Studies On The Effect And Mechanism Of The Tolypocladium Sinense Extract On Osteoporosis Induced By Ovariectomy

Objectives: To study the prevention and treatment effects of Tolypocladium sinense extract(TSE)on osteoporosis in mice,and to explore the preliminary molecular mechanism to provide experimental evidence for a new drug for the treatment of osteoporosis.Methods:(1)The effect of TSE on ovariectomized mice: Based on the above in vitro experiments,bilateral oophorectomy was used to simulate postmenopausal osteoporosis.Gavage was started on the 7th day after ovariectomy in mice,every day once,continuous intragastric administration for 6 weeks.After execution by carbon dioxide asphyxiation,both femurs,tibias and peripheral blood were removed;one femur was subjected to Micro CT scan analysis,and the other femur was used for H&E staining and TRAP staining;ELISA Detect the content of BALP,ACP5 and BGP in peripheral blood.(2)CCK-8 method to determine the effect of TSE on the activity of bone marrow macrophages(BMMs)and RAW264.7 cells: Macrophages were extracted from the bone marrow of C57BL/6 mice,and BMMs and RAW264.7 cells were incubated with different concentrations of TSE for 48 h At and 96 h,the CCK-8 method was used to determine the TSE concentration that was non-cytotoxic to BMMs and RAW264.7.(3)The effect of TSE on RANKL-induced osteoclast differentiation:During the process of osteoclast differentiation,TSE was added to intervene treatment,and the number of osteoclasts was analyzed by Tartrate-resistant acid phosphatase(TRAP)staining.(4)The effect of TSE on the formation of F-actin ring and bone resorption function of RANKL-induced osteoclasts: plant the nearly mature osteoclasts on the bone plate and observe the area of bone lacuna;use DAPI and phalloidin to treat osteoclast actin the ring(F-actin belt)was stained to observe the number of nuclei in the ring and the morphology of the ring.(5)The effect of TSE on ROS during osteoclast differentiation: use flow cytometry to detect the fluorescence intensity of DCF after RANKL stimulation and TSE intervention.(6)The effect of TSE on RANKL-induced osteoclast-specific gene expression: QRT-PCR was used to detect the expression of DCSTAMP,ATP6V0d2,TRAP,CTSK,NFATc1,and c-Fos.(7)The effect of TSE on the expression of MAPK pathway and NF-κB pathway protein in osteoclast differentiation:Western-Blot was used to detect ERK,p-ERK,JNK,p-JNK,P38,p-P38,P65,p-P65,p-The relative expression levels of IκBα and IκBα proteins.(8)The effect of TSE on the osteogenic differentiation of mesenchymal stem cells(MSCs): First,determine the concentration of TSE that is non-cytotoxic to MSCs by the CCK-8 method,and then incubate the cells with osteogenic medium and different concentrations of TSE for 14 days and 21 days.Alkaline phosphatase staining and alizarin red staining were used to analyze alkaline phosphatase activity and the area and number of calcium nodules.(9)The effect of TSE on specific gene expression during osteogenic differentiation: QRTPCR was used to detect the expression of ALP,Runx2,osteopon,osteocal and 2-col on the 3rd and 7th day of osteogenic induction.(10)The effect of TSE on the expression of Wnt pathway protein during osteogenic differentiation: On the 14 th day of osteogenic differentiation,QRT-PCR was first used to detect the expression of Axin-2,β-catenin,GSK3β,LEF-1,and Wnt3a;In the process of bone differentiation,after TSE has an effect on the expression of Wnt pathway related genes,continue to use Western-Blot to detect the relative protein expression of Axin-2,β-catenin,GSK3β,LEF-1,and Wnt3 a.Results:(1)Micro CT scan results show that TSE treatment can significantly improve the bone microenvironment of mouse femurs,increase the number,thickness,and overall bone volume of bone trabeculae;HE and TRAP staining results show that TSE treatment can increase the bone volume of mice.Reduce the number of osteoclasts and the area of osteoclasts.ELISA results showed that TSE increased the concentration of ALP and BGP in peripheral blood and decreased the concentration of ACP5.(2)When the concentration is below 0.5 mg/m L,TSE has no cytotoxicity to BMMs;when the concentration is below 0.25 mg/m L,TSE has no cytotoxicity to RAW264.7.(3)Compared with the RANKL group,at a concentration of 0.125 mg/m L,TSE significantly inhibited the differentiation of osteoclasts in a dose-dependent manner;and it hindered the formation of osteoclasts in the early stage of differentiation.(4)Compared with RANKL group,TSE significantly inhibited the formation of F-actin ring and reduced the area of bone lacuna.(5)The flow cytometry results showed that after RANKL stimulation,the level of ROS in BMMs increased significantly,and this level decreased as the concentration of TSE increased.(6)PCR results showed that TSE inhibited the expression of DC-STAMP,V-ATPase,TRAP,CTSK,NFATc1,and c-Fos.(7)Western-Blot results show that TSE down-regulates the ratios of p-JNK/JNK,pERK/ERK,and p-P38/P38,but has no significant effect on the ratios of p-P65/P65,pIκBα/IκBα,and down-regulates NFATc1,c-Fos Relative protein expression.(8)TSE has no cytotoxicity to MSCs when the concentration is below 0.25 mg/m L,and promotes the differentiation and mineralization of osteoblasts.(9)PCR results showed that TSE up-regulated the expression of ALP and Runx2 on the 3rd day,and had no significant effect on the expression of osteopon,osteocal,2-col;on the 7th day,the expression of ALP,osteocal,and osteopon was up-regulated,and the expression of osteocal was down-regulated.The expression of lipid-related gene 2-col has no significant effect on the expression of Runx2.(10)TSE up-regulates the expression of Wnt3 a,β-catenin and LEF-1 genes,and down-regulates the expression of GSK3β and Axin-2 genes;at the protein level,TSE up-regulates the relative expression of Wnt3 a,β-catenin and LEF-1 proteins,and down-regulates GSK3β and Axin-2 Relative protein expression.(10)Micro CT scan results show that TSE treatment can significantly improve the bone microenvironment of mouse femurs,increase the number,thickness,and overall bone volume of bone trabeculae;HE and TRAP staining results show that TSE treatment can increase the bone volume of mice.Reduce the number of osteoclasts and the area of osteoclasts.ELISA results showed that TSE increased the concentration of ALP and BGP in peripheral blood and decreased the concentration of ACP5.Conclusion: TSE inhibits the differentiation and function of osteoclasts by downregulating the phosphorylation levels of JNK,ERK and P38 in the MAPK pathway;at the same time,it activates the Wnt pathway to promote the differentiation and mineralization of osteoblasts;It has a good therapeutic effect on postmenopausal osteoporosis mice.