| ObjectiveDiabetic nephropathy as a common and difficult to treat chronic microvascular complication of diabetes mellitus,is one of the leading causes of end-stage renal disease(ESRD).Its incidence is rising year by year worldwide over the past decades,has becoming one of the major causes of disability and death in patients with diabetes mellitus.The pathogenesis of DN is complex.Recently,it has been confirmed that inflammatory plays an important role in the occurrence and development of DN.A variety of inflammatory signaling pathways mediate the occurrence and development of DN,including TLR4 and its downstream NF-κB signaling pathways involved in inflammatory response,innate immunity and other biological effects.Stimulation such as high glucose and advanced glycation end products can activate TLR4,then activate the downstream NF-κB and other inflammatory pathways,which induced the expression of inflammatory cytokines and massive releasing inflammatory factor,eventually led to the occurrence of inflammation.Therefore,inhibition of inflammatory signaling pathway can delay the progression of DN.MicroRNA(miRNA)is a class of 21-25 nucleotide small RNAs which is excised from a stable hairpin-like secondary structure.miRNA belong to a novel family of highly conserved,short,non-coding,single-stranded RNA molecules that regulate transcriptional and post-transcriptional gene expression,and involve in a series of physiological and pathological processes.Recent studies have shown that miRNA is involved in the occurrence and development of DN,and can inhibit the expression of inflammatory cytokines to improve the progress of DN,but the exact mechanism is not clear.It has anti-inflammatory and immunosuppressive immune effects,which can not only act on T cells,dendritic cells,but also inhibit the secretion of inflammatory cytokines,adhesion molecules and chemokines.Study results showed that triptolide treatment effectively reduced albuminuria,and ameliorated podocyte foot process effacement and glomerular hypertrophy in diabetic rats.While the effect and possible mechanisms of Triptolide on renal tubuloinsterstitial injury in DN have not been fully established.Recent studies have shown that triptolide can exert pharmacological effects by regulating miRNA.Whether there is a correlation between Triptolide and miRNA in the inflammatory response of DN is also a question we need to discuss.The aim of this study was to investigate whether triptolide can reduce the inflammatory response of DN through miRNA,thus delaying the progress of DN.MethodsRat kidney tubular epithelial cells(NRK52E cells)were maintained in low glucose DMEM medium containing 10% FBS at 37℃ in a humidified incubator containing 5% CO2.NRK52 E cells were replaced culture medium every 2-3 days.When cultured to 80% fusion,digest NRK52 E cells with trypsin for passage or experiment.The logarithmic growth phase NRK-52 E cells were inoculated in 6 Hole cell culture plate for experiments at 1×105/ml density.Before each intervention,starved cells by replacing the culture medium with serum-free medium containing 0.2%BSA to ensure the cell synchronization.(1)The NRK-52 Ecells were divided into NG group(DMEM low glucose medium containing 5.5 mmol/L glucose),MA group(DMEM containing 5.5 mmol/L glucose and 19.5 mmol/L mannitol),HG group(DMEM containing 25 mM glucose,and HG+TP group(5 ng/m L triptolide in DMEM containing 25 mM glucose).Cells in each group were cultured in DMEM medium for 24 h.Using qRT-PCR and western-blot to detecte the mRNA and protein expression of TLR4,NF-κB,TGF-β1 and ICAM-1.The miRNA chip was used to screen the differentially expressed miRNA between the three groups and validated by qRT-PCR.(2)High glucose-induced NRK-52 E cells were transfected with mi R-224-3p mimics,divided into NG group,HG group,HG+miR-NC group(HG+mimics NC)、and HG+ mi R-224-3pm group(HG+miR-224-3p mimics).After transfection cells in each group were cultured for 48 h.qRT-PCR and western-blot wasused to detecte the mRNA and protein expression of TLR4,NF-κB,TGF-β1 and ICAM-1.(3)Luciferase reporter gene plasmid and miR-224-3p mimics were transfected into 293 T cells,and luciferase assays were performed to test whether miR-224-3p directly regulate the TLR4 expression by targeting the 3’UTR of TLR4 mRNA.(4)high glucose cultured NRK-52 E cells by Triptolide intervention were transfected with mi R-224-3p control and inhibitor,divided into n NG group,HG group,HG+TP group,HG+TP+mi R-i NC group(HG+ triptolide +inhibitor NC)、HG+TP+mi R-224-3pi group(HG+triptolide+mi R-224-3p inhibitor).After transfection 24 h add triptolide,cells were totol cultured for 48 h.q RT-PCR and western-blot wasused to detecte the m RNA and protein expression of TLR4,NF-κB,TGF-β1 and ICAM-1.Results(1)After NRK-52 E cells cultured in high glucose for 24 h,RT-PCR and western blotting showed that comparing with the NG group the m RNA and protein expression levels of TLR4,NF-κB,TGF-β1 and ICAM-1 significantly incressed in HG group(P<0.05),and comparing with the HG group the m RNA and protein expression levels of TLR4,NF-κB,TGF-β1 and ICAM-1 significantly decressed in HG+TP group(P<0.05).mi RNA chip analysis showed that mi R-224-3p expression was down-regulated clearly in the HG group comparing with the NG group(P<0.05),while in the HG+TP group mi R-224-3p expression was up-regulated obviously.We subsequently increased our sample size and utilized q PCR to verify the differences in mi R-224-3p expression in each group.Our results were consistent with the mi RNA microarray analysis data.(2)High glucose-induced NRK-52 E cells transfected with mi R-224-3p mimics and cultured for 48 h,RT-PCR and western blotting showed that comparing with the NG group the m RNA and protein expression levels of TLR4,NF-κB,TGF-β1 and ICAM-1 significantly incressed in HG group(P<0.05),and comparing with the HG group the m RNA and protein expression levels of TLR4,NF-κB,TGF-β1 and ICAM-1 significantly decressed in HG+ mi R-224-3pm group(P<0.05).(3)When mi R-224-3p overexpression vector was cotransfected with TLR4-3′UTR luciferase reporter construct,TLR4 luciferase activity of TLR4-3′UTR was significantly decreased,In contrast,no significantly changes in luciferase activity were observed in the mut-TLR4-3′UTR upon TLR4 overexpression or negative control.(4)HG+TP group NRK-52 E cells transfected with mi R-224-3p inhibitor,RT-PCR and western blotting showed that comparing with the NG group the m RNA and protein expression levels of TLR4,NF-κB,TGF-β1 and ICAM-1 significantly incressed in HG group(P<0.05),comparing with the HG group the m RNA and protein expression levels of TLR4,NF-κB,TGF-β1 and ICAM-1 significantly decressed in HG+TP group(P<0.05),and comparing with the HG+TP group the m RNA and protein expression levels of TLR4,NF-κB,TGF-β1 and ICAM-1 significantly incressed in HG+TP+ mi R-224-3pi group(P<0.05).Conclusions:(1)Triptolide attenuated high glucose-induced inflammation in NRK-52 E cells via decreasing the expression of TLR4,NF-κB,TGF-β1 and ICAM-1.(2)mi R-224-3p binding site within the 3’UTR of TLR4 inhibits TLR4 expression.Triptolide contributes to decrease TLR4 expression by upregulating mi R-224-3p to inhibit the inflammatory reaction in high glucose-induced NRK-52 E cells,moreover downregulate mi R-224-3p expression reduced the anti-inflammatory effects of triptolide. |
PDF Full Text Request