)stimulation of primary mouse renal tubular epithelial cells(mTECs)and mouse-derived normal liver cells(AML12)culture,explore the mechanism of Tangshen Formula ’s "simultaneous treatment of liver and kidney"at the cellular level.ObjectiveTo evaluate the effect of TSF on liver and kidney damage in db/db mice,and to reveal the mechanism of TSF for improving diabetic liver and kidney damage through lipid deposition regulated by nuclear receptor FXR pathway.MethodsPart 1:To study the mechanism of TSF regulating kidney lipid metabolism through nuclear receptor FXR pathway.(1)Experiment 1:Pharmacodynamic observation of TSF on kidney damage of spontaneous type 2 diabetic db/db mice.After being adaptively fed to 8-week-old male db/db mice and db/m mice with the same genetic background for 2 weeks,they were randomly divided into normal control group db/m,model control group db/db,TSF treatment group:db/db+TSF(2.4 g/kg/d),positive drug Irbesartan treatment group:db/db+ Apro(22.5 mg/kg/d).Gavage was started at 10 weeks of age,and the animal’s body weight was recorded once a week.Blood was collected from the tail tip every three weeks to measure fasting blood glucose.Before and 12 weeks after the administration,the animals were placed in a metabolic cage to collect 24-hour urine to determine urine protein.After the administration for 12 weeks,blood was collected by cardiac puncture under anesthesia to determine renal function,blood lipids and blood glucose levels.At the same time,the animals were sacrificed and kidney tissues were taken,and biochemical,molecular biology and histopathological tests were performed to evaluate the improvement effect of the drugs on kidney injury.(2)Experiment 2:To study the regulation mechanism of TSF on renal lipid deposition mediated by the nuclear receptor FXR pathway of db/db mouse.On the basis of experiment 1,take the mouse kidney cortex from the normal control group db/m,model control group db/db,and TSF treatment group.Enzymatic colorimetric method was used to determine the levels of cholesterol and triglycerides in the kidney cortex of each group of mice.Filipin staining and oil red O staining were performed in renal tissue to observe the effect of TSF on kidney lipid metabolism in db/db mice.Western Blotting,real-time PCR,and immunofluorescence were used to detect the effects of TSF on the expression levels of FXR,FXR downstream target gene ApoE,and FXR upstream transcription co-activator PGC-la protein and mRNA expression levels in the kidney cortex of each group of mice.(3)Experiment 3:To study the regulation mechanism of TSF on lipid deposition mediated by the nuclear receptor FXR pathway of renal tubular epithelial cells.Using sodium palmitate to stimulate in vitro renal tubular epithelial cells(mTECs)model,the CCK8 method was used to detect the growth inhibition rate of different concentrations of TSF on mTECs cells,and to screen the effective concentration of TSF.Filipin staining,oil red O staining and enzymatic colorimetric method were used to determine the changes in cholesterol and triglyceride content in mTECs cells after the intervention of TSF,so as to evaluate the effect of TSF on lipid deposition in mTECs cells.Western Blotting and real-time PCR were used to detect the effects of TSF on ApoE,FXR,PGC-1α protein and mRNA expression levels in mTECs stimulated by PA.TSF interfered with mTECs cells which were transfected with SiRNA targeting FXR,and explored the molecular mechanism of TSF to regulate the lipid metabolism of mTECs cells through the nuclear receptor FXR.Part 2:To study the mechanism of TSF regulating liver lipid metabolism through nuclear receptor FXR pathway.(1)Experiment 1:Pharmacodynamic observation of TSF on liver damage of spontaneous type 2 diabetic db/db miceAfter being adaptively fed to 8-week-old male db/db mice and db/m mice with the same genetic background for 2 weeks,they were randomly divided into normal control group db/m,model control group db/db,and TSF treatment group:db/db+TSF(2.4 g/kg/d),positive drug fenofibrate treatment group:db/db+Fen(30 mg/kg/d).Gavage was started at 10 weeks of age,and the animal’s body weight was recorded once a week.Blood was collected from the tail tip every three weeks to measure fasting blood glucose.After 12 weeks of administration,blood was collected by cardiac puncture under anesthesia to determine liver function,blood lipids and blood glucose levels.At the same time,the animals were sacrificed and liver tissues were taken for biochemical,molecular biology,and histopathological tests to evaluate the improvement effect of the drugs on kidney injury.(2)Experiment 2:To study the regulation mechanism of TSF on liver lipid deposition mediated by the nuclear receptor FXR pathway of db/db mouse.On the basis of the previous experiment,took the mouse liver tissues of the normal control group db/m,model control group db/db,and TSF treatment group.Performed Filipin staining and Oil Red O staining to observe the effects of TSF on liver cholesterol and triglyceride metabolism in db/db mice.Western Blotting and real-time PCR were used to detect the effects of TSF on the protein and mRNA expression levels of FXR,FXR downstream target gene ApoE,and FXR upstream transcription co-activator PGC-1α of each group of mice.(3)Experiment 3:To study the regulation mechanism of TSF on lipid deposition mediated by the nuclear receptor FXR pathway of mouse hepatocyte.Fillibin staining and oil red O staining were performed on the mouse-derived normal hepatocyte model stimulated by sodium palmitate to determine the changes in cholesterol and triglyceride content in AML 12 cells after the intervention of TSF,so as to evaluate t effect of TSF on lipid deposition in AML12 cells.Western Blotting and real-time PCR were used to detect the effects of TSF on ApoE,FXR,PGC-la protein and mRNA expression levels in AML12 cells stimulated by PA,and explored the molecular mechanism of TSF to regulate the lipid metabolism of AML12 cells through the nuclear receptor FXR.ResultsPart 1::(1)It was proved that TSF could alleviate kidney damage in db/db mice.TSF could reduce serum creatinine(SCr)and urine protein levels in db/db mice.Masson and PAS staining of the kidney of db/db mice revealed that the glomerulus collagen increased,the area of the glomerular increased,the glycogen increased in the mesangial area,and the mesangial matrix proliferated significantly.After the intervention of TSF,the pathological manifestations of PAS and Masson staining of kidney tissue in db/db mice were improved.TSF could slowe down the weight gain of db/db mice,and lower blood lipids and blood sugar.(2)TSF could improve lipid deposition observed by Filipin staining and oil red O staining,and decrease the levels of cholesterol and triglyceride in the kidney cortex.TSF could increase ApoE,FXR,PGC-1α protein and mRNA expression in the kidney cortex of db/db mice.(3)The CCK8 method determined the intervention concentration of TSF was 500 μg/mL.Filipin staining,oil red O staining and enzymatic colorimetric method found that TSF reduced the levels of cholesterol and triglycerides in mTECs cells.TSF could increase ApoE,FXR,PGC-1α protein and mRNA expression in mTECs cells.After FXR-SiRNA was transfected into mTECs cells,it significantly weakened the effect of TSF on up-regulating the expression of FXR and ApoE protein and mRNA.It suggested that TST might improve renal lipid deposition through PGC-1α/FXR/ApoE signaling pathway.Part 2:(1)It was proved that TSF could alleviate liver damage in db/db mice.TSF could reduce serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels in db/db mice.HE staining of liver tissue of db/db mice showed that hepatocytes were irregular in shape and showed diffuse steatosis,with more cytoplasmic vacuoles,some fused into large lipid droplets,and liver cords were arranged disorderly.After the intervention of TSF,the pathological manifestations of HE staining of liver tissue in db/db mice were improved.(2)TSF could improve lipid deposition observed by Filipin staining and oil red O staining.TSF could increase ApoE,FXR,PGC-1α protein and mRNA expression in the liver tissue of db/db mice.(3)Filipin staining and oil red O staining found that TSF reduced the levels of cholesterol and triglycerides in AML12 cells.TSF could increase ApoE,FXR,PGC-la protein and mRNA expression in AML12 cells.It suggested that TSF might improve liver lipid deposition by regulating the PGC-1α/FXR/ApoE signaling pathway.ConclusionThrough in vivo and in vitro experiments,we found that TSF could improve liver and kidney damage and regulate lipid metabolism disorders in db/db mice.Its mechanism of action was related to its regulation of the PGC-1α/FXR/ApoE pathway,and the nuclear receptor FXR played a key role in the regulation of liver and kidney lipid metabolism by TSF.