| Background and Objective:Diabetic kidney disease(DKD)is a chronic kidney disease related to aging,and has become the main cause of end-stage renal disease(ESRD).In the context of China’s increasing ageing,the economic and social problems caused will become more serious.However,the current treatment of DKD still lacks effective drugs,mainly controls blood glucose,blood lipids,blood pressure and other risk factors.Studies have shown that longevity-related nutrition-sensitive signaling molecules mTOR,AMPK and SIRT1 etc.not only participate in multiple pathogenesis related to DKD;meanwhile,they independently and synergistically induce renal autophagic activity,showing multiple therapeutic effects ameliorating renal cell damage and fibrosis to treat DKD.Professor Hongfang Liu,my tutor,based on Zhang Jingyue’s theory of "Zhen Yin Jing Qi",found that the loss of kidney essence is an important pathogenesis feature of DKD from clinical syndrome research,and proposed the pathogenesis theory of "Jing Sun Luo Bi" in DKD and established the treatment method of "Tian Jing Tong luo",and made the effective formula QiDiTangShen granules(QDTS)for the treatment of DKD.Previous clinical and experimental studies have confirmed that it can reduce proteinuria in patients with DKD and model rats.However,the mechanism of treatment of DKD with the treatment method of"Tian Jing Tong luo"(QDTS granules)is not yet clear.Considering the relationship between kidney essence and aging in traditional Chinese medicine,this study explored the mechanism of the treatment method of "Tian Jing Tong luo"(QDTS granules)to reduce proteinuria and reduce renal damage based on the regulation of autophagy activity by longevity signal pathway.Methods:1.Pharmacodynamic investigation:db/db mice were used as objects.At their 14th week old,they were randomly divided into three groups,namely the model group(db/db),the valsartan group(db/db+V),and the QDTS Granule group(db/db+Q),C57BL/6J mice of the same age were used as normal controls.The items including blood glucose,body weight,and 12h urinary microalbumin were checked regularly.Blood was collected to measure blood lipid,liver function,renal function,and insulin level.2.Pathological examination:Renal tissues were taken for HE staining,hexaamine silver staining,and MASSON staining.Immunohistochemistry was used to detect the expression of glomerular podocyte-specific proteins Nephrin and WT1,as well as fibrosisrelated molecules such as transforming growth factorβ(TGF-β),collagen Ⅰ(Col1)and smooth muscle actin α(α-SMA).The integrity of the glomerular basement membrane filtration barrier were investigated by transmission electron microscopy(TEM),3.The expression of autophagy-related marker molecules Atg14,Lc3-II,Beclinl,and SQSTM/p62 in mouse kidney was detected by WB,and the protein expression of autophagy marker molecule Lc3-Ⅱ was verified by immunofluorescence.The number and volume of autophagic vesicles and intracellular mitochondria in mouse renal tubular epithelial cells were analyzed by transmission electron microscopy.Related indicators of renal apoptosis were detected by detecting apoptosis-related proteins Bcl-2,BAX,and TUNEL fluorescence.4.Prediction of signal pathway:The differentially expressed renal miRNAs regulated by QDTS were screened by the Illumina second-generation sequencing technology,and then verified by real-time quantitative PCR technology.The network of target genes of differentially expressed miRNAs was constructed to predict the signal pathways mainly regulated by QDTS Granules with the method of network pharmacology technology.5.Analysis of pharmaceutical ingredients and their targets:The main drug components of QDTS were analyzed by high performance liquid chromatography combined with mass spectrometry.The possible effective drug ingredients of QDTS were screened by oral bioavailability(>30%)and drug-likeness(>0.18).By examining published studies,analyze whether the active ingredients of QDTS have a regulatory effect on the relevant regulatory molecules in the predicted main signal pathway?6.Validate the expression of signal molecules:The expression levels of the signal molecules and related active sites in mouse kidney tissues were detected by Western blot(WB).Result:1.After gavage treatment,QDTS granules gradually reduced the urinary albumin level in db/db mice for 12 h,and the urinary protein level stabilized after 8 weeks of administration,and there was a significant statistic between the urinary protein level and the model group.There was a significant difference(p<0.001),but there was no statistically significant difference compared with the valsartan group(p>0.05).Compared with the model group mice,the traditional Chinese medicine QDTS granules significantly improved the insulin resistance of db/db mice(p<0.05),but it had a significant effect on blood glucose,body weight,aspartate/alanine aminotransferase,serum creatinine,and urea nitrogen.,Blood triglyceride and cholesterol were not significantly different(p>0.05).2.Pathological examination found that compared with the model group,the QDTS Granules significantly reduced the degree of fibrosis in renal MASSON staining(p<0.01).This was related to fibrosis-related molecules TGF-β,α-SMA,and COL1.The protein and mRNA expression levels were consistent(p<0.001).Transmission electron microscopy showed that compared with the model group,the integrity of the vascular endothelium,basement membrane thickness,and podocyte process of the glomerular basement membrane of the mice in the QDTS granule group were all improved,which was in contrast to the immune group.The expression of Nephrin protein in podocytes of QDTS granule group was significantly increased(p<0.01),while the expression of WT1 protein was not significantly different in db/db mice(p>0.05).3.It was found from checking the renal autophagy related proteins that the expression of Atg14,beclin1 and autophagic marker protein Lc3-II in kidneys of db/db mice were significantly reduced(p<0.05),compared with normal mice.The accumulation of Autophagic transport substrate p62 increased(p<0.001).QDTS Granules up-regulated the protein expressions of Atg14(p<0.001),beclin1(p<0.05)and Lc3-II(p<0.001)in kidneys of db/db mice,while reduced the expression of p62(p<0.01).QDTS promoted the synthesis of renal autophagic vesicles and reduced mitochondrial damage of renal tubular epithelial cells in db/db mice under TEM.With the improvement of autophagy activity,compared with the model group,the expression of pro-apoptotic protein BAX(p<0.01)in the group of QDTS granules decreased,while the expression of the inhibitory protein Bcl-2 increased(P<0.001);and TUNEL fluorescence found that compared with the model group,the ratio of TUNEL positive cells in the QDTS granule group was significantly reduced(p<0.001).4.The predictive analysis of the action pathways and targets of QDTS Granules found that QDTS granules up-regulated 3 miRNAs(1955-5p,615-3p and 193b-p,p<0.001)and down-regulated one miRNA(181-2-3p,p<0.05).Based on the network regulation analysis of its target genes,it was found that QDTS granule mainly regulates the longevity regulation signaling pathway of mice,which mainly includes three signal molecules of mTOR,AMPK and SIRT1.Through liquid-mass spectrometry analysis,78 drug ingredients with the highest content in QDTS Granules were identified,and 13 effective drug ingredients were screened out.According to literature analysis,8 of them were reported to up-regulate AMPK(8/13),7 lower mTOR(7/13)and 5 higher SIRT1(5/13).5.Through verification analysis,compared with the model group,QDTS Granules upregulated SIRT1 and AMPK protein expression and the AMPK(Thr172)/AMPK phosphorylation ratio(p<0.05)in the kidneys of mice.mTOR(Ser2448)/mTOR phosphorylation ratio(p<0.001).Conclusion:1.QDTS granules,which contains the main treatment method of replenishing renal essence,reduced urinary albumin and treated DKD without lowering blood sugar.2.By analyzing differentially expressed miRNAs and effective pharmaceutical ingredients,the treatment method of "Tian Jing Tong luo"(QDTS granules)is predicted to induce and enhance renal autophagic activity by regulating the expression of the longevity regulating pathway nutrition sensitive signal molecules mTOR,AMPK and SIRT1,play a role in improving insulin resistance,reducing kidney damage,and reducing proteinuria.3.QDTS granules,with "Tian Jing Tong luo" as the main treatment,activates autophagy induced by nutrient-sensitive signaling molecules of the longevity regulation pathway,to a certain extent,it explains the mechanism of tonifying kidney and filling essence in delaying kidney aging. |
