Exploring Shen Kang Decoction Inhibition Of AGEs-induced EMT In Renal Tubular Epithelial Cells Based On JMJD1A/NR4A1 Signaling Pathway

BackgroundDiabetic nephropathy(DN)is one of the most common and serious microvascular complications in diabetic patients.Sheng-Kang Decoction has anti-inflammatory and antioxidant as well as hypoglycemic effects,but the exact mechanism of action is not clear.Studies have shown that epithelial-mesenchymal transition(EMT)occurs in renal tubular epithelial cells as an important mechanism for the development of renal tubular interstitial fibrosis,and the JMJD1A/NR4A1 signaling pathway is closely related to the development of EMT.Therefore,this experiment was conducted to explore the prevention and treatment mechanism of DN from the perspective of this pathway with Sheng-Kang Decoction.Methods1.An in vitro model was established using advanced glycation end products(AGEs)stimulation of renal tubular epithelial cells(HK-2)to determine the concentration of AGEs stimulation as well as the duration of action.2.Fluorescence quantitative PCR,protein blotting(Western blot)and flow cytometry were used to detect the EMT and apoptosis of HK-2 cells induced by AGEs in Shen-Kang Decoction drug-containing serum.3.RNA-seq was used to screen epigenetic factor JMJD1A.4.Fluorescence quantitative PCR,protein blotting(Western blot),and immunofluorescence techniques were used to detect the expression levels of JMJDIA and fibrogenic factors in HK-2 cells induced by AGEs.5.JMJD1A silencing/overexpressing HK-2 stably transfected cell lines were constructed using lentivirus transfection;and the expression levels of JMJD1A and fibrogenic factors in JMJD1A silencing/overexpressing HK-2 stably transfected cell lines induced by AGEs were detected by fluorescence quantitative PCR and Western blot with Shen-Kang Decoction drug-containing serum.6.Screening for the epigenetic factor JMJD1A downstream transcription factor NR4A1 using RNA-seq.7.Western blot was used to detect the expression of NR4A1 in AGEs induced HK-2 and the expression level of EMT-related factors after the adoption of NR4A1 inhibitor.8.The JMJD1A/NR4A1 signaling pathway as well as CTGF,TGF-β1,Vimentin,a-SMA,Collagen I,FN mRNA and protein expression in HK-2 cells were detected by applying Shen-Kang Decoction drug-containing serum.Results1.Different concentrations of AGEs induced an increase in CTGF,TGF-β1 expression in HK-2 cells at 48 and 72 hours;400ug/ml of AGEs on HK-2 cells for 72 hours increased the level of apoptosis;HK-2 cells showed myofibroblast structure with prolonged stimulation time,suggesting that AGEs can induce EMT in HK-2 cells.fluorescence quantification PCR showed that the expression levels of CollagenⅠ、Vimentin、CTGF were reduced in the Shen-Kang Decoction drug-containing serum group compared with the AGEs group,and the differences were statistically significant.2.The epigenetic factor JMJD1A was screened from ctrl group,AGEs group and AGEs+SKF group by high-throughput sequencing technology.3.The results of fluorescence quantitative PCR,Western blot,and immunofluorescence showed that the expression levels of JMJD1A,CTGF,TGF-β1,Vimentin,a-SMA,Collagen I,and FN were increased in the AGEs group compared with the Ctrl group,and the gene expression was decreased after the intervention of the Shen-Kang Decoction drug-containing serum.4.JMJD1A silencing HK-2 stable cell was successfully constructed;the expression of fibrogenic factors after JMJD1A silencing was detected by applying fluorescence quantitative PCR,Western blot,and immunofluorescence techniques,and the results showed that the expression levels of CTGF,Vimentin,and TGF-β1 were significantly down-regulated compared with the null group,and the differences were statistically significant.5.The results of fluorescence quantitative PCR showed that JMJD1A expression was up-regulated,suggesting the successful construction of JMJD1A overexpression HK-2 stable cell line;the expression of fibrogenic factors after JMJD1A overexpression was detected by applying fluorescence quantitative PCR,Western blot,and immunofluorescence techniques,and the results showed that the expression levels of CTGF,Vimentin,and TGF-β1 were significantly down-regulated compared with the null group.were significantly down-regulated compared with the null group,and the differences were statistically significant.6.High-throughput sequencing technology was applied to screen the downstream transcription factor NR4A1 of JMJD1A by Ctrl_AGEs group,SKF_AGEs group and shJMJD1 A_SKF_AGEs group;the results of fluorescence quantitative PCR and Western blot assay showed that NR4A1 downregulation in AGEs-induced HK-2 cells could increase the expression level of CTGF,Vimentin,Collagen Ⅰ expression levels.7.Shen-Kang Decoction drug-containing serum can reduce CTGF,TGF-β1,Vimentin,a-SMA,Collagen I,FN expression by down-regulating the expression level of JMJD1A/NR4A1.ConclusionIn summary,we found that JMJD1A/NR4A1 signaling pathway is involved in the EMT process induced by AGEs in HK-2 cells.Shen-Kang Decoction drug-containing serum inhibited the fibrosis process of renal tubular epithelial cells by regulating the JMJD1A/NR4A1 signaling pathway,which has certain reference value and significance for the treatment of renal diseases.