| objective:The effects of different concentrations of PM2.5 exposure on bone microstructure,osteoblast-osteoclast co-culture system and OPG/RANKL pathway in ovariectomized SD rats were studied to explore the effect of PM2.5 on postmenopausal osteoporosis and the role of OPG/RANKL pathway in it.Methods:1.Effects of PM2.5 exposure on bone microstructure of ovariectomized SD female rats(1)24 SPF female healthy SD rats were randomly divided into three groups:low dose group,middle dose group,high dose group and control group.PM2.5 suspension was given by airway drip once every 3 days.(2)SD rats were instilled with PM2.5 of different concentrations(35、70、150 ug/m~3),while those in the control group were infused with normal saline.(3)Castration was performed at the end of 5 months after drip.Femur,tibia and lumbar vertebrae were taken at the end of 9 months after drip,and the bone microstructure of femur,tibia and lumbar vertebrae were detected by microcomputerized tomography(micro-CT).2.Effects of PM2.5 exposure on osteoblast-osteoclast co-culture system and OPG/RANKL pathway(1)The osteoblast-osteoclast co-culture system was established in Transwell chamber.(2)Different concentrations of PM2.5(0、50、100、200 ug/ml)before and after menopause were co-cultured with osteoblasts and osteoclasts in vitro by(+/-)estrogen simulation.(3)CCK8 method was used to detect the proliferation of osteoblasts for 24 hours and48 hours,and Western-blot technique and real-time quantitative PCR technique were used to detect the expression of ERα,Ah R,OPG,RANKL protein and m RNA of osteoblasts.Results:1.Effects of PM2.5 exposure on bone microstructure of ovariectomized SD female rats(1)Compared with the control group,trabecular bone mineral density(Tb.BMD),bone volume fraction(BV/TV),structural model index((SMI)),trabecular thickness(Tb.Th),trabecular number(Tb.N)and trabecular separation(Tb.Sp)of tibia,femur and lumbar vertebrae in PM2.5 exposure group decreased,and trabecular separation degree(Tb.Sp)increased.In the PM2.5 exposure group,the changes of BV/TV,Tb.Th,Tb.N and Tb.Sp of tibial trabeculae were significantly different from those of the control group,and the changes of Tb.BMD,BV/TV,SMI,Tb.N and Tb.Sp of femoral trabeculae were significantly different from those of the control group.The changes of SMI,Tb.Th,Tb.N and Tb.Sp in lumbar trabeculae were significantly different from those in the control group(P<0.05).(2)Compared with the control group,the bone marrow cavity of tibia in PM2.5exposure group was obviously empty and trabeculae were absent,the number of bone trabeculae in word triangle of femur was sparse and the gap was enlarged,and the integrity of lumbar trabecular structure was destroyed and rod-shaped structure was increased in PM2.5 exposure group compared with the control group.2.Effects of PM2.5 exposure on osteoblast-osteoclast co-culture system and OPG/RANKL pathway(1)No matter in estrogen group or estrogen-free group,when PM2.5 was exposed to osteoblast-osteoclast co-culture system for 24h and 48h,the OD value of osteoblasts decreased and cell viability decreased with the increase of exposure concentration.In the estrogen-free group,compared with the control group,the OD value of the 25ug/ml exposure group decreased,the difference was not statistically significant(P>0.05),and the OD value of the other groups decreased compared with the control group,the difference was statistically significant(P<0.001);In the estrogen group,compared with the control group,the OD value of each group decreased,with statistical significance(P<0.001).(2)After PM2.5 was exposed to osteoblast-osteoclast co-culture system for 24 hours,compared with the control group,the expression of RANKL increased and the expression of ERa decreased in both estrogen group and non-estrogen group.In the non-estrogen group,compared with the control group,the expression of Ah R increased(P<0.05),but there was no significant difference in estrogen group(P>0.05).In the non-estrogen group,compared with the control group,the expression of OPG decreased.In the estrogen group,compared with the control group,the expression of OPG in the 50 ug/ml PM2.5 decreased.There was no significant difference in the expression of OPG between the 100 ug/ml PM2.5 exposure group and the control group.The expression of OPG in the 200 ug/ml PM2.5 exposure group increased.(3)Compared with the control group,the m RNA expression of RANKL and Ah R of osteoblasts in the estrogen group and non-estrogen group increased 24 hours after PM2.5 exposure,and the m RNA expression level of 200 ug/ml exposure group was the most significant(P<0.05).The m RNA expression level of 200 ug/ml exposure group was the most significant(P<0.05);OPG),the m RNA expression level of 50ug/ml and 100 ug/ml exposure group was not significantly different from that of the control group,but that of 200 ug/ml exposure group was significantly lower(P<0.05).Conclusion:1.PM2.5 exposure could change the bone microstructure of ovariectomized female SD rats:Tb.BMD decreased,BV/TV decreased,SMI increased,Tb.Th decreased,Tb.N decreased and Tb.Sp increased in tibia,femoral ward triangle and lumbar trabeculae of ovariectomized female rats.Trabecular structure was destroyed and the number decreased,changing from lamellar structure to rod-shaped structure.It can be seen that PM2.5 exposure can aggravate postmenopausal bone mass loss.2.PM2.5 exposure can inhibit the proliferation of osteoblasts in osteoblast-osteoclast co-culture system.3.PM2.5 exposure for 24 h increased the expression of RANKL,Ah R protein and m RNA,and decreased the expression of OPG and ERa protein and m RNA in osteoblasts in osteoblast-osteoclast co-culture system.4.PM2.5 exposure may cause or aggravate postmenopausal osteoporosis through OPG/RANKL pathway. |
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