| Background:Chronic kidney disease(CKD)has become an important public health problem in China and even the world,which seriously affects and endangers human health and life.Zhen Wu Decoction(ZWD),which was first recorded in Treatise on Cold Injuries,has the effect of warming Yang and improving water.It is a classic prescription for treating the syndrome of kidney Yang deficiency and failure and excessive water qi.It is also one of the commonly used prescriptions for treating chronic kidney disease.Previous studies have found that ZWD has a protective effect on renal function,but its mechanism remains unclear.Aims:Through in vivo and in vitro experimental studies,combined with clinical blood sample verification,it is clear that ZWD restricts the formation of tubular fibers in renal tubules by activating mitochondrial biological energy,so as to have a protective effect on the kidney and further inhibit CKD.Methods:1.In vivo experiment:Firstly,the unilateral ureter obstraction(UUO)model of C57BL/6 mice was taken as the study object to explore the action mechanism of ZWD in treating CKD.High(25.2g/kg),medium(12.6g/kg)and low(6.3g/kg)ZWD groups and positive control group(30mg/kg)were set up,respectively,for continuous gavage for 7 days,to explore the dose-dependent effect of ZWD on renal fibrosis.The effects of ZWD on mitochondrial bioenergy-related genes(TFAM,NRF2,STING,OXPHOS)were detected by Western blot.The structural changes of mitochondria induced by ZWD were observed by biological transmission electron microscopy.Secondly,high dose of folic acid induced renal fibrosis model of C57BL/6 mice,combined with NRF2 inhibitor ML385,which was injected intrabitoneal continuously for 7 days to investigate whether NRF2 activity affected the anti-fibrosis effect of ZWD.Finally,UUO induced mouse CKD model with intraperitoneal injection of STING inhibitor C-176.ZWD combined with inhibitor C-176 was set to be administered continuously for 7 days to explore whether ZWD affects fibrosis development by regulating STING pathway.2.In vitro experiment:TGF-β1 induced fibrosis model of HK2(human renal tubular epithelium)cells to further verify the effect of mitochondrial injury on renal tubular epithelial cells.TGF-β1 and siRNA(small interfering RNA)induced fibrosis and mitochondrial damage in HK2 cells.The regulatory mechanism of NRF2 on STING pathway was investigated,and how NRF2 affected mitochondrial biogenesis.3.Validation of clinical blood samples:QPCR was used to detect the expression level of mitochondria-related genes(TFAM,NRF2,STING)in blood samples of patients with chronic kidney disease,to further confirm the existence of such gene changes in patients with CKD and whether they were consistent with the above animal and cell experiments.Results:Animal experiments:1.We found that ZWD can effectively reduce UUO-induced renal damage and inhibit renal interstitial fibrosis.ZWD promotes oxidative phosphorylation(OXPHOS)by increasing the expression of mitochondrial transcription factor A(TFAM)and mitochondrial respiratory chain subunits.2.Electron microscope results showed that ZWD could also reduce mitochondrial membrane rupture and ridge disturbance,and improve mitochondrial biogenesis.3.The results of mitochondrial DNA extraction kit showed that ZWD inhibited mitochondrial DNA(mtDNA)leakage into the cytoplasm.4.Western blot showed that ZWD increased the total NRF2 expression,inhibited the transfer of NRF2 from cytoplasm to nucleus,and inhibited the NRF2-dependent STING pathway.5.Western blot and immunohistochemical analysis showed that Zhenwu Decoction could slightly improve renal tubule cell fibrosis and mitochondrial damage caused by high-dose folic acid induction combined with NRF2 inhibitor ML385,and inhibit the activation of STING pathway.6.We found that the anti-fibrosis effect of ZWD was mainly through protecting the integrity of damaged mitochondria and inhibiting the subsequent abnormal activation of the stimulant interferon gene(STING),while the combined intervention of ZWD and STING inhibitor C-176 did not produce additional renal protection.Cell experiments:1.TGF-β1 induced fibrosis in HK2 cells.Both Western blot and immunofluorescence showed increased expressions of NRF2 and STING in the nucleus,but decreased expressions of mitochondrial energy metabolism-related genes TFAM and OXPHOS.2.Mito-Tracker and mitochondrial copy number detection both showed that the number of mitochondria decreased after fibrosis in HK2 cells.3.Mitochondrial ROS and membrane potential tests also showed TGF-β1-induced oxidative stress and decreased mitochondrial membrane potential.4.Knockdown of NRF2 gene expression in HK2 cells by small interfering RNA(siRNA)revealed fibrosis-like changes and mitochondrial dysfunction in HK2 cells.5.STING inhibitor C-176 can reduce fibrosis and mitochondrial ROS production caused by NRF2 expression inhibition and improve mitochondrial membrane.Validation of clinical specimens:Blood samples from patients with chronic kidney disease were analyzed by QPCR method,and the mRNA expression levels of nuclear factor(erythrocyte derivation 2)related factor 2(NRF2)and TFAM were significantly decreased,while STING expression was increased.These molecular expression changes were consistent with UUO-induced mouse CKD model.Conclusion:1.ZWD improves mitochondrial biogenesis by maintaining mitochondrial membrane integrity and enhancing OXPHOS activity.2.ZWD improves mitochondrial bioenergy and inhibits fibrosis by increasing TFAM expression and inhibiting STING pathway dependent on NRF2 expression restriction.3.ZWD inhibits STING activation by restricting mtDNA leakage into cytoplasm,and enhances the expression of TFAM and OXPHOS by promoting the activation of NRF2.Altogether,ZWD represses renal fibrosis by maintaining mitochondrial integrity and optimizing bioenergetic capacity through NRF2 and TFAM activation.These findings provide unambiguous evidence that ZWD maintains mitochondria integrity and improves OXPHOS which represents an innovative insight of "strengthening Yang-Qi" theory. |
PDF Full Text Request