Effect Of PM2.5 Exposure On Bone Metabolism In Postmenopausal Women And Its Mechanism

Objective:Animal experiment:to explore the relationship between PM2.5 and postmenopausal bone metabolism by observing the effects of PM2.5 exposure on bone mineral density(BMD),femoral bone morphology and biomechanics of lumbar vertebrae(L3-L5)in ovariectomized rats with osteoporosis.Cell experiment:the co-culture system of osteoblasts and osteoclasts was established in Transwell chamber.After exposure to different concentrations of PM2.5,the effects of PM2.5 exposure on cytotoxicity,oxidative damage and classical antioxidant pathway of Nrf2/ARE in the co-culture system were studied,and the mechanism of PM2.5 in bone metabolism was discussed.Finally,it provides experimental basis for the relationship between PM2.5 and bone health in postmenopausal women.Methods:Animal experiment1.One week later,the female SD rats(SPF grade)were randomly divided into three groups:sham operation group,operation group and PM2.5 group,ovariectomized group and PM2.5 group at the end of 5 months,and parovary adipose tissue was removed in sham operation group.2.After castration,the rats were given tracheal suspension drip until the end of 9 months,and the bone mineral density((bone mineral density,BMD)of lumbar vertebrae(L3-L5)in each group was measured at the end of the 8th month.3.Under the aseptic condition of airway drip at the end of September,bilateral femurs were taken out for bone histomorphology(HE staining)and bone biomechanical index detection.Cell experiment4.Establishment of co-culture system of PM2.5 and osteoblast-osteoclasts in vitro using Transwell chamber.5.After co-culture system was exposed to different concentrations of PM2.5 for 48 hours,CCK-8 kit was used to detect the survival rate of osteoblasts in co-culture system.6.After 48 hours of exposure,the content of oxidative stress index MDA in cell suspension was determined by thiobarbituric acid(TBA)method,and the activity of serum antioxidant enzyme superoxide dismutase(SOD)was determined by water-soluble tetrazolium(WST-1)method.7.The protein expression levels of nuclear factor E2 related to oxidative stress Nrf2 signal pathway(Nrf2),heme oxygenase-1(HO-1)and quinone oxidoreductase 1(NQO1)in co-cultured cells were detected by Western blotting(Western blot).Results:Animal experiment1.The results showed that the bone mineral density of lumbar vertebrae in the operation group was 0.223 ± 0.032,which was significantly lower than that in the sham operation group,indicating that the model was successful,and the bone mineral density in the PM2.5 operation group was significantly different from that in the operation group.2.HE staining of bone tissue under light microscope:bone trabeculae were closely arranged with good continuity and no fat vacuole structure was seen in bone marrow cavity in sham operation group;bone trabecular fracture,reduced trabecular number,thinning,loose arrangement and fat vacuole structure in bone marrow cavity were observed in operation group and sham operation group;large-scale bone trabecular fracture,larger spacing,significant decrease in trabecular number and obvious disorder were found in PM2.5 group.3.The results of three-point bending test showed that the elastic load,ultimate load and elastic modulus in the operation group were significantly lower than those in the sham operation group,which further indicated that the model was successful,and the above mechanical parameters in the high concentration PM2.5 operation group were significantly lower than those in the operation group(P<0.05).Cell experiment4.Identification of osteoclasts by TRAP staining:the cytoplasm of some cells turned burgundy or light purple,enlarged,cytoplasmic expansion,vacuolation,and contained multiple nuclei,which were the hallmarks of mature osteoclasts,indicating that RAW264.7 could be successfully induced into osteoclasts.5.Compared with the control group,the survival rate of osteoblasts decreased when the concentration of PM2.5 was 25 μ g/mL,but there was no significant difference compared with the control group.When the concentration of PM2.5 was 50,100,200 μ g/mL,the survival rate of osteoblasts decreased gradually,which was statistically significant compared with the control group.6.With the increase of PM2.5 concentration,the content of MDA increased.The content of MDA in 100,200 μg/mL groups was significantly higher than that in the control group(P<0.01).The activity of);SOD decreased gradually and the ability of antioxidation decreased.The activity of SOD in 50,100,200 μ g/mL groups was significantly higher than that in the control group(P<0.05).The activity of GSH-Px in 100,200 μ g/mLPM2.5 groups was lower than that in the control group,and the difference was statistically significant(P<0.05).7.The protein levels of Nrf2 signal pathway in osteoblasts detected by Western Blot showed that the protein levels of Nrf2,HO-1 and NQO1 decreased in a concentration-dependent manner with the increase of PM2.5 concentration,and the protein levels of Nrf2,HO-1 and NQO1 in 50 μ g/mLPM2.5 group,NQO1100 group and 200 μg/mLPM2.5 group decreased compared with the control group(P<0.05or P<0.01).Conclusion:1.PM2.5 aggravates the decrease of BMD and bone biomechanics in ovariectomized SD rats,further osteoporosis of bone tissue structure,and accelerates bone loss in ovariectomized rats.2.PM2.5 could break the balance of oxidative stress in the co-culture system,increase the production of MDA,also decrease the activity of SOD and GSH-Px,finally decrease the survival rate of osteoblasts.3.When exposed to 4.PM2.5,the antioxidant signal pathway of Nrf2 is inhibited,and the expression of Nrf2 and downstream genes HO-1 and NQO1 is down-regulated,which weakens the antioxidant capacity of the body and promotes the increase of oxidative stress of osteoblasts,and finally affects bone metabolism.