Based On The Regulation Of LKB1/AMPK/Sirt1 Signaling Pathway To Explore The Mechanism Of Jiedu Tongluo Baoshen Formula On Diabetic Kidney Disease

Objective: To observe the mechanism of Jiedu Tongluo Baoshen formula(JTBF)on diabetic kidney disease by regulating the LKB1/AMPK/Sirt1 signaling pathway.Methods: 1.In vivo experiment: 100 SPF male SD rats were purchased,and 10 were randomly selected as the control group and fed with conventional feed.The remaining 90 rats were fed with high-fat diet for 6 weeks,and then given STZ(40 mg/kg)intraperitoneal injection to induce DKD rat model.The rats after successful modeling were randomly divided into model group,irbesartan group,JTBF formula low,medium and high dose groups,15 rats in each group.After successful modeling,drugs were given by gavage from the 10 th week for 8 consecutive weeks.During the administration period,the changes of fasting blood glucose and body weight of rats in each group were monitored weekly,and24-hour urine was collected in metabolic cages every 4 weeks,and the urine volume was recorded.,detect 24-hour urine protein quantification.At the end of the 8th week of administration,after collecting 24 hours of urine,the rats in each group were anesthetized and killed by sodium pentobarbital.The pathological changes of kidney tissue were observed by H&E,PAS,Masson staining and transmission electron microscope.RT-q PCR,immunohistochemistry and Western blot methods were used to detect the changes of FN,Col IV,α-SMA and LKB1/AMPK/Sirt1 signaling pathway protein levels in renal tissue of DKD rats with interstitial fibrosis.RT-q PCR and Western blot methods were used to detect the expression levels of autophagy-related proteins Beclin-1,LC3 and P62 in kidney tissue.2.In vitro experiments: NRK-52 E cells were stimulated with high glucose in vitro,and the optimal modeling concentration,modeling time and the intervention concentration of JTBF formula of NRK-52 E cells were detected by CCK8 method,and the modeling conditions were screened to be 30 mmol/L After 48 hours of intervention,JTBF formula200 μg/ml was used for intervention.Western blot was used to detect interstitial fibrotic proteins FN,Col IV,α-SMA,E-cad,N-cad,autophagy-related proteins Beclin-1,LC3II/LC3 I,P62,and the pathway LKB1/AMPK/ Sirt1-related protein expression.And according to different groups,when JTBF formula intervenes in NRK-52 E cells,the autophagy inhibitor chloroquine and Sirt1 inhibitor are intervened respectively.Western blot was used to detect the protein expressions of FN,Col IV,α-SMA,Beclin-1,LC3II/LC3 I,P62,p-LKB1,p-AMPK and Sirt1.Results: 1.In vivo experiment:(1)General morphology: Compared with the control group,the rats in the model group had dull hair,mental fatigue,weight loss,and symptoms of polydipsia,polyphagia,and polyuria in varying degrees.Compared with the model group,the body weight of the rats in the JTBF formula treatment group increased,the urine output decreased,and various symptoms were improved.(2)Biochemical detection of blood and urine: Compared with the control group,the 24-hour urine volume and urine protein quantification in the model group were significantly increased(p < 0.001);Fasting blood glucose and glycosylated hemoglobin in rats were significantly increased(p < 0.01);Blood urea nitrogen was significantly increased(p < 0.001),while serum creatinine was not significantly increased(p > 0.05);There was no significant change in alanine aminotransferase and aspartate aminotransferase(p > 0.05);Triglyceride and cholesterol were significantly increased(p < 0.001).Compared with the model group,the 24-hour urine volume and urine protein quantification of the rats in each administration group were significantly decreased(p < 0.001);There was no significant change in fasting blood glucose and glycosylated hemoglobin in each administration group(p > 0.05);There was no significant change in blood urea nitrogen in the irbesartan group,the low-dose and medium-dose groups of JTBF(p > 0.05),but it decreased in the high-dose group of JTBF(p< 0.05).There was no significant change in the creatinine administration group(p > 0.05);There was no significant change in the alanine aminotransferase and aspartate aminotransferase administration groups(p > 0.05);There was no significant change in triglyceride and cholesterol in the irbesartan group,JTBF low-dose and medium-dose groups(p > 0.05).There was no significant change in triglyceride and cholesterol in irbesartan group,JTBF low-dose and medium-dose groups(p > 0.05).(3)Pathological changes of kidney tissue: kidney appearance: compared with the control group,the kidneys of the model group were slightly larger,and the kidneys of the rats in the high-dose JTBF group were slightly smaller than those in the model group.H&E staining: Compared with the control group,the glomeruli in the model group showed dilation,local glomerular atrophy and sclerosis,significant proliferation of mesangial cells,widening of the mesangial area,and a small number of glomeruli appeared with balloon wall adhesion and renal tubules.Epithelial cells show vacuolar degeneration;Compared with the model group,the pathological changes of the kidneys in the high-dose JTBF group were significantly less than those in the model group.PAS staining: Compared with the control group,the glomerular basement membrane in the model group was significantly thickened,the mesangial matrix was proliferated,the positive substances in the mesangial area were significantly increased,the renal capsule was enlarged,and some renal tubular epithelial cells were vacuolated.Compared with the model group,the basement membrane thickening and mesangial matrix hyperplasia in the JTBF group were reduced.Masson staining:Compared with the control group,blue fiber deposition was seen in the glomerulus of the model group,collagen fibers in the glomerular mesangial matrix and in the gap between the glomerulus and the renal tubule were significantly increased,and glomerulosclerosis appeared.Compared with the model group,the vacuolar degeneration of the renal tubular epithelial cells was reduced in the high-dose JTBF group,and the collagen fibers in the glomerular and tubular spaces were significantly reduced.Transmission electron microscope:Compared with the control group,the model group showed moderate edema of podocytes,moderate to severe swelling of mitochondria,moderate to severe expansion of rough endoplasmic reticulum,hypertrophy and mild expansion of Golgi apparatus,and no typical autophagolysozyme was found in this field of view.The body is present,the thickness of the basement membrane is relatively uniform,the foot processes are fused,and the endothelial cells are edematous.Compared with the model group,the foot processes in the JTBF group were slightly fused,the basement membrane thickening was not obvious,a large number of autophagic lysosomes were seen,the mitochondria were slightly swollen,the rough endoplasmic reticulum was mildly expanded,and the Golgi apparatus was slightly hypertrophic.(4)RT-q PCR results: Compared with the control group,the m RNA expressions of interstitial fibrosis proteins FN,Col IV and α-SMA in the model group were significantly increased(p < 0.05).The m RNA expressions of autophagy-related proteins Beclin-1 and LC3II/LC3 I were significantly decreased(p < 0.05),and the expression of P62 m RNA was increased(p < 0.05).Compared with the model group,the expressions of FN,Col IV and α-SMA m RNA decreased in each administration group(p< 0.05),among which the JTBF high-dose group decreased the most(p < 0.01).The m RNA expressions of autophagy-related proteins Beclin-1 and LC3II/LC3 I did not change significantly in the irbesartan group and the low-dose JTBF group(p > 0.05),but showed a significant upward trend in the medium-dose and high-dose JTBF groups(p < 0.01).There was no significant change in P62 m RNA in the irbesartan group(p > 0.05),but a significant decrease in the JTBF group(p < 0.01).(5)Immunohistochemical results: FN: Compared with the control group,in the model group,the expression of FN in the dilated basement membrane of the glomerulus was significantly increased,and the distribution of FN protein expression was also seen in the tubulointerstitium.Compared with the model group,the distribution of FN in the high-dose JTBF group was significantly reduced,the distribution in the glomerulus decreased,and the distribution in the tubulointerstitium decreased.Col IV: In the high-dose JTBF group,the distribution of Col IV was significantly reduced,the distribution on the glomerulus was reduced,and the distribution in the tubulointerstitium decreased.α-SMA: Compared with the control group,the glomeruli in the model group were dilated,the cut surface was larger,and the degree of dilatation was significantly higher than that in the normal control group.Compared with the model group,the degree of glomerular dilatation in the high-dose JTBF group was significantly reduced,the distribution of α-SMA positive protein in the glomerulus was significantly reduced,and the degree of renal tubular dilatation was reduced,and the glomerular afferent arterioles were α-SMA positive express.(6)Western blot results: Compared with the control group,the expressions of interstitial fibrosis proteins FN,Col IV and α-SMA in the model group were significantly increased(p < 0.01);the expressions of autophagy-related proteins Beclin-1,LC3II/LC3 I The expression of P62 protein level increased(p < 0.01);the expressions of pathway proteins p-LKB1,p-AMPK and Sirt1 were significantly decreased(p < 0.01).Compared with the model group,the expressions of FN,Col IV,and α-SMA in each treatment group were significantly decreased(p < 0.01);there was no significant change in Beclin-1 in the irbesartan group,JTBF low-dose and medium-dose groups(p <0.01),but the expression in the high-dose JTBF group was significantly increased(p <0.001);the expression of LC3II/LC3 I had no significant change in each treatment group(p > 0.05);the expression level of P62 protein in each treatment group In the irbesartan group,the expression of p-LKB1 protein did not change significantly(p > 0.05).There was no significant change in the group(p > 0.05),but it was significantly increased in the middle and high dose groups of JTBF(p < 0.01).2.In vitro experiments: Compared with the control group,the expression levels of FN,Col IV,α-SMA,N-cad,and P62 in the model group were increased,and the expression levels of E-cad,Beclin-1,LC3II/LC3 I,p-LKB1,p-AMPK,Sirt1 protein expression levels were decreased.Compared with the model group,the expression levels of FN,Col IV,α-SMA,N-cad,and P62 in the JTBF group were decreased,and the expression levels of E-cad,Beclin-1,LC3II/LC3 I,p-LKB1,p-AMPK,Sirt1 were decreased.protein expression levels were increased.Compared with the JTBF group,the expression level of P62 in the JTBF+CQ group was increased,and the protein expression levels of Beclin-1 and LC3II/LC3 I were decreased.Compared with the JTBF group,the protein expression levels of p-LKB1,p-AMPK,and Sirt1 in the JTBF+Sirt1 inhibitor group decreased,the protein expression levels of Beclin-1,LC3II/LC3 I decreased,and the expression level of P62 increased.Conclusion: 1.JTBF may reduce 24-hour urine volume,24-hour urine protein quantification,Scr,BUN,TG,TC in diabetic kidney disease rats,and it is safe and reliable.2.JTBF may reduce the thickening of glomerular basement membrane,tubular vacuolar degeneration and collagen fibers in the renal tubular space in diabetic nephropathy rats,and enhance renal autophagy activity.3.JTBF may enhance renal autophagy and improve renal fibrosis through LKB1/AMPK/Sirt1 signaling pathway.4.JTBF may reduce the interstitial fibrosis of renal tubular epithelial cells under high glucose stimulation,and enhance the autophagy activity of renal tubular epithelial cells through the LKB1/AMPK/Sirt1 signaling pathway to reduce renal interstitial fibrosis.