Molecular Mechanisms Of Yishen Qingli Huoxue Decoction In Improving Renal Aging And Fibrosis Based On The Regulation Of M~6A Methylation-mediated Autophagic Flux

Research background:Cellular senescence is a state of cycle arrest,phenotypic changes and active secretion of inflammatory factors,which can promote the occurrence and development of various aging-related diseases.Renal tubular epithelial cellular senescence is an important factor in promoting the progression of renal fibrosis(RF).However,the molecular regulatory mechanisms remain unclear.Autophagic flux injury blocks autophagosome degradation and leads to cellular senescence accelerating.Transcription factor EB(TFEB)is a key gene which regulates autophagic flux and plays a central role in the upstream of the"autophagosome-lysosome" pathway.Methylation of 6-methyladenosine(m6A)widely exists in various biological processes.The latest research shows that m6A has a variety of biological regulatory effects on aging-related diseases.However,the molecular regulation mechanism has not been known yet and there is also a lack of interventions of traditional Chinese medicine and western medicine.Based on the concept of "kidney protection and aging fighting",Professor Sun Wei comprehensively summarized the pathogenesis of kidney aging as "kidney deficiency,dampness and blood stasis",created the "Yishen Qingli Huoxue" method and applied his prescription("Yishen Qingli Huoxue decoction")to improve renal aging and RF.However,so far,the related molecular mechanisms of Yishen Qingli Huoxue decoction have been still unclear.Research purposes:1.To clarify the effects of Yishen Qingli Huoxue decoction in improving renal aging and RF;2.To reveal the molecular mechanisms of Yishen Qingli Huoxue decoction in delaying renal aging and RF through the regulation of m6A methylation-mediated autophagic flux.Research method:1.Network pharmacology research:Firstly,using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)database and literature search to collect the ingredients and targets of Yishen Qingli Huoxue decoction,and using Cytoscape software to construct the active ingredients-targets network of single herbs and drug-ingredient-target network of Yishen Qingli Huoxue decoction.Secondly,the RF(or CKD)related molecular targets are collected in the GeneCards database,and the common targets of traditional Chinese medicine prescriptions and diseases are screened through the Xiantao Shengxin analysis website.By analyzing the GO/KEGG pathway enrichment of common targets of prescriptions and diseases,a protein interaction network was constructed and key targets were screened.Finally,molecular docking verification of important active ingredients and key targets were carried out using the website of Protein Data Bank(PDB).2.Experimental research:In vivo,"Unilateral nephrectomy + D-gal intraperitoneal injection" was used to establish the renal aging and RF models,and 30 rats were randomly divided into 4 groups.The specific groups and interventions are as follows:6 rats in the sham operation group were given intraperitoneal injection of normal saline 6 ml/kg daily;12 rats in the D-gal group were intraperitoneally injected with D-gal 300mg/kg daily;6 rats in the Yishen Qingli Huoxue decoction group were given by gavage of Yishen Qingli Huoxue decoction granule suspension 2.745g/kg combined with intraperitoneal injection of D-gal 300mg/kg daily;6 rats in the vitamin E(VE)group were given by gavage of 30mg/kg VE combined with intraperitoneal injection of D-gal 300mg/kg daily.Before modeling and after drug intervention,the general condition,renal function and body weight of rats were regularly observed.In addition,Sirius red staining was used to observe the level of collagen deposition in rat kidneys;Immunohistochemistry or immunofluorescence staining were used to observe the anti-aging protein Klotho,autophagy-associated protein microtubule-associated protein 1 light chain 3(LC3),autophagy protein Beclinl and autophagy adaptor protein(Sequestosome-1,SQSTM1),as well as methyltransferase-like 3(Methyltransferase-like 3,METTL3)distribution and abundance in kidney;Senescence-related β-galactosidase(Senescence-associated β-galactosidase,SA-β-gal)staining was used to analyze the number of senescent cells;Western Blot(WB)was used to detect Klotho,senescence cyclins P27 and P16,senescence-associated secretory phenotype(SASP)inflammatory factors interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and transforming growth factor-β(TGF-β),RF markers collagen-Ⅰ(Col-Ⅰ),α-Smooth muscle actin(α-SMA),methyltransferase METTL14 and wilms tumor 1-associated protein(WTAP)in rats’ kidneys;qRT-PCR and WB were used to observe the expression levels of TFEB and METTL3;Transmission electron microscopy was used to observe the number of autophagosomes in cells;EpiQuikTM m6A RNA methylation quantitative kit was used to detect the overall level of m6A in rats’ kidney tissues.In vitro,using D-gal to construct NRK-52E cellular senescence and RF models,the specific groups and interventions are as follows:control serum group(10%blank serum group),model group(D-gal 200mM),low-dose of Yishen Qingli Huoxue decoction-containing serum group(D-gal 200mM+5%Yishen Qingli Huoxue decoction-containing serum),middle-dose of Yishen Qingli Huoxue decoction-containing serum group(D-gal 200mM+10%medicated serum of Yishen Qingli Huoxue decoction-containing serum),and VE group(D-gal 200mM+VE 50μg/mL).Effects of D-gal,Yishen Qingli Huoxue decoction-containing serum and VE on the activity of NRK-52E cells were detected by CCK-8.Observation of senescence changes in NRK-52E cells was detected by SA-β-gal staining.The protein expression levels of Klotho,P27,P16,IL-6,TNF-α,TGF-β,Col-Ⅰ,α-SMA,METTL14 and WTAP in NRK-52E cells were detected by WB.The expression levels of TFEB and METTL3 were observed by qRT-PCR and WB.Klotho silenced cell lines,TFEB overexpressed cell lines,and METTL3 silenced cell lines were constructed.After the cell lines were constructed,qRT-PCR and WB were used to detect the results of cell line construction.The EpiQuikTM m6A RNA Methylation Quantification Kit was used to detect the overall level of intracellular m6A,and the EpiQuikTM m6A Methylated RNA RIP Kit was used to detect the binding level of TFEB to m6A.Results:1.Network pharmacology:Yishen Qingli Huoxue decoction in treating RF is a multi-component and multi-target network regulation mechanism.Pathway enrichment analysis of common targets of prescriptions and diseases found that multiple cellular oxidative stress-related pathways,inflammation and inflammatory factor-related pathways,and senescence or cellular senescence-related pathways were enriched,which was highly consistent with the disease characteristics of RF.KEGG analysis enriched cellular senescence,phosphatidylinositol-3 kinase(PI3K),protein kinase B(AKT),mitogen-activated protein kinase(MAPK)etc.All these signaling pathways play important roles in RF.Molecular docking results suggested that the active ingredients of Yishen Qingli Huoxue decoction,including quercetin,kaempferol and baicalein,could effectively bind to their target genes TP53 and AKT1.2.Experimental study:In vivo:1)For the rats’renal aging and RF model induced by"unilateral nephrectomy+D-gal intraperitoneal injection",Yishen Qingli Huoxue decoction and VE can maintain basal body weight,reduce BUN level and the number of blue-positive senescent cells in renal tissues,up-regulate Klotho protein expression,down-regulate P27,P16,IL-6,TNF-α,TGF-β,Col-Ⅰ,and α-SMA protein expressions,and inhibit the renal tubular peripheral collagen deposition;2)Yishen Qingli Huoxue decoction can repair the autophagic flux injury in rats’ kidney tissues induced by "unilateral nephrectomy+D-gal intraperitoneal injection",and restore the protein expressions of LC3 Ⅱ,Beclinl and phosphorylated SQSTM1 to the basic levels;3)Yishen Qingli Huoxue decoction inhibits the activity of METTL3,reduces the m6A methylation modification level of TFEB mRNA,increases the expression of TFEB at the transcriptional and protein levels,and improves the level of autophagic flux,thereby delaying aging and fighting RF.In vitro:1)D-gal induces senescence and RF in NRK-52E cells,and the intervention of D-gal 200mM for 24h is the most significant;2)Yishen Qingli Huoxue decoction-containing serum and VE can up-regulate the protein expression of Klotho,reduce the protein expressions of P27,P16,Col-I and α-SMA,inhibit the release of SASP factors,and reduce the positive staining rate of SA-β-gal.The treatment of serum containing Yishen Qingli Huoxue decoction was mostly significant at 10%concentration for 24 hours,and the VE at 50μg/mL was the most significant at 24 hours;3)In NRK-52E cells with Klotho protein knockdown,the expression levels of Col-Ⅰ and TGF-β proteins were increased,but did not change significantly after the intervention of Yishen Qingli Huoxue decoction-containing serum;4)Yishen Qingli Huoxue decoction-containing serum and VE can repair D-gal-induced autophagic flux injury and accelerate the degradation of LC3 Ⅱ,Beclin1,and p-SQSTM1 proteins;5)Yishen Qingli Huoxue decoction-containing serum and VE can eliminate intracellular m6A methylation modification,reduce the binding amount of TFEB mRNA and m6A,and restore the mRNA transcription level and protein expression level of TFEB;6)The inhibition of intracellular m6A methylation modification by Yishen Qingli Huoxue decoction-containing serum relies on METTL3 protein.By reducing the activity of METTL3,inhibiting intracellular m6A methylation modification and upregulating the protein expression level of TFEB,it can play a role in repairing impaired autophagic flux and delaying aging and fighting RF.Conclusion:1)"Unilateral nephrectomy+D-gal intraperitoneal injection" can establish renal aging and RF animal model,and D-gal intervention at 200mM for 24h can establish NRK-52E cellular senescence and RF cell model;2)Yishen Qingli Huoxue decoction and its drug-containing serum can delay renal aging and fighting RF in vivo and in vitro;3)Yishen Qingli Huoxue decoction and its drug-containing serum can repair damaged autophagic flux in vivo and in vitro;4)The potential molecular mechanisms of Yishen Qingli Huoxue decoction and its drug-containing serum are accomplished by regulating the level of m6A methylation on TFEB mRNA related to autophagic flux to delay renal aging and fighting RF;5)Yishen Qingli Huoxue decoction-containing serum inhibits intracellular m6A methylation modification is accomplished by METTL3 protein.