| BACKGROUND&OBJECTIVEStem cells have a good effect on DMED rats,and the decrease of CCSM cells is an important reason for DMED.Is there any other way of cell death in the penile tissue of DMED patients?The main purpose of this study is to explore whether there is ferroptosis in the penis of DMED rats,and to verify whether ADSCs reverses ferroptosis through some mechanism.To provide ideas for the new research direction and treatment of DMED.METHODS1.Primary ADSCs cells were extracted,surface markers were identified by flow cytometry,differentiation ability was identified by osteogenic and adipogenic induction experiments;DMED rat model was established and treated with ADSCs cells.smooth muscle content was detected by HE and Masson staining.Ferroptosis was detected by WB,and GSH/GSSG was determined.2.The primary CCSM cells were extracted and the purity was identified by immunofluorescence.The ferroptosis model of CCSM cells was screened and constructed by CCK8.The control group and the experimental group were screened and analyzed by cytokine chip.3.DMED rats were injected into corpus cavernosum and divided into PBS,ADSCs,NRP1 and ADSCs+NRP1 groups.Determination of ICP/MAP in each group.Immunofluorescence was used to detect NRP1,GPX4,SLC7A11 and ASCL4.Determination of GSH/GSSG in each group.RESULTS1.Flow cytometry showed that the cultured ADSCs with high expression of CD29,CD90 and low expression of CD45,conformed to the characteristics of ADSCs.Osteogenesis and adipogenesis showed that ADSCs have differentiation ability.HE and Masson staining showed that the content of penile smooth muscle of DMED rats decreased.WB suggested that the expression of GPX4 and SLC7A11 decreased significantly,ASCL4 expression increased significantly in DMED rats.GSH/GSSG decreased significantly in DMED rats.All of the above can be significantly reversed by ADSCs cell therapy.2.Immunofluorescence shows that high purity CCSM cells were extracted,and the best conditions for ferroptosis of CCSM cells induced by erastin were 5umol/L and 24 hours by CCK-8.The ferroptosis model of CCSM cells was successfully constructed.WB showed that the expression of GPX4 and SLC7A11 decreased significantly and the expression of ASCL4 increased significantly after treatment of CCSM cells,which accorded with the characteristics of ferroptosis.Co-culture with ADSCs can significantly inhibit ferroptosis,suggesting that ADSCs can secrete some factors to inhibit ferroptosis.Cytokine chip screening found 48 differential factors(18 low expression,30 high expression).Bioinformatics analysis found that NRP1 may be related to the improvement of ED after ADSCs treatment,so it was selected for further animal experiments.3.ICP/MAP suggested that intracavernous injection of ADSCs and NRP1 could improve the effect of DMED.The effect of NRP1 group was weak,while that of ADSCs+NRP1 group was obvious.Immunofluorescence showed that ADSCs+NRP1 could overexpress NRP1,and reverse the expression of GPX4,SLC7A11 and ASCL4 in vivo.GSH/GSSG suggested that ADSCs,NRP1 and ADSCs+NRP1 could increase the content of GSH in penile tissue,especially in ADSCs+NRP1 group.CONCLUSION1.Ferroptosis of CCSM cells exists in the corpus cavernosum of DMED rats was confirmed for the first time.ADSCs cell therapy can inhibit ferroptosis of CCSM cells and improve ED.2.We successfully constructed a model of ferroptosis in CCSM cells induced by erastin.Co-culture with ADSCs could significantly inhibit ferroptosis in CCSM cells.Through cytokine chip screening and analysis,it is found that NRP1 may be related to the improvement of ED after ADSCs treatment.3.Through further animal experiments,we found that ADSCs+NRP1 can reverse ferroptosis and enhance the antioxidant capacity of CCSM cells.Therefore,we believe that NRP1,as an important cytokine secreted by ADSCs,can inhibit ferroptosis in CCSM cells,and may play an important role in the treatment of DMED with ADSCs,but its specific mechanism needs to be further explored. |
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