Bushen Jianpi Huoxue Recipe Treats PMOP Through Inhibiting Ferroptosis By Downregulating TLR4 Signal

Objective1.To explore the role of serological markers of ferroptosis in the pathogenesis of postmenopausal osteoporosis(PMOP)through clinical studies,so as to provide direction and basis for effective prevention and treatment of PMOP.2.Based on our clinical research,we applied bioinformatics methods combined with animal experiment to verify TLR4 signaling regulates ferroptosis involved in PMOP pathogenesis.3.Verification of Bushen Jianpi Huoxue Recipe in the treatment of PMOP by inhibiting ferroptosis via down-regulating TLR4 signaling.Methods1.Clinical studyA total of 80 postmenopausal women were divided into osteoporosis group(n=60)and non-osteoporosis group(n=20)according to the diagnostic criteria of osteoporosis.The general data and serum levels of GPX4,GSH and MDA were compared between the two groups.Spearman correlation analysis was used to analyze the correlation between each variable and bone mineral density.Random forest algorithm was used to evaluate the importance of each variable on the incidence of postmenopausal osteoporosis.ROC curve further confirmed the predictive efficacy of variables for PMOP.Finally,the PMOP prediction model is constructed.2.Bioinformatics combined with animal experiment to identify key signals of ferroptosis in PMOP(1)Bioinformatic analysisA qualified data set was obtained by searching GEO database and gene expression matrix was standardized.Firstly,differentially expressed gene analysis and weighted gene co-expression module analysis were carried out respectively to obtain differentially expressed genes and the genes in the module highly correlated with disease occurrence.Then,ferroptosis-related genes were obtained from FerDb database and GeneCards database.Finally,the differentially expressed genes,genes in the module highly related to disease occurrence and genes related to ferroptosis were intersected to obtain the ferroptosis-related genes that highly related to disease occurrence.Finally,GO functional enrichment analysis and KEGG pathway analysis were performed.(2)Construction of PMOP rat modelTwenty-four 6-month-old SD rats were randomly divided into 3 groups with 8 rats in each group:control group(CON group),SHAM group,and model group(OVX group).Ovariectomized rats were used to establish PMOP model.After 12 weeks,samples were collected and the following items were detected:①Bone mineral density was performed on the living rats;②Left femurs of the rats were isolated and distal femurs were scanned by microCT.③After②completion,HE staining was performed to observe the bone morphology of distal femurs.④Right femurs and left and right tibias of rats were isolated and cryopreserved.(3)Identification of ferroptosis phenomenon and key signaling in PMOP① RT-qPCR detected the expressed level of the nine disease highly relevant ferroptosis-related genes:ACVR1B,ANXA2,FSCN1,KRT18,MUC1,PANX2,SLC38A1,TF,TLR4.②Protein expression levels of ferroptosis markers ACSL4,GPX4,SLC7A11 and FTH1 in bone tissue was detected by Western Blot.3.Bushen Jianpi Huoxue Recipe treats PMOP by down-regulating TLR4 signal to inhibit ferroptosis in bone tissue(1)Inhibition of TLR4 signal saved MC3T3-E1 cells from ferroptosis induced by Erastin①The cell activity of MC3T3-E1 treated with Erastin and TLR4 inhibitor TAK-242 was detected by CellTiter-Glo assay at 1,3,5,and 7days after intervention;②MC3T3-E1 cells were treated with the screened drug concentration for 24h;③After intervention,total protein was extracted and the expression levels of ferroptosis markers GPX4,SLC7A11 and FTH1 as well as TLR4 were detected by Western Blot.(2)Bushen Jianpi Huoxue Recipe can prevent bone loss and effectively treat PMOPSixty 6-month-old SD rats were randomly divided into 6 groups with 10 rats in each group:control group(CON group),SHAM group,model group(OVX group),OVX+Bushen Jianpi Huoxue Recipe group(BSF group),OVX+Ferrostatin-1 group(FER group)and OVX+Alendronate sodium group(ALN group).Ovariectomized rats were used to establish postmenopausal osteoporosis model,and drug intervention started after 12 weeks.Samples were collected after another 12 weeks and the following items were detected:①Bone mineral density was performed on the living rats;② Left lower limbs of the rats were isolated and distal femurs and proximal tibias were scanned by microCT.③After ②completion,HE staining was performed to observe the bone morphology of distal femur.④Right lower limbs of the rats were isolated and cryopreserved.⑤Based on the microCT scanning results of②,the mechanical properties of distal cancellous bone of femur were analyzed by finite element method.(3)Bushen Jianpi Huoxue Recipe enhanced the expression of osteogenic protein in PMOP ratsThe expression level of Runx2 and Osterix in bone tissue were detected by Western Blot.(4)Bushen Jianpi Huoxue Recipe down-regulated TLR4 signal and inhibited ferroptosis in bone tissue of PMOP rats.The expression levels of TLR4 protein,ferroptosis markers GPX4,SLC7A11 and FTH1 were detected by Western Blot.Results1.Clinical study(1)There were no significant differences in height,weight and BMI between the two groups(P>0.05).The BMD,GPX4 and GSH levels in the non-osteoporosis group were higher than those in the osteoporosis group,while the age and MDA levels were lower than those in the osteoporosis group(P<0.05).(2)Spearman correlation analysis showed that with the increase of GPX4 level(R=0.42,P<0.05)and GSH level(R=0.43,P<0.05),the degree of osteoporosis decreased.With the increase of MDA level(R=-0.30,P<0.05),the degree of osteoporosis increased.With the decrease of GPX4 level(R=-0.30,P<0.05)and GSH level(R=0.42,P<0.05),MDA level increased.The levels of GPX4 and GSH showed a positive trend(R=0.43,P<0.05).(3)Random forest analysis showed that age,GPX4,GSH and MDA levels played an important role in the pathogenesis of PMOP.At the same time,ROC curve also confirmed this result,and found that the combined diagnosis of these four indicators can better predict the occurrence of PMOP.Based on these four variables,the nomogram we constructed can help identify high-risk PMOP populations.2.Bioinformatics combined with animal experiment to identify key signals of ferroptosis in PMOP(1)Bioinformatic analysisA total of 1078 differentially expressed genes were detected by differential expression analysis,including 505 up-regulated genes and 573 down-regulated genes.A number of 924 highly disease related genes were identified by WGCNA analysis.A total of 683 ferroptosis-related genes were identified by FerDb and GeneCards databases.Nine ferroptosis-related genes were obtained from the three:ACVR1B,ANXA2,FSCN1,KRT18,MUC1,PANX2,SLC38A1,TF,and TLR4.GO function results mainly revealed that the function of the nine key genes mainly affected the basic life activity forms of cells and acted on different cell microstructure.The results of KEGG pathway enrichment analysis highlight the important mediating role of TLR4 signaling in ferroptosis in PMOP.(2)Construction of PMOP rat modelAfter bilateral ovariectomy,the bone density of lumbar vertebrae decreased significantly,the bone microstructure of distal femur was destroyed,the number of bone trabeculae decreased significantly,and the bone marrow cavity was seriously fatty,indicating that PMOP rat model was successfully constructed.(3)Identification of ferroptosis phenomenon and key signaling in PMOPBy Western Blot,we found that ferroptosis-related proteins SLC7A11,GPX4 and FTH1 were significantly down-regulated and ACSL4 was significantly up-regulated in OVX group,indicating that ferroptosis occurred in bone tissue of postmenopausal osteoporosis rats.Second,we used RT-qPCR to find that TLR4 signal was highly expressed in OVX group rat bone tissue.3.Bushen Jianpi Huoxue Recipe treats PMOP by down-regulating TLR4 signal to inhibit ferroptosis in bone tissue(1)Inhibition of TLR4 signal saved MC3T3-E1 cells from ferroptosis induced by ErastinMC3T3-E1 cells were treated with Erastin to construct the ferroptosis model of postmenopausal osteoporosis.TLR4 protein expression was upregulated by Erastin.And the expression of SLC7A11,GPX4 and FTH1 protein was significantly up-regulated after the intervention of TAK-242,a TLR4 inhibitor.(2)Bushen Jianpi Huoxue Recipe can effectively protect bone loss and treat PMOPAfter treated by BSF,the overall bone mineral density,lumbar vertebra bone mineral density,right femur bone mineral density and right hip bone mineral density were significantly improved,and the bone microstructure of distal femur and proximal tibia were improved,the number of bone trabeculae was increased,and the fat infiltration in pulp cavity was alleviated.The effects of BSF were equivalent to Ferrostatin-1,the ferroptosis inhibitor and alendronate sodium.By finite element analysis,we found BSF can improve the mechanical parameters of bone trabeculae of cancellous bone in castrated rats,and the effect is similar to that of Ferrostatin-1 and alendronate sodium.(3)Bushen Jianpi Huoxue Recipe enhanced the expression of osteogenic protein in PMOP ratsWestern Blot showed that BSF,Ferrostatin-1 and alendronate sodium can increase the expressions of RUNX2 and Osterix proteins in the bone tissue of OVX-rats.(4)Bushen Jianpi Huoxue Recipe down-regulated TLR4 signal and inhibited ferroptosis in bone tissue of PMOP rats.Western Blot showed that BSF could significantly improve the expression of SLC7A11,GPX4 and FTH1,and down-regulate the expression of TLR4 in bone tissues of OVX-rats.Conclusion1.Age combined with peripheral blood GSH,GPX4 and MDA levels can well predict the occurrence of postmenopausal osteoporosis,and ferroptosis is an important pathogenesis of postmenopausal osteoporosis clinically.2.Bioinformatics analysis and in vivo experiments confirmed that TLR4-regulated ferroptosis mediated the occurrence of postmenopausal osteoporosis.3.Bushen Jianpi Huoxue Recipe can effectively treat postmenopausal osteoporosis by down-regulating the TLR4 signaling and inhibiting ferroptosis of bone tissue.