| Background:The kidney is an important target organ of hypertensive injury.When hypertensive kidney injury occurs,autophagy is mobilized for timely removal of damaged organelles and accumulated metabolites in the kidney cells and maintain the normal function of cells.However,with the progression of hypertensive kidney injury,the autophagy function becomes gradually abnormal,outstanding minent by impaired autophagy in renal tubules.Cordyceps sobolifera mycelium(CSM)is commonly used in the treatment of kidney diseases,and previous studies showed that its mechanism may be related to the regulation of SIRT1 expression and the regulation of autophagy.We hypothesized that CSM could regulate the acetylation level of TFEB by increasing SIRTI expression,thereby promoting lysosome biogenesis,dredging autophagy progression,and subsequently lightening hypertensive kidney injury.Objective:Autophagy plays an important role in the progression of chronic kidney disease(CKD),and regulating autophagy is expected to be a new idea to delay the progression of CKD.The proportion of kidney diseases caused by hypertension in CKD is increasing.Chinese medicine CSM is often used in the treatment of hypertensive nephropathy,and has achieved good curative effect,but its internal mechanism is not clear.In this study,we investigated the intrinsic mechanism of CSM delaying the progression of hypertensive nephropathy based on autophagy and lysosomal function.Methods:This study investigated thoroughly the molecular mechanism of hypertensive nephropathy from animal model and cell model.Spontaneous hypertensive rats(SHR)were selected as study subjects,and normal rats(Wistar Kyoto rat,WKY)were used as control.The spontaneously hypertensive rats were randomly divided into model group,CSM group,CSM group + nicotinamide(SIRT1 inhibitor)group and losartan(angiotensin Ⅱ receptor antagonist)group.The animals were killed at 36 weeks to collect tissue samples.Histomorphological changes and renal fibrosis were observed by HE and MASSON staining.The expression of autophagy-related proteins and the SIRT1-TFEB pathway was detected by Western Blot.We used TUNEL and detected the key apoptosis protein Claved-Caspase-3 by Western Blot to evaluate the cell apoptosis in each group.In vitro experiments 1,human renal tubular epithelial cells(HK-2)were divided into four groups:CON(negative control)group,ANG(angiotensin Ⅱ 10-7M)group,CSM(cicada containing serum+angiotensin Ⅱ)group,and LLO(LLOME(lysosomal membrane agent)+cicada containing serum+ angiotensin Ⅱ)group.After HK-2 cells were stimulated with Ang(10-7 mmol/mL)for 72 h,the following indicators were detected in each group of cells:①level of autophagy bubble generation:the protein expression of Beclinl,ATG 5 and ATG 7 was detected by Western Blot,the induction level of autophagosome was observed;and the induction level of autophagosome was observed;the ratio of LC3 Ⅱ to Ⅰ was measured by Western Blot to evaluate the membrane formation of autophagy bubbles;immunofluorescence co-localized LC3 and P62,and the wrapping of autophagy substrate was observed.②Autophagy bubble degradation level:the protein expression amount of LC3 and P62 were detected by Western Blot to evaluate the efficiency of autophagy turnover;"stubRFP-senseGFP-LC3" fluorescent labeled lentivirus transfection was constructed to detect autophagy flow and evaluate the clearance ability of autophagic bubble.In vitro experiment 2,human renal tubular epithelial cells(HK-2)were divided into four groups:CON(negative control)group,ANG(angiotensin Ⅱ 10-7 M)group,CSM(cicada containing serum+angiotensin Ⅱ)group,and TRE(TREHALOSE(trehalose,TFEB agonist)+AngⅡ)group.The following indexes of cells in each group were detected:①Lysosomal synthesis:Nuclear translocation of TFEB was observed by immunofluorescence and TFEB mediated lysosomal synthesis;immunofluorescence observed the distribution and quantity of lysosomal membrane marker protein LAMP 1 by immunofluorescence.②Lysosomal function:measure the number of bioactive lysosomes by Lyso Tracker,reflect the lysosomal biosynthesis;test the lysosomal enzymes CTSB,CTSD,CTSL,and evaluate the lysosomal digestion capacity.③SIRT1-TFEB pathway related detection:Western Blot detected the protein expression of SIRT1,cytoplasmic TFEB and TFEB in the nucleus,and measured the level of acetylated TFEB in the presence of immunoprecipitation,reflecting the nuclear translocation of TFEB and SIRT1-mediated TFEB.Finally,Image J software was used to analyze the pictures,and Graph Pad Prism software analyzed and collated the data and made statistical maps.Results:In animal experiments,compared with the normal group,model group’s blood pressure was significantly increased(P<0.01),but there was no significant difference between those treatment groups(P>0.05).HE staining showed that the model group had suboptimal morphological integrity compared with the normal group,structural damage was visible,and morphological damage was improved in the cicada group and losartan group.The MASSON staining results indicated that compared with the normal group,the collagen area in the tubular tissue of the model group was significantly increased(P<0.01),and the collagen area in the hypertensive rats was reduced after cicada treatment,with a statistically significant difference(P<0.01).TUNEL results suggested that the prportion of apoptosis in model group was higher than that in normal group(P<0.05),and the apoptosis in hypertensive rats was reduced after treattmen(P<0.05),it was consistent of the Western Blot results of Claved-Caspase-3.The Western Blot results showed that the levels of Beclinl,ATG 5 decreased and ATG 7 increased in the model group(P>0.05);compared with the normal group,LC3Ⅱ/Ⅰ and P62 levels increased in the kidney(P<0.05,P<0.01),while LC3Ⅱ/Ⅰand P62 in the CSM group were significantly decreased compared with those in the model group(P<0.01).Compared with the normal group,the expression of SIRT 1 in the kidney tissue was significantly decreased(P<0.01),and the expression of SIRT 1 and TFEB was increased compared with the model group(P<0.05,P<0.01).In Cell experiment 1,after 72 h of Ang Ⅱ stimulation of HK-2 cells,Western Blot found that ATG 5 protein expression increased in the ANG group compared with the CON group(P<0.05),indicating increased level of autophagosome induction;ATG 5 protein expression was decreased in CSM group compared with the ANG group(P<0.05);compared with CON group,the ANG group ratio increased(P<0.01),suggesting increased autophagy.Immunofluorescence co-localization LC3 and P62 found that compared with the CON group,a large number of positive spots appeared in the ANG group,while the colocalization spots in the CSM group decreased compared with the ANG group,and the LLO group showed a trend of increasing.Western Blot To detect the protein expression of LC3 and P62,we found that P62 increased in ANG group compared with CON group(P<0.05),indicating accumulation of autophagy substrate;LC3 increased(P<0.05),indicating increased number of autophagic bubbles.The detection of autophagic flow by stubRFP-senseGFP-LC3 suggested that the clearance was decreased in the ANG group compared with the decrease in the LLO group.In cell experiment 2,the nuclear transposition of TFEB by immunofluorescence suggested that TFEB transposition to nucleus increased in the ANG group,but decreased TFEB expression(P<0.01);TFEB decreased and increased TFEB expression in the CSM group compared to ANG group(P<0.01);TRE group and increased TFEB expression(P<0.01).LAMP 1 distribution and expression were observed by immunofluorescence,which indicated that LAMP 1 expression was decreased in ANG group compared to CON group(P<0.01);LAMP 1 expression was increased in CSM group compared to ANG group(P<0.01);and TRE group compared to ANG group(P<0.01).Lyso Tracker The results showed that the ANG group decreased compared to the CON group(P<0.01);the CSM group increased compared to the ANG group(P<0.01);the TRE group increased more than the ANG group(P<0.01),indicating increased lysosomal biosynthesis.The results of lysosomal cathepsin CTSB,CTSD and CTSL suggested that CTSB and CTSL expression were significantly decreased in ANG group compared with CON group(P<0.01),suggesting decreased lysosome digestion capacity in ANG group;CTSB and CTSL in CSM and TRE group improved compared with ANG group(P<0.05);CTSD expression in each intervention group compared with CON group,but there was no significant difference in CTD between ANG and CSM group and TRE group(P>0.05).Western Blot Detection of protein expression of SIRT 1,cytoplasmic TFEB and TFEB showed that SIRT 1 expression decreased in ANG group and TFEB(P<0.01);compared with ANG group,SIRT 1 expression was increased in ANG group(P<0.01);TFEB in IRE group(P<0.05),but SIRT I expression was not change compared with ANG group.Western Blot The level of acetylated TFEB showed that the proportion of acetylated TFEB to total TFEB increased in the ANG group compared to the CON group(P<0.01);the proportion of acetylated TFEB in the CSM group(P<0.01);and the proportion of acetylated TFEB compared to the ANG group(P<0.01).Conclusion:1.Cordyceps sobolifera myceliums regulating autophagy through the SIRT1-TFEB pathway alleviated renal injury in hypertensive rats.2.Cordyceps sobolifera myceliums promote the conversion of autophagic bubble from hypertensive renal tubular epithelial cells to autophagy-lysosome and improve lysosomal function through the SIRT1-TFEB pathway,thus delaying the progression of hypertensive kidney injury. |
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