The Protective Mechanism Of Human Umbilical Cord Mesenchymal Stem Cells On Ferroptosis Of Renal Tubular In MRL/lpr Mice

Objective: To investigate the role of ferroptosis in renal injury of MRL / lpr lupus mice;to explore the therapeutic effect of human umbilical cord derived mesenchymal stem cells(UC-MSC)on MRL / lpr lupus mice and its effect on ferroptosis;To verify if uc-msc play a related biological function by secreting UC-MSC-MVs,produce therapeutic effect on MRL/lpr lupus mice,and can affect the ferroptosis phenomenon of lupus mice and HK-2 cell ferroptosis model.Materials and methods: The first part: the contents of GSH and MDA in renal tissue of mrl/lpr lupus mice and C57BL/6 mice were detected;TUNEL staining was used to detect cell death,and DAB enhanced Prussian blue staining was used to detect iron deposition.The expression of GPX4 and FTH1 in kidney tissues of mice was detected by immunohistochemistry,PCR and Westernblot.After intraperitoneal administration of iron death inhibitor liproxstatin-1,the contents of GSH and MDA in MRL/lpr lupus mice were compared,and the infiltration and damage degree of inflammatory cells in kidney tissue were compared.The second part: the human umbilical cord mesenchymal stem cells were identified,and the tail vein was injected with MSCs to treat MRL/lpr lupus mice;the spleen index,urine albμmin creatinine ratio and C3,C4,Ig G,ds DNA and ANA in serum of mice were measured by ELISA;The improvement of disease activity in rats was evaluated by HE,Masson and PAS staining.The expression of FTH1,GPX4 and NRF2 were measured by WB,PCR and immunohistochemistry.The cell death was detected by TUNEL staining.Prussian blue staining was used to evaluate the improvement of iron death.The third part: the starvation method was used to induce human umbilical cord mesenchymal stem cells to release particles,and the particles were extracted by gradient centrifugation;the particle structure was observed by transmission electron microscopy,and the diameter and concentration of the particles were identified by the tracking analysis of nanoparticles;the surface markers of the particles were identified by western blot,and the preparation of MSCs was used to treat the mice with MRL/lpr mice.The disease activity of mice was evaluated by measuring the spleen index,urinary albumin creatinine ratio,ELISA and the content of ds DNA,ANA,C3,C4,Ig G,flow cytometry,immunohistochemistry and PCR Whether the degree of renal tissue was improved;the inflammatory infiltration and damage of renal tissue were evaluated by HE staining,Masson staining and PAS staining;the expression levels of NRF2,GPX4,FTH1 in mouse kidney were detected by immunohistochemistry,PCR and Westernblot technique.TUNEL staining was used to detect the cell death,and the iron deposition was determined by using the method of DAB strengthening Prussian blue,and the improvement of iron death was evaluated.The transcription and protein expression of NRF2,GPX4 and FTH1 were detected by PCR and WB,and the apoptosis was detected by flow cytometry.Results: Compared with C57BL/6 mice,MRL/lpr mice had lower GSH level,higher MDA level,increased cell death and iron deposition in kidney tissue,and lower expression of GPX4 and FTH1.After treatment with ferroptosis inhibitor liproxstatin-1,GSH expression increased,MDA expression decreased,renal inflammatory cell infiltration and renal tubular injury improved in lupus mice.MSC can improve the disease activity of MRL/lpr lupus mice,reduce the spleen index,urinary microalbumin creatinine ratio,up regulate the expression of breg cells in spleen,reduce the infiltration of inflammatory cells in kidney tissue and renal tubular injury.MSC can improve the ferroptosis of renal tissue,increase the content of GSH,reduced the content of MDA.MSC can improve the expression of NRF2,increase the expression of GPX4 and decrease the expression of MDA.MVs extracted from MSC can also reduce the disease activity,increase the expression of breg cells in spleen,reduce the infiltration of inflammatory cells in kidney tissue and the damage of renal tubules,improve the iron death of renal cells,reduce the consumption of GSH,reduce the content of MDA,increase the expression of NRF2,and increase the expression of GPX4 and FTH1.In cell experiment,after the particles intervened in the erastin induced iron death model of HK-2 cells,the concentration of GSH increased,the concentration of MDA decreased,and the expression levels of NRF2,GPX4 and FTH1 increased,and also the apoptosis of cells was reduced.Conclusion: The study found that ferroptosis in MRL/lpr lupus mice may participate in the development of the disease;human cord mesenchymal stem cells and their particles can effectively improve the disease activity and renal injury of MRL/lpr lupus mice.MSCs may secrete particles in the way of paracrine,thus play a role in the treatment of MRL/lpr mice,the mesenchymal stem cell particles retain the biological functions of the parent cells from which they are derived.Mesenchymal stem cells and their secreted particles can effectively improve the ferroptosis of renal cells.It may be one of the mechanisms for improving renal tubule damage in lupus nephritis.In cell experiment also further confirmed that microparticles can improve the ferroptosis model of HK-2 cells induced by erastin;stem cells and microparticles can up regulate NRF2,reduce the depletion of GPX4,reduce the body’s lipid peroxidation level,increase the FTH1 content and antioxidant level,so as to play a protective role on the kidney,especially can effectively improve the damage of renal tubules,that is,UC-MSC may protect renal tubμles by inhibiting ferroptosis throμgh their MVs.