Role And Its Mechanism Of Melatonin On Mesangial Cells Stimulated By High Glucose Based On TLR4 Signaling Pathway

Background and purpose With the increase in the number of diabetic patients,the incidence of diabetic nephropathy(DN)has also been on the rise.It has now become an important cause of end-stage renal disease in China.Since diabetic nephropathy has complicated metabolic disorders,once it develops into end-stage renal disease,it is more difficult to treat than other kidney diseases.Therefore,timely prevention and treatment is of great significance for delaying diabetic nephropathy.Mesangial cells serve as the intrinsic cells of the kidney and have the functions of secreting cell matrix,producing cytokines,phagocytosis and elimination of macromolecular substances and similar to smooth muscle cell contraction.They play an important role in the development of diabetic nephropathy.TLRs are a class of receptors in the innate immune system that initiate inflammatory responses by detecting dangerous molecular patterns associated with pathogens.Studies have shown that TLR4 signaling plays an important role in diabetic nephropathy.Activation of TLR4 signaling pathway can initiate a series of inflammatory responses,and promote the production of inflammatory cytokines in immune cells or other innate cells in the kidney,thereby inducing the activation of TGF?1 signaling pathway.Promotes fibrosis in the kidneys,further exacerbating kidney damage.Melatonin(MT)is a quinone produced by the circadian rhythm of the pineal gland.It is one of the most effective antioxidants known,and it also has certain anti-inflammatory properties.In our study,MT was used to stimulate the mesangial cells stimulated by high glucose,and the relationship between TLR4 signaling pathway and renal inflammation and fibrosis was observed at the cellular and molecular level,and the mechanism of the development of TLR4 signaling pathway in DN was discussed.The intervention of MT and TLR4 signaling pathway provides new ideas for the prevention and treatment of DN.MethodsSV 40 purchased from the cell bank of the Chinese Academy of Sciences.The CCK-8 kit was used to examine the effects of MT intervention and high glucose stimulation on SV 40 activity.The experimental conditions were optimized by observing the changes of TLR4 protein expression under different high glucose stimulation time,different concentrations of high glucose stimulation,and different concentrations of MT intervention.Experimental group: Mannitol,normal control group(LG),normal control + MT group(LG+MT),high glucose group(HG),high glucose + 10 ?M MT group(HG +10 ?M MT),high glucose group(HG),high glucose + 100 ?M MT group(HG + 100 ?M MT),high glucose group(HG),high glucose + 1000 ?M MT group(HG + 1000 ?M MT),high glucose + TLR4 Inhibitor group(HG+TAK242)Cells and cell supernatants were collected at the corresponding stimulation time points.EdU kit was used to detect hyperglycemic-stimulated MT intervention,and the effect of TLR4 inhibitor on the proliferation of SV40.The expression of TLR4,ColIV and FN and the nuclear transfer of NF-?Bp65 in each group were observed under laser confocal microscope;ELISA kit The expression of IL-1?,TNF-?,MCP-1,ColIV and Fn in the cell culture supernatant of each group was detected;The expression of TLR4,IL-1?,TNF-?,MCP-1,Col IV and Fn mRNA was detected by Quantitative Real-time PCR;Western blot was used to detect total protein TLR4 and MyD88 in each group.Protein expression of Trif,p-IRF3,IRF3,I-?B,pI-?B,TGF?1,Smad3,pSmad3,ColIV and Fn.Results(1)Compared with the control group,the experimental use of MT had no significant effect on the high sugar stimulated SV40 activity(P>0.05).(2)The EDU method showed that high glucose can stimulate the proliferation of mesangial cells,MT intervention and TLR4 inhibitors can inhibit the proliferation of mesangial cells caused by high glucose stimulation.(3)Confocal laser scanning microscopy showed that there was no significant difference in the expression of TLR4,ColIV and Fn between the mannitol group and the LG+MT group,and there was no difference in nuclear transfer of NF-?B p65;high glucose stimulated the cell TLR4,ColIV and Fn.The expression of NF-?Bp65 was significantly increased,and the nuclear transfer phenomenon of NF-?B p65 was significantly increased.Both MT intervention and TLR4 inhibitors were downregulated by high glucose stimulation,and the inhibitory effect of MT was concentration-dependent.;(4)The results of ELISA showed that there was no significant difference in the levels of IL-1?,TNF-?,MCP-1,ColIV and Fn in the mannitol and LG+MT cell culture media compared with the LG group.(P<0.05)Compared with HG group,the levels of IL-1?,TNF-?,MCP-1,Col IV,and Fn in MT medium and TLR4 inhibitor group were significantly decreased(P<0.05),and MT intervention The degree is concentration-dependent.PCR assay results showed that compared with LG group,(5)there was no significant difference in mRNA expression of TLR4,IL-1?,TNF-?,MCP-1,Col IV and Fn in mannitol group and LG+MT group.(P<0.05)Compared with HG group,MT intervention and TLR4 inhibitor use could down-regulate the mRNA expression(P<0.05).(6)Western blot showed that compared with LG group,there was no significant difference in the expression of TLR4,MyD88,Trif,p-IRF3,pI-?B,TGF?1,Smad3,pSmad3,ColIV and Fn in mannitol group and LG+MT group.The expression of these proteins was significantly increased in the group(P<0.05).Compared with the HG group,MT intervention and use of TLR4 inhibitors significantly decreased the expression of the above proteins(P<0.05),and the extent of MT intervention was concentration-dependent.ConclusionsIn high glucose environment,the expression of TLR4 and downstream signals of SV40 is up-regulated,leading to increased expression of inflammatory factors,which leads to the activation of TGF?1 signaling pathway,resulting in increased secretion of extracellular matrix ColIV and Fn.MT intervention and application of TLR4 inhibitors could inhibit activation of these signaling pathways and down-regulation of extracellular matrix secretion in mesangial cells.