Mechanism Of Kidney-tonifying And Bone Marrow-nourishing Traditional Chinese Medicine In Preventing And Treating PMOP Based On The Regulation Of TGF-β1/Smads Pathway By Mir-133a-3p

Objective1.To screen differentially expressed genes related to the TGF-β1/Smad pathway in bone marrow stem cells(BMSCs)of postmenopausal osteoporosis(PMOP)patients through bioinformatics analysis,and to explore the correlation between the TGF-β1/Smads pathway and PMOP through clinical samples.2.To clarify the targeted regulatory relationship between miR-133a-3p and TGF-βRI,and to further explore the role of miR-133a-3p in regulating TGF-β1/Smads pathway in osteogenic differentiation of BMSCs by establishing lentiviruses with overexpression/silenced expression.3.To observe the effect of medicated serum containing kidney-tonifying and bone marrow-nourishing traditional Chinese medicine(TCM)on the osteogenic differentiation of BMSCs,and to investigate the role of TGF-β1/Smads pathway regulated by miR-133a-3p in promoting osteogenesis by kidney-tonifying and bone marrow-nourishing TCM.4.To clarify the osteoprotective effect of kidney-tonifying and bone marrow-nourishing TCM in preventing and treating PMOP,and to validate the involvement of miR-133a3p/TGF-β1/Smads signaling axis in the regulation process of bone protection.Methods1.Study on the correlation between TGF-β1/Smads pathway and PMOP based on clinical samplesHigh-throughput sequencing chips of BMSCs of PMOP patients and postmenopausal non-osteoporosis(OP)patients were obtained from the GEO database for bioinformatics analysis,to screen differentially expressed genes related to TGF-β1/Smads pathway and to analyze the mRNA expressions of differentially expressed genes.Meanwhile,bone tissue samples were collected clinically to detect the protein expressions of the screened differentially expressed genes using Western Blot,with the purpose to verify the reliability of the differentially expressed genes and to test the expressions of these genes in PMOP.2.The mechanism of miR-133a-3p regulating TGF-β1/smads pathway in promoting osteogenic differentiation of BMSCs(1)Targeted regulatory relationship between miR-133a-3p and TGF-βRIThe binding sites between the 3’-UTR region of the TGF-βRI and miR-133a-3p were predicted using an online bioinformatics tool.Then,the targeted binding relationship between miR-133a-3p and TGF-βRI was detected by dual luciferase reporter gene assay.(2)Role of miR-133a-3p in promoting osteogenic differentiation of BMSCs by regulating TGF-β1/smads pathwayAfter the construction of lentiviruses with overexpression/silenced expression,293T cells were packaged with these lentiviruses for the transfection of BMSCs.Then cells were divided into the negative control group(NC),miR-133a-3p overexpression group(vec-miR133a-3p),and silenced expression of the miR-133a-3p group(miR-133a-3p sponge).Simultaneously,a fresh osteogenic induction solution was added for cell culture.The relevant indexes were detected:①detection of ALP activity after the culture of BMSCs for 14 days;②alizarin red staining was performed after the culture of BMSCs for 21 days;③ the mRNA expressions of miR-133a-3p,TGF-β1,TGF-βRI,Smad2,Smad3 and Smad4 as well as Runx2,OPN,OCN were detected by qRT-PCR after the culture of BMSCs for 7 days;and④the protein expressions of TGF-β1 and TGF-βRI were detected by Western Blot.3.Mechanism of medicated serum containing kidney-tonifying and bone marrow-nourishing TCM in promoting osteogenesis in BMSCs through the regulation of TG F-β1/Smads pathway by miR-133a-3p(1)Effect of the medicated serum containing kidney-tonifying and bone marrownourishing TCM on osteogenic differentiation of BMSCsThe representative formula of kidney-tonifying and bone marrow-nourishing TCM,as well as calcitriol,and physiological saline were administrated by gavage in SD rats at equal volumes to prepare medicated serum containing kidney-tonifying and bone marrownourishing TCM,medicated serum containing calcitriol,and drug-free blank serum.Then,BMSCs were divided into the traditional induction group,blank control group(Control),kidney-tonifying and bone marrow-nourishing TCM group(BSTS),and calcitriol group(Cal).After the addition of an equal volume of BMSCs osteogenic induction medium,except for the traditional induction group,the other groups were supplemented with the prepared different medicated serums(equal volume of 12%)for cell culture.The relevant indexes were detected:①detection of ALP activity after the culture of BMSCs for 7 days and 14 days;and②alizarin red staining was performed after the culture of BMSCs for 21 days.(2)The role of medicated serum containing kidney-tonifying and bone marrownourishing TCM in promoting osteogenesis in BMSCs through the miR-133a-3p/TGF-β1/Smads signaling axisAccording to the method mentioned in(1),BMSCs were grouped and intervened with the prepared medicated serums.After cultivation,cells were collected for relevant testing:①the mRNA expressions of miR-133a-3p,TGF-β1,TGF-βRI,Smad2,Smad3 and Smad4 as well as Runx2,OPN,OCN were detected by qRT-PCR;and ②the protein expressions of TGF-β1,TGF-βRI,p-Smad2,p-Smad3 and Smad4 as well as Runx2,OPN,OCN were detected by Western Blot.4.Prevention and treatment of PMOP using kidney-tonifying and bone marrownourishing TCM through the regulation of TGF-β1/Smads by miR-133a-3pSD rats were divided into a sham operation group(Sham),model group(OVX),kidneytonifying and bone marrow-nourishing TCM group(BSTS),and alendronate sodium group(ALN).After successful modeling,samples were taken after 8 weeks of drug intervention.The relevant indexes were detected:①detection of the bone mineral content(BMC)and bone mineral density(BMD)of the femur of rats in each group;②micro CT scanning of the distal femur to calculate bone microstructure related indicators:trabeculae number(Tb.N),trabeculae separation(Tb.Sp),trabeculae thickness(Tb.Th),and relative bone volume(BV/TV);③observation of bone tissue morphology and structure by hematoxylin-eosin(HE)staining for the pathological study of the distal femur after scanning in ②;④the mRNA expressions of miR-133a-3p in bone tissues of each group was detected by qRT-PCR;and ⑤the protein expressions of TGF-β1,TGF-βRI,p-Smad2,p-Smad3 and Smad4 as well as Runx2,OPN,OCN were detected in bone tissues of each group by Western Blot.Results2.Study on the correlation between TGF-β1/Smads pathway and PMOP based on clinical samples(1)Eight high-throughput sequencing chips of BMSCs of postmenopausal female patients were obtained from the GEO database,including 4 PMOP patients and 4 postmenopausal non-OP patients.Through bioinformatics analysis and R language methods,there were differences in expressions of TGF-β1,Smad2,Smad3,Smad4,Runx2,and OPN genes,all of which were downregulated in the PMOP group(P<0.05).(2)Bone tissue samples were collected clinically for Western Blot.Compared with the control group(postmenopausal non-OP),the expression levels of TGF-β1,Smad2,Smad3,Smad4,and Runx2 proteins were significantly reduced in the PMOP group(P<0.05).Moreover,the expression of TGF-βRI was obviously downregulated,despite no statistical difference.2.The mechanism of miR-133a-3p regulating TGF-β1/smads pathway in promoting osteogenic differentiation of BMSCs(1)Targeted regulatory relationship between miR-133a-3p and TGF-βRI①The binding sites between the 3’-UTR region of the TGF-βRI gene and miR-133a-3p were discovered using an online bioinformatics tool.②Dual luciferase reporter gene assay confirmed that miR-133a-3p could targetedly regulate TGF-βRI expression.TGF-PRI was a target gene for miR-133a-3p,and miR-133a3p could inhibit TGF-βRI expression.(2)Role of miR-133a-3p in promoting osteogenic differentiation of BMSCs by regulating TGF-β1/smads pathway①ALP activity:Compared with the NC group,ALP activity was significantly decreased in the vec-miR-133a-3p group(P<0.05)and obviously increased in the miR-133a-3p sponge group(P<0.01).②Alizarin red staining:Compared with the NC group,BMSCs in the vec-miR-133a3p group showed a lighter color of alizarin red and significantly reduced calcium salt deposition;while those in the miR-133a-3p sponge group had a significant increase in calcium salt nodules.Meanwhile,compared with the vec-miR-133a-3p group,BMSCs in the miR-133a-3p sponge had the darker color of alizarin red and a significant increase in calcium salt deposition.③qRT-PCR results:Compared with the NC group,the expression of miR-133a-3p was highly increased(P<0.01),while those of TGF-β1,TGF-βRI,Smad2,Smad3 and Smad4 as well as Runx2,OPN,OCN were significantly decreased in vec-miR-133a-3p group(P<0.01).Meanwhile,compared with the NC group,the miR-133a-3p sponge group showed obviously downregulated miR-133a-3p expression(P<0.01),while remarkably upregulated expressions of TGF-β1,TGF-βRI,Smad2,Smad3 and Smad4 as well as Runx2,OPN,OCN(P<0.01).④Western Blot results:Compared with the NC group,the protein expressions of TGFβ1 and TGF-βRI were significantly downregulated in vec-miR-133a-3p(P<0.01),but obviously upregulated in miR-133a-3p sponge group(P<0.01).3.Mechanism of medicated serum containing kidney-tonifying and bone marrow-nourishing TCM in promoting osteogenesis in BMSCs through the regulation of TG F-β1/Smads pathway by miR-133a-3p(1)Effect of the medicated serum containing kidney-tonifying and bone marrownourishing TCM on osteogenic differentiation of BMSCs①ALP activity:After culture for 7 days and 14 days,ALP activity in the Control group was significantly reduced compared with the traditional induction group(P<0.01);while compared with the Control group,BSTS group and Cal group showed significantly enhanced ALP activity(P<0.01).②Alizarin red staining:BSTS group and Cal group showed the darkest color of alizarin red staining and significant calcium salt deposition,while the Control group had the least calcium salt deposition.Meanwhile,the Control group had significantly reduced calcium salt deposition compared with the traditional induction group;while in relative to the traditional induction group and the Control group,BSTS group and Cal group revealed significant staining and increased calcium deposition.(2)The role of medicated serum containing kidney-tonifying and bone marrownourishing TCM in promoting osteogenesis in BMSCs through the miR-133a-3p/TGF-β1/Smads signaling axis①qRT-PCR results:Compared with the traditional induction group,the Control group showed significantly upregulated expression of miR-133a-3p(P<0.01),but downregulated expressions of TGF-β1,TGF-βRI,Smad2,Smad3 and Smad4 as well as Runx2,OPN,OCN.Simultaneously,compared with the Control group,BSTS group and Cal group were detected with obviously downregulated expression of miR-133a-3p(P<0.01),while highly upregulated expressions of TGF-β1,TGF-βRI,Smad2,Smad3 and Smad4 as well as Runx2,OPN,OCN(P<0.01).②Western Blot results:Compared with the traditional induction group,the Control group had decreased protein expressions of TGF-β1,TGF-βRI,p-Smad2,p-Smad3 and Smad4 as well as Runx2,OPN,OCN(P<0.01).Meanwhile,compared with the Control group,BSTS group and Cal group showed obviously increased protein expressions of TGF-β1,TGF-βRI,p-Smad2,p-Smad3 and Smad4 as well as Runx2,OPN,OCN(P<0.01).4.Prevention and treatment of PMOP by kidney-tonifying and bone marrow-nourishing TCM through the regulation of TGF-β1/Smads by miR-133a-3p①BMC and BMD:BMC and BMD were significantly reduced in the OVX group than those in the Sham group(P<0.01).While compared with the OVX group,BSTS group and ALN group showed obviously increased BMC and BMD(all P<0.01).Inter-group comparison between the BSTS group and ALN group revealed no significant difference in BMC and BMD(all P>0.05).②Bone microstructure:OVX group had significantly decreased Tb.N,Tb.Sp and BV/TV than those in the Sham group(all P<0.01).Meanwhile,in relative to the OVX group,the BSTS group and ALN group were detected with obviously increased Tb.N,Tb.Sp and BV/TV(all P<0.05).While no significant difference in Tb.N,Tb.Sp and BV/TV was observed in the inter-group comparison between the BSTS group and ALN group(all P>0.05).③HE staining:The bone trabeculae in the Sham group were abundant,and arranged tightly and orderly.In the OVX group,bone trabeculae were sparsely distributed,with obvious fractures and disordered arrangement,and obviously widened spacing between bone trabeculae.Compared with the OVX group,the BSTS group and ALN group had a significant increase in the number of trabeculae and in trabecular thickness,but a reduction in trabecular spacing.④qRT-PCR results:The expression of miR-133a-3p was detected to be highly increased in the OVX group compared with the Sham group(P<0.01),while it was obviously decreased in the BSTS group compared with OVX group(P<0.01).⑤Western Blot results:Compared with the Sham group,OVX group had significantly downregulated protein expressions of TGF-β1,TGF-βRI,p-Smad2,p-Smad3,Smad4 and Runx2(P<0.01).While compared with the OVX group,the BSTS group showed highly increased protein expressions of TGF-β1,TGF-βRI,p-Smad2,p-Smad3,Smad4 and Runx2(P<0.01).Conclusion1.There are low expressions of TGF-β1/Smads pathway related genes as well as Runx2,OPN in bone tissue of PMOP patients.TGF-β1/Smads pathway may be involved in the pathogenesis of PMOP.2.miR-133a-3p can regulate the expression of TGF-βRI targetedly.Both overexpression and silenced expression of miR-133a-3p can regulate the expressions of TGFβ1/Smads pathway related genes and osteogenic differentiation of BMSCs.miR-133a3p/TGF-β1/Smads signaling axis may be an important regulatory mechanism that affects the progression of PMOP.3.The application of kidney-tonifying and bone marrow-nourishing TCM can promote the osteogenic differentiation of BMSCs.The mechanism may be that it can upregulate the expression of TGF-β1/Smads pathway related genes by downregulating the expression of miR-133a-3p,thus promoting osteogenesis.4.The application of kidney-tonifying and bone marrow-nourishing TCM can increase bone density and improve bone microstructure in the modeled rats with PMOP,thereby achieving the prevention and treatment of PMOP.Collectively,miR-133a-3p/TGF-β1/Smads signaling axis may be an important target pathway for the treatment of PMOP with kidneytonifying and bone marrow-nourishing TCM.