| Objective:To observe the effect of Juanbi Capsule on TLR4 /NF-?B signal pathway and its related factors expression in postmenopausal knee osteoarthritis animal model,and to explore the possible mechanism of Juanbi Capsule in the treatment of postmenopausal knee osteoarthritis.Methods:(1)Thirty 5-month-old female non pregnant guinea pigs were selected to establish the animal model of primary knee osteoarthritis.The fibroblast like synoviocytes(FLS)and subchondral osteoblasts(OB)of 5-month-old KOA guinea pigs were isolated by trypsin and type I collagenase combined digestion.The cells were cultured in vitro and passed on.The cells were stained by inverted microscope and he staining,the basic morphology of the cells was observed;the fibroblast like synoviocytes(FLS)were identified by immunohistochemistry,CD44,CD55,vinmentin,CD90 and CD68;the subchondral osteoblasts(OB)were identified by ALP and alizarin red staining.(2)Thirty-two 5-month-old female knee osteoarthritis model guinea pigs were randomly divided into three groups: Juanbi Capsule group,western medicine group,model group and sham operation group.On this basis,the compound animal model of postmenopausal knee osteoarthritis was established by bilateral ovariectomy.The rats in the Juanbi Capsule group were given the water solution of Juanbi Capsule and the rats in the western medicine group were given the water solution of Diacerein Capsule,the rats in the model group and the sham operation group were given the same amount of pure water.At the end of gastric perfusion,blood was collected from the abdominal aorta to prepare the serum containing drugs in the groups of Juanbi Capsule,western medicine(Diacerein Capsule),model group and sham operation group.At the end of the cell intervention,the cell culture medium in the culture hole at the end of the last day of the intervention was collected,centrifuged and the supernatant was retained;the total RNA in fibroblast like synoviocytes and subchondral osteoblasts was extracted by Trizol method;the total protein in fibroblast like synoviocytes and subchondral osteoblasts was extracted by enhanced RIPA lysate.The levels of estrogen in serum and TNF-? in supernatant of model group and sham operation group were measured by ELISA,TLR4 and TRAF6 gene expression levels were detectedby RT-PCR,NF-?Bp65 protein expression levels were detected by Western blot.Use the path builder tool,spss22.0,graphpad prism 8.0.2,Image J software to experiment drawing,establish basic database,image processing and statistical analysis processing.Results:(1)The primary fibroblast like synovial cells(FLS)can be further purified by in vitro culture and passage.When the FLS is cultured to the third generation,the FLS is mostly spindle shaped,irregular spindle shaped,polygonal,triangle like,star shaped,with vortex like aggregation and growth.The oval nucleus is located in the center of the cell,which is morphologically consistent with the characteristics of synthetic fiber like synovial cells(FLS).The surface specific markers were identified by immunohistochemistry,immunofluorescence CD44,CD55,vinmentin and CD90.A large number of positive substances were found in the cytoplasm and nuclear membrane of these group.There was no significant positive expression in the cytoplasm and nuclear membrane of CD68 group and PBS control group.It was further confirmed that the cells were fibroblast like synoviocytes(FLS);the subchondral osteoblasts(OB)gathered and distributed in a linear pattern some cells are connected by synapses,and oval nuclear are distributed along the long axis of cells.In addition,a large number of alkaline phosphatase deposits and calcified nodules were observed in the subchondral osteoblasts(OB)by ALP and alizarin red staining.(2)The level of estrogen in the serum of the model group and the sham operation group was measured by ELISA.The level of estrogen in the serum of the model group was significantly lower than that of the sham operation group(P < 0.05),which proved that the compound animal model of postmenopausal knee osteoarthritis was successfully established.The expression of TNF-? in the supernatant of fibroid synoviocytes and subchondral osteoblasts was measured by ELISA.The results showed that :compared with the sham operation group,the expression level of TNF-? in the model group was significantly up-regulated(P < 0.05);compared with the model group,the expression level of TNF-? in the Juanbi Capsule group was significantly down regulated(P< 0.05),and the expression level of TNF-? in the western medicine group(Diacerein Capsule)was significantly down regulated(P < 0.05);compared with the western medicine group(Diacerein Capsule),the expression level of TNF-? in the Juanbi Capsule group had no significant change(P > 0.05).(3)RT-PCR results of fibroblast like synovial cells and subchondral osteoblasts showed that: compared with sham operation group,TLR4 and TRAF6 gene expression level in model group was significantly increased(P <0.05);compared with model group,TLR4 and TRAF6 gene expression level in Juanbi Capsule group was significantly decreased(P < 0.01),TLR4 and TRAF6 gene expression level in western medicine group was significantly decreased(P < 0.01);compared with western medicine group,there was no significant difference in TLR4 and TRAF6 geneexpression in Juanbi Capsule group(P > 0.05).(4)Fibroblast like synoviocytes and subchondral osteoblasts western the results showed that:compared with the sham operation group,the protein expression level of NF-?Bp65 in the model group was significantly up-regulated(P < 0.01);compared with the model group,the protein expression level of NF-?Bp65 in the Juanbi Capsule group was significantly down regulated(P < 0.05),and the protein expression level of NF-?Bp65 in the western medicine group(Diacerein Capsule)was significantly down regulated(P < 0.05);compared with the western medicine group(Diacerein Capsule),there was no significant difference in NF-?Bp65 protein expression in Juanbi Capsule group(P > 0.05).Conclusions:(1)The fibroblast like synoviocytes(FLS)and subchondral osteoblasts(OB)of the KOA model of female guinea pigs were successfully isolated and cultured,and further purified and identified to provide an in vitro cell model for the follow-up study of KOA mechanism and pathway.(2)By inhibiting TLR4 and TRAF6 gene expression in fibroblast like synoviocytes(FLS),inhibiting NF-?Bp65 protein expression in FLS,reducing TNF-? and other inflammatory factors expression level outside FLS,regulating the transmission process of innate immune response,thus slowing down the immune inflammatory response of synovium and delaying the articular cartilage and subchondral bone degeneration,and finally achieve the purpose of prevention and treatment of postmenopausal knee osteoarthritis.(3)By inhibiting the expression of TLR4 and TRAF6 genes in chondroblasts(OB),inhibiting the expression of NF-?Bp65 protein in chondroblasts(OB),reducing the expression of TNF-? and other inflammatory factors outside of chondroblasts(OB),regulating the transmission process of innate immune response,thus slowing down the immune inflammatory response of synovium and delaying the articular cartilage and subchondral bone degeneration,and finally achieve the purpose of prevention and treatment of postmenopausal knee osteoarthritis. |
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