| ObjectiveBushen Huatan Decoction was used to intervene ovariectomized rats.The changes of intestinal mucosal barrier function,the expression of pro-inflammatory and anti-inflammatory factors in serum and bone tissue,and the effects of Bushen Huatan Decoction on IL-6/JAK2/STAT3 and IL-2/JAK1/STAT5 signaling pathway,CD4~+T cell subsets,helper cell 17(Th17)/regulatory T cell(Treg)were observed to explore the mechanism of Bushen Huatan Decoction in the prevention and treatment of postmenopausal osteoporosis.MethodsNinety 6-month-old female SD rats were random Ly divided into sham operation group(Sham),model group(OVX),estradiol valerate group(EV),low-dose Bushen Huatan recipe group(OVX+BL,BL),medium dose Bushen Huatan recipe group(OVX+BM,BM)and high-dose Bushen Huatan recipe group(OVX+BH,BL),Except sham group,all the other groups were operated to make postmenopausal osteoporosis model.OVX group and Sham group were given the same volume of normal saline by gavage,once a day,12 weeks later.Experiment 1:Hematoxylin eosin staining,tartrate resistant acid phosphatase staining were used to observe the pathological morphology of bone tissue;the bone mass and bone microstructure of rats were detected by micro computed tomography,and the changes of bone mass and bone trabecula were observed.Experiment 2:Observation of pathological changes of colon by hematoxylin eosin staining;the contents of IL-6,IL-2,IL-10 and IL-17 in colon tissue were detected by enzyme-linked immunosorbent assay,and the expressions of occludin,claudin-1 and ZO-1 m RNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting.Experiment 3:The levels of IL-6 and TNF-?in serum were detected by ELISA,the m RNA expressions of IL-6,IL-6R,JAK2 and STAT3 in bone tissue were detected by PCR,and the protein expressions of IL-6,IL-6R,JAK2,P-JAK2,STAT3 and P-STAT3 in bone tissue were detected by Western blot.Experiment 4:The levels of IL-2 and TGF-?1 in serum were detected by enzyme-linked immunosorbent assay,the m RNA expressions of IL-2,IL-2R,JAK1 and STAT5 in bone tissue were detected by PCR,and the protein expressions of IL-2,IL-2R,JAK1,P-JAK1,STAT5 and P-STAT5 in bone tissue were detected by Western blot.Experiment 5:the levels of Foxp3,ROR?t,IL-10 and IL-17 in serum were detected by enzyme-linked immunosorbent assay.The m RNA expressions of Foxp3,ROR?t,IL-10 and IL-17 in bone tissue were detected by real-time fluorescent quantitative PCR.The protein expressions of Foxp3,ROR?t,IL-10 and IL-17 in bone tissue were detected by Western blot.The number of Th17,Treg cells and Th17/Treg ratio in bone tissue were detected by flow cytometry.ResultsExperiment 1:In sham group,the structure of femur was complete,the trabecula was rich,and the adipocytes were few.In OVX group,the bone microstructure of femur was destroyed and a large number of vacuolated adipocytes appeared in the medullary cavity.In EV group,the number of adipocytes in medullary cavity decreased,the reticular structure was slightly repaired,and the structure was still incomplete.Compared with OVX group,the trabecular morphology of BL?BM and BH group was significantly improved.Staining:compared with sham group,the number of OC in OVX group was significantly increased;compared with OVX group,the number of OC in EV?BM and BH groups decreased significantly(P<0.01,P<0.05);while the number of OB increased significantly in BM and BH groups,but there was no significant difference in EV?BL group.Bone microstructure:compared with sham group,in OVX group,BMD,TMD,BMC,TMC,BV/TV,Tb.N,Tb.Th reduce and Conn.D.?Tb.Sp rise(P<0.01).Compared with OVX group,in the group of EV?BL?BM?BH,the levels of BMD,TMD,BMC,TMC,BV/TV,Tb.N,Tb.Th was significantly high,Conn.D.?Tb.Sp was significantly low(P<0.05,P<0.01).In the three-dimensional reconstruction,the structure of bone trabeculae in sham group was more uniform,arranged orderly,connected in a network,and the shape was relatively complete;in OVX group,the structure of bone trabeculae was significantly thinner,broken,and thinned,and the bone cortex was thinner,with poor structure and typical osteoporosis like changes.Experiment 2:In sham group,the structure of colonic mucosa was relatively complete,no obvious degeneration,necrosis or abscission of epithelial cells,dense arrangement of intestinal glands,abundant goblet cells in lamina propria,normal morphology and quantity,no obvious infiltration of inflammatory cells.Compared with sham group,the integrity of colonic mucosa in OVX group was damaged,the epithelial cells were degenerated,necrotic or exfoliated,the lumen of intestinal gland in lamina propria was expanded,and inflammatory cells infiltrated into the muscularis mucosa.Compared with OVX group,EV?BM and BH groups had clear structure of colonic mucosa,regular arrangement of intestinal glands,decreased infiltration of inflammatory cells,increased number of goblet cells and improved morphology.The structure of BL group was not improved significantly.compared with sham group,the concentrations of IL-6 and IL-17 in OVX group were significantly increased,while the concentrations of IL-2 and IL-10 were significantly decreased(P<0.01).Compared with OVX group,the concentrations of IL-6 and IL-17 in EV,BM and BH groups were significantly decreased(P<0.01),while the concentrations of IL-6 in BL group were increased(P<0.05),and there was no significant difference in the concentrations of IL-17;the concentrations of IL-2 and IL-10 in EV,BM and BH groups were significantly increased(P<0.01,P<0.05),while the concentrations of IL-2 in BL group were increased(P<0.05),and there was no significant difference in the concentrations of IL-10.PCR and WB:compared with sham group,the m RNA and protein expressions of occludin,claudin-1 and ZO-1 in OVX group were significantly decreased(P<0.01);compared with OVX group,the m RNA expressions of occludin,claudin-1and ZO-1 in BH group were increased(P<0.01,P<0.05),the m RNA expressions of ZO-1 in EV group were increased(P<0.05),the m RNA expressions of claudin-1 and ZO-1 in BM group were increased(P<0.05),and the m RNA expressions of occludin,claudin-1 and ZO-1 in EV,BM and BH groups were increased(P<0.05),The protein expression was significantly increased in BL group(P<0.01,P<0.05).Experiment 3:Compared with sham group,the levels of IL-6 and TNF-?in serum of OVX group were significantly increased(P<0.01);the m RNA expressions of IL-6,IL-6R,JAK2 and STAT3 in bone tissue were significantly increased(P<0.01),and the protein expressions of IL-6,IL-6R,JAK2,P-JAK2,STAT3 and P-STAT3 were significantly increased(P<0.01).Compared with OVX group,the levels of IL-6 and TNF-?in serum,the m RNA expressions of IL-6,IL-6R,JAK2 and STAT3 in bone tissue,and the protein expressions of IL-6,IL-6R,JAK2,p-JAK2,STAT3and p-STAT3 in bone tissue were significantly decreased(P<0.01 or P<0.05).Compared with BL group,the serum levels of IL-6 and TNF-?in BH group were significantly lower(P<0.01).The m RNA expressions of IL-6,IL-6R,JAK2 and STAT3 in bone tissue were significantly decreased(P<0.01),and the protein expressions of IL-6,IL-6R,p-JAK2 and p-STAT3 were significantly decreased(P<0.01).Experiment 4:Compared with sham group,the serum levels of IL-2 and TGF-?1 in OVX group were significantly decreased(P<0.01);the m RNA and protein expressions of IL-2,IL-2RA and il-2RB in bone tissue were significantly decreased,while Jak1 and STAT5 in OVX group were significantly decreased Compared with OVX group,the levels of IL-2 and TGF-?1 in serum of each intervention group were significantly increased(P<0.01),and the m RNA expressions of IL-2,IL-2RA and Il-2RB in bone tissue of EV,BM and BH groups were significantly increased(P<0.01)The expression of IL-2RA m RNA was significantly decreased in BL group(P<0.01,P<0.05).The protein expressions of IL-2,IL-2Ra and il-2rb in bone tissue of EV,BL,BM and BH groups were increased(P<0.01,P<0.05),and the protein expressions of p-JAK1 and p-STAT5 were decreased(P<0.01,P<0.05).There was no significant difference in Jak1and STAT5 protein.Compared with BL group,the serum levels of IL-2 and TGF-?1 in BH group were significantly increased(P<0.01).In BH group,the m RNA expression of IL-2 and IL-2RB increased significantly,while the m RNA expression of JAK1 and STAT5 decreased significantly(P<0.01,P<0.05).The protein expressions of IL-2,IL-2RA and IL-2RB were significantly increased,while the protein expressions of P-JAK1 and p-STAT5 were significantly decreased(P<0.01,P<0.05).Experiment 5:Compared with sham group,Foxp3 and IL-10 in OVX group were significantly reduced,and the expression of Foxp3,IL-10 and protein in bone tissue were significantly decreased(P<0.01),and ROR?t,IL-17 m RNA and protein were significantly increased(P<0.01);compared with OVX group,the concentrations of Foxp3 and IL-10 were significantly increased(P<0.01),and the concentrations of ROR?t and IL-17 were significantly decreased(P<0.01,P<0.05).The results showed that Foxp3and IL-10mrna in EV,BM and BH group were significantly increased(P<0.01,P<0.05),but there was no significant difference in BL group.The m RNA of ROR?t and IL-17 m RNA in EV,BL,BM and BH group was significantly decreased(P<0.01,P<0.05).The expression of Foxp3 and IL-10 in bone tissue of EV,BM and BH group was significantly increased(P<0.01,P<0.05),while that of BL group was higher(P<0.05),and the difference of IL-10 protein was not statistically significant;the expression of ROR?t and IL-17 in EV,BM and BH group was significantly decreased(P<0.01,P<0.05).There was no significant difference in BL group.Flow analysis:compared with sham group,the number of CD4~+CD25~+Foxp3(Treg)cells in OVX group was significantly decreased(P<0.01),the number of CD4~+IL-17A(Th17)cells increased significantly(P<0.05),and the ratio of Th17/Treg was significantly increased(P<0.01).Compared with OVX group,the number of CD4~+CD25~+Foxp3(Treg)cells in EV,BL,BM and BH group increased significantly(P<0.01),and the number of CD4~+IL-17A(Th17)cells decreased significantly in EV,BM and BH group(P<0.01,P<0.05),and the th17/treg ratio decreased significantly(P<0.01).ConclusionBushen Huatan prescription may repair the intestinal mucosal barrier function,regulate bone immune environment,adjust the balance of Th17/Treg,and may bidirectional regulate bone formation and bone resorption to prevent and treat PMOP. |
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