| Research backgroundRheumatoid arthritis(Rheumatoid Arthritis,RA)is mainly characterized by inflammation ofjoint synovium and progressive destruction of cartilage and bone.With the use of magnetic resonance imaging,more and more studies have found that RA bone marrow edema(Bone Marrow Edema,BME)is an important part of inflammatory reaction,so this study takes this as a starting point to observe the effect of bone marrow fluid on CD4+T lymphocyte differentiation and osteoclast(Osteoclasts,OC)differentiation and fuction.ObjectiveIn this study,from the point of view of bone marrow microenvironment/BMECD4+T lymphocyte-Th17/Treg on the differentiation and function of OC,to explore the mechanism of Yishen Juanbi Pill(YSJB)in improving bone destruction of RA and the role of kidney deficiency in the occurrence and development of RA.Methods1 Preparation and detection of YSJB bone marrow fluidSixty 8-week-old female SD rats were randomly divided into 8 groups:blank control group,sham operation(Sham)group,Sham+collagen induced arthritis(CIA)group,Sham+CIA+Yi shen Juan bi pill(YSJB)group,ovariectomized(OVX)group,OVX+CIA group,OVX+CIA+YSJB group and OVX+CIA+methotrexate(MTX)group.The mice were immunized 28 days after castration,and intragastric administration began 14 days after the first immunization.The rats in OVX+CIA+MTX group were injected intraperitoneally with MTX at a dose of 1.5 mg/kg body weight The rats in Sham+CIA+YSJB group and OVX+CIA+YSJB group were given YSJB twice a day for 5 days at a dose of 2.60 g/kg body weight.The rats in the blank control group,Sham group,OVX group,Sham+CIA group and OVX+CIA group were fed with the same volume of pure water.The preparation method of medicated bone marrow fluid:the cleaned bilateral femurs and tibia of rats in each group were washed with α-MEM medium,then centrifuged 5min at the speed of 330g,and the supernatant was frozen at-80℃.2 Effect of Yishen Juanbi Pill Bone Marrow fluid on OC cultured in VitroUnder aseptic conditions,t the bone marrow cells of femur and tibia of 6-weekold C57BL/6 mice were cultured in a-MEM complete medium for 24 hours.The unattached cells were resuscitated in α-MEM complete medium containing 25 ng/mL M-CSF,and inoculated in 96-well culture plate and 96-well culture plate with bone slices(4×104/well)and 6-well culture plate(8×105/well).After 72 hours,the supernatant was discarded and cultured to induce with the differentiation medium(10%FBS+100 U/mL penicillin+100 μg/mL streptomycin+25 ng/mL M-CSF+50 ng/mL RANKL α-MEM medium).At the same time,5%bone marrow fluid was added and changed every other day.After 4 days of drug intervention,the differentiation of OC was observed by TRAP staining.The expression of NFATC-1,CTSK and MMP-9 mRNA and NFATC-1,CTSK and MMP-9 protein in OC was detected by qPCR and Western-Blot method,and the area of bone resorption lacunae was detected by toluidine blue staining after 8 days of drug intervention.3 Effect of Yishen Juanbi Pill bone marrow fluid on the differentiation of CD4+T lymphocytesUnder aseptic condition,the spleen of 6-week-old C57BL/6 mice of SPF grade was removed and the cell suspension was prepared.CD4+T cells were obtained by magnetic beads sorting.The sorted CD4+T cells were adjusted to a concentration of 1×106 cells/ml.α-MEM complete medium containing CD28 antibody was resuscitated and inoculated in a 48-well plate coated with Anti-CD3 and incubated at 37℃ for 2 h.After 2 hours,5%bone marrow fluid was added,and after 2 days of culture,the cells of each group were collected,and the expression levels of related transcriptional regulatory factors(RORγt,TNF-α,IL-17,Foxp3,TGF-β,IL10)mRNA in CD4+T cells were detected by qPCR.The protein levels of TNF-α,IL-17A,IL-10 and TGF-β in the culture supernatant of each group were detected by ELISA method,and the differentiation of CD4+T lymphocytes into Th17 cells and Treg cells was analyzed.4 Effect of Yishen Juanbi Pill Bone Marrow fluid on CD4+T-OC Co-culturesystemBone marrow-derived OC was isolated according to the above method,and after 72 hours of culture,it was replaced with α-MEM differentiation medium.At the same time,5%bone marrow fluid and 2×104 spleen CD4+T lymphocytes activated according to the above method were added,and the fluid was changed every other day.After 4 days of induction,the OC was observed and counted by TRAP staining,the protein expression of NFATC-1,CTSK and MMP-9 of OC was detected by immunohistochemical method.The supernatant of cell culture was collected and the levels of cytokines such as TNF-α,IL17A,TGF-β and IL-10 were detected.After 8 days of induction,the area of bone resorption lacunae was detected by toluidine blue staining.5 Effect of tetrahydropalmatine(THP)on OC differentiation and functionThe drug components in YJSB bone marrow solution were detected by high performance liquid chromatography and mass spectrometry OC derived from bone marrow was isolated and cultured according to the method according to the above method.72 hours later,different concentrations of THP(final concentration of 0.625,1.5625,3.125,6.25,12.5 μg/ml)were added,and a blank control group(Ctrl)without THP was set up.The fluid was changed every 2 days.After 24 days of induction,the OC was observed and counted by TRAP staining,the mRNA levels of NFATC-1,CTSK and MMP-9 in OC were detected by qPCR square method,the expression of NFATC1,CTSK and MMP-9 protein in OC was detected by Western-Blot method,and the area of bone resorption lacunae was detected by toluidine blue staining after 8 days of induction.Results1 Effect of Yishen Juanbi Pill bone marrow fluid on differentiation and function of OC1.1 Effect of Yishen Juanbi Pill Bone Marrow liquid on OC differentiation and Bone erosion caused by OCCompared with Sham group,the number of OC and the area of bone resorption lacunae in Sham+CIA group,OVX group and OVX+CIA group increased significantly.there was no significant difference between OVX group and Sham+CIA group,and the number of OC and the area of bone resorption lacunae in OVX+CIA group increased significantly compared with OVX group and Sham+CIA group(P<0.01).The number of OC and the area of bone resorption lacunae in Sham+CIA+YSJB group was significantly lower than that in Sham+CIA group,and the number of OC and the area of bone resorption lacunae in OVX+CIA+YSJB group and OVX+CIA+MTX group was significantly lower than that in OVX+CIA group(P<0.01).1.2 Effect of Yishen Juanbi Pill Bone Marrow fluid on the expression of OC NFATC-1,CTSK and MMP-9 mRNACompared with Sham group,the expression of NFATC-1 mRNA in Sham+CIA group tended to increase,but there was no statistical difference.The expression of NFATC-1,CTSK and MMP-9 mRNA in OVX group and OVX+CIA group increased significantly(P<0.05,P<0.01).The expression of NFATC1,CTSK and MMP-9 mRNA in OVX+CIA group was significantly higher than that in Sham+CIA group and OVX group(P<0.01),Compared with Sham+CIA group,the expression of NFATC-1 mRNA in Sham+CIA+YSJB group decreased,but there was no statistical difference,and the expression of CTSK and MMP-9 mRNA decreased significantly(P<0.05,P<0.01).Compared with OVX+CIA group,the expression of NFATC-1 and CTSK mRNA in OVX+CIA+YSJB group and OVX+CIA+MTX group decreased significantly.1.3 effect of Yishen Juanbi Pill bone marrow fluid on the expression of OC NFATC1,CTSK and MMP-9 proteinsCompared with Sham group,the expression of NFATC-1 and MMP-9 protein increased in Sham+CIA group tended to increase,but there was no statistical difference;The expression of CTSK protein in Sham+CIA group was significantly higher than that in control group(P<0.05);The expression of MMP-9 protein in OVX group tended to increase,but there was no statistical difference;The expression of NFATC-1 and CTSK protein was significantly increased in OVX group(P<0.05,P<0.01).The expression of NFATC-1、CTSK and MMP-9 protein was significantly increased in OVX+CIA group(P<0.01).Compared with Sham+CIA group,the expression of NFA TC-1,CTSK and MMP9 protein in OVX+CIA group was significantly higher than that in OVX+CIA group(P<0.05).Compared with OVX group,the expression of NFATC-1,CTSK and MMP-9 protein in OVX+CIA group was significantly higher than that in OVX+CIA group(P<0.05).The expression of NFATC-1 protein in Sham+CIA+YSJB group was significantly lower than that in Sham+CIA group P<0.05).The expression of CTSK and MMP-9 protein had a downward trend,but there was no statistical difference.Compared with OVX+CIA group,the expression of NFATC-1,CTSK and MMP9 protein in O VX+CIA+YSJB group and OVX+CIA+MTX group further decreased(P<0.05,P<0.01).2 Effect of Yi shen Juanbi Pill bone marrow fluid on the differentiation of CD4+T lymphocytes2.1 Effect of Yi shen Juanbi Pill Bone Marrow liquid on mRNA expression of CD4+T Lymphocyte related Transcriptional Regulatory factors(RORyt,TNF α,IL-17,Foxp3,TGF-β,IL-10)mRNACompared with Sham group,the expression of RORyt,TNF-α and IL-17 mRNA in Sham+CIA group increased,but there was no statistical difference.The expression of RORγt,TNF-α and IL-17 mRNA in OVX group and OVX+CIA group increased significantly(P<0.05,P<0.01).The expression of Foxp3,TGF-β and IL-10 mRNA in Sham+CIA group and OVX group had a downward trend,but there was no statistical difference.The expression of Foxp3 mRNA in OVX+CIA group decreased significantly(P<0.01).The expression of TGF-β and IL-10 mRNA in OVX+CIA group had a downward trend,but there was no statistical difference.Compared with Sham+CIA group,the expression of RORyt,TNF-α and IL-17 m RNA in OVX+CIA group increased significantly(P<0.05,P<0.01);The expression of Foxp3 mRNA in OVX+CIA group decreased significantly(P<0.01).TGF-β and IL10 mRNA in OVX+CIA group had a downward trend,but there was no statistical difference.The expression of RORyt and TNF-α mRNA in OVX+CIA group was significantly higher than that in OVX group(P<0.01).The expression of IL-17 mRNA in OVX+CIA group showed an upward trend,but there was no statistical difference.Foxp3,TGF-βand IL-10 mRNA in OVX+CIA group had a downward trend,but there was no statistical difference.Compared with Sham+CIA group,the expression of RORγt,TNF-α and IL-17 mRNA in Sham+CIA+YSJB group decreased,but there was no statistical difference.Foxp3,TGF-β and IL-10 mRNA had an upward trend,but there was no statistical difference.Compared with OVX+CIA group,the expression of RORγt、TNF-α、IL-17 mRNA in OVX+CIA+YSJB group and OVX+CIA+MTX group decreased significantly(P<0.01).Foxp3 mRNA in OVX+CIA+YSJB group had an increasing trend,but there was no statistical difference;The expression of IL-10 and TGF-β mRNA increased further(P<0.05,P<0.01).The expressions of Foxp3,TGF-β and IL-10 mRNA were further increased in OVX+CIA+MTX group(P<0.01).2.2 Effect of Yishen Juanbi Pill Bone Marrow liquid on the contents of cytokines(TNF-α,IL-17,TGF-β,IL-10)secreted by CD4+T lymphocytesCompared with Sham group,the contents of TNF-α、IL-17A protein in cell culture supernatant of Sham+CIA group,OVX group and OVX+CIA group increased significantly(P<0.05,P<0.01).The contents of TGF-β and IL-10 protein in the culture supernatant of Sham+CIA group,OVX group and OVX+CIA group decreased significantly(P<0.01).Compared with Sham+CIA group,the contents of TNF-α and IL-17A protein in cell culture supernatant of OVX+CIA group showed an upward trend,but there was no statistical difference.The contents of TGF-β protein in the culture supernatant of OVX+CIA group was significantly decreased(P<0.01).The contents of IL-10 protein in the culture supernatant of OVX+CIA group decreased,but there was no statistical difference.Compared with OVX group,the contents of TNF-α and IL-17A protein in cell culture supernatant of OVX+CIA group showed an upward trend,but there was no statistical difference.The contents of TGF-β and IL-10 protein in the culture supernatant of OVX+CIA group decreased significantly(P<0.05,P<0.01).The contents of TNF-α and IL-17A protein in the supernatant of Sham+CIA+YSJ B group was significantly lower than that of Sham+CIA group(P<0.05,P<0.01).The contents of TGF-β protein in the supernatant of cell culture increased significantly(P<0.05);The contents of IL-10 protein in the supernatant of cell culture showed an upwa rd trend,but there was no statistical difference.Compared with OVX+CIA group,the contents of TNF-α and IL-17A protein in cell culture supernatant of OVX+CIA+YSJB group and OVX+CIA+MTX group decreased significantly(P<0.05).The contents of TGF-β protein in the culture supernatant of OVX+CIA+YSJB group and OVX+CIA+MTX group was significantly increased(P<0.01);The contents of IL-10 protein in the culture supernatant of OVX+CIA+YSJB group showed an upward trend,but there was no statistical difference.The contents of TGF-β protein in the culture supernatant of OVX+CIA+MTX group was significantly increased(P<0.05).3 Effect of Yishen Juanbi Pill bone marrow fluid on CD4+T-OC co-culture system3.1 Effect of Yishen Juanbi Pill bone marrow fluid on OC differentiation and Bone resorption function in CD4+T-OC co-culture systemCompared with Sham group,the number of OC in Sham+CIA group,OVX group and OVX+CIA group increased significantly(P<0.01).The number of OC in OVX group was significantly higher than that in Sham+CIA group,but there was no significant difference in bone resorption lacunae.Compared with Sham+CIA group,the number of OC and the area of bone resorption lacunae in OVX+CIA group were significantly increased(P<0.01).Compared with OVX group,the number of OC and the area of bone resorption lacunae in OVX+CIA group were significantly increased(P<0.01).The number of OC and the area of bone resorption lacunae in Sham+CIA+YSJB group were significantly lower than those in Sham+CIA group(P<0.01).The number of OC and the area of bone resorption lacunae in OVX+CIA+YSJB group and OVX+CIA+MTX group decreased significantly compared with OVX+CIA group(P<0.01).3.2 Effect of Yishen Juanbi Pill bone marrow fluid on the expression of OC-related proteins in CD4+T-OC co-culture systemCompared with Sham group,the expression of NFATC-1,CTSK and MMP-9 in Sham+CIA group and OVX group had an upward trend,but there was no statistical difference,the expression of NFATC-1 and CTSK protein in OVX+CIA group was significantly higher than that in OVX+CIA group(P<0.05),and the expression of MMP-9 protein in OVX+CIA group had an upward trend,but there was no statistical difference.Compared with Sham+CIA group and OVX group,the expression of NFATC-1 and MMP-9 protein in OVX+CIA group increased,but there was no significant difference,while the expression of CTSK protein increased significantly in OVX+CIA group.Compared with Sham+CIA group,the expression of NFATC-1 protein in Sham+CIA+YSJB group decreased significantly,the expression of CTSK and MMP-9 protein decreased,but there was no significant difference.Compared with OVX+CIA group,the expression of MMP-9 protein in OVX+CIA+YSJB group decreased,the expression of NFATC1 and CTSK protein decreased significantly in OVX+CIA+YSJB group,the expression of NFATC-1,CTSK and MMP-9 protein decreased significantly in OVX+CIA+MTX group(P<0.05,P<0.01).3.3 Effects of Yishen Juanbi Pill bone marrow fluid on the contents of TNF-α,IL17,TGF-β and IL-10 proteins by CD4+T lymphocytes in co-culture systemCompared with Sham group,the contents of TNF-α and IL-17A protein in cell culture supernatant of Sham+CIA group,OVX group and OVX+CIA group were significantly increased(P<0.05,P<0.01).The content of TGF-β protein in cell culture supernatant of Sham+CIA group,OVX group and OVX+CIA group decreased,but there was no statistical difference,while the content of IL-10 protein in cell culture supernatant of Sham+CIA group,OVX group and OVX+CIA group decreased.(P<0.01).Compared with Sham+CIA group,the contents of TNF-α protein in cell culture supernatant of OVX+CIA group increased,but there was no statistical difference,the contents of IL-17A protein in cell culture supernatant of OVX+CIA group increased significantly(P<0.05),while the contents of TGF-β and IL-10 protein in cell culture supernatant of OVX+CIA group tended to decrease,but there was no statistical difference.Compared with OVX group,the contents of TNF-α protein in cell culture supernatant of OVX+CIA group increased,but there was no statistical difference.The contents of IL-17A protein in cell culture supernatant of OVX+CIA group increased significantly,while the contents of TGF-β and IL-10 protein in cell culture supernatant of OVX+CIA group tended to decrease,but there was no statistical difference.Compared with Sham+CIA group,the contents of TNF-α protein in the supernatant of Sham+CIA+YSJB group decreased,but there was no statistical difference,the contents of IL-17A protein in the supernatant of cell culture decreased significantly(P<0.01),and the contents of TGF-β and IL-10 protein in the supernatant of cell culture increased,but there was no statistical difference.Compared with OVX+CIA group,the contents of TNF-α and IL-17A protein in cell culture supernatant of OVX+CIA+YSJB group and OVX+CIA+MTX group decreased significantly(P<0.01),the contents of TGF-β protein in cell culture supernatant of OVX+CIA+YSJB group increased,but there was no statistical difference,and the contents of IL-10 protein in cell culture supernatant of OVX+CIA+YSJB group increased significantly(P<0.01).The contents of TGF-β and IL-10 protein in the culture supernatant of OVX+CIA+MTX group was significantly increased(P<0.01).4 Effect of levo-tetrahydropalmatine(THP)on differentiation and function of OC4.1 Detection of components in Yishen Juanbi Pill bone marrow fluidTHP and aristolochic acid were detected in the bone marrow fluid of Yishen Juanbi Pill,in which the content of THP was 0.6 ng/mL,and aristolochic acid existed matrix interference.Therefore,this experiment explored the effect of THP on the differentiation and function of OC.4.2 Effect of THP on differentiation and absorption of OCWith the increase of the concentration of THP,the number of OC and the area of bone resorption lacunae decreased gradually in a dose-dependent manner.Compared with Ctrl group,THP of 0.625,1.5625,3.125,6.25,12.5μg/ml could significantly inhibit the differentiation and absorption of OC(P<0.01).4.3 Effect of THP on the expression of OC-related transcription factors(NFATC1,CTSK and MMP-9)mRNA and(NFATC-1,CTSK and MMP-9)proteinCompared with Ctrl group,there was no statistical difference in the expression of NFATC-1 and CTSK mRNA in THP 0.625 μg/mL group,but NFATC-1,CTSK,MMP-9 mRNA and the protein expression of NFATC-1,CTSK and MMP-9 in other groups decreased significantly in a dose-dependent manner.(P<0.01).ConclusionThe bone marrow fluid of CIA,kidney deficiency and kidney deficiency CIA rats can not only directly promote the differentiation and absorption of OC,but also promote the differentiation of CD4+T lymphocytes to Th 17,and indirectly promote the differentiation and absorption of OC,and the bone marrow fluid of CIA rats with kidney deficiency can promote the differentiation and absorption of OC more obviously.Both bone marrow fluid of CIA rats and bone marrow fluid of kidney deficiency CIA rats interfered by YSJB could inhibit the differentiation and absorption of OC in single culture and co-culture,and the effect of bone marrow fluid of kidney deficiency CIA rats interfered by YSJB was more obvious.THP may be one of the main active substances of YSJB.Its mechanism is that the bone marrow fluid of YSJB rats can down-regulate the expression of TNF-α and IL-17 in CD4+T lymphocytes,promote the secretion of TGF-β and IL-10,restore the balance of Th17/Treg,and then inhibit the differentiation and function of OC.This study not only provides an experimental basis for the treatment of RA from the kidney,but also provides a further basis for the theory of kidney governing water and bone marrow generation. |
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