The Mechanism Of Pulsed Electromagnetic Field To Prevent Osteoporosis By Inhibiting The Activation Of NLRP3 Inflammasome

The first chapter:Systematic evaluation of the biological effects of PEMF in the treatment of osteoporosis in vitro and vivoObjective:To systematically review the literature PEMF were used to treat osteoporosis in vitro and in vivo,and to provide evidence-based medical evidence for basic research.Materials and Methods:Search the databases of PubMed,Embase and Web of Science,and include relevant literatures PEMF were used to treat osteoporosis in vitro and in vivo,extract the data and use the SYRCLE bias risk assessment tool and ARRIVE guidelines to evaluate the quality of the methods and the quality of reports.Results:(1)Twenty-four studies on rodent osteoporosis animal were included,of which 23 studies showed the positive effect of PEMF on osteoporosis,and only one study failed to demonstrate the anti-osteoporosis effect of PEMF.Twenty studies in vitro on osteogenic cells have shown that PEMF can promote osteoblastogenesis.Ten studies on osteoclast-related cells all reported the positive effects of PEMF in inhibiting osteoclastogenesis.(2)The parameters of PEMF were highly heterogeneous,including frequency and intensity,However,the frequency of PEMF in rodent osteoporosis animal is mainly concentrated in 15Hz(10 articles),8Hz(2 articles),50Hz(2 articles),7.5 Hz(2 articles).The intensity range used is between 0.1mT-2mT and 3.8mT-4mT.The research frequency of osteogenic-related cells is mainly concentrated in 15Hz(7 articles),50Hz(6 articles),75Hz(5 articles),and the commonly used intensity is concentrated on 0.1mT-2mT and 2mT-1T.The most frequently used frequencies in osteoclastogenesis-related cell research are 15 Hz(4 articles),8 Hz(2 articles),50 Hz(2 articles),and 75 Hz(1 article).The intensity range used is between 0.1mT-1mT.(3)The methodological quality of the included rodent osteoporosis animal is fair.The total score of the 20 studies is between 5-7,and the average report quality is 16.8.However,due to the large heterogeneity of the methods and data reported between the studies,it is impossible to conduct a meta-analysis,and only do a systematic review.Conclusions:Without considering the heterogeneity of PEMF parameters,PEMF can improve the bone loss of rodent osteoporosis animal,promote osteoblastogenesis and inhibit osteoclastogenesis in vitro.However,PEMF intervention programs are highly heterogeneous,and it is necessary to design and explore standard and more optimized PEMF parameters in order to better explore its biological effects and potential treatment mechanisms.The second chapter:Verification of the biological effects of potentially more optimized PEMF parameters which were used to treat osteoporosis in vitro and in vivoObjective:To determine the optimal parameters of PEMF in the treatment of osteoporosis.Materials and Methods:(1)Combined with the mathematical prediction model of parameter analysis established by the research group in the earlier stage,computer mathematical model simulation prediction data,system evaluation results and the existing basic research data of the research group,the potential more optimized PEMF parameter in the treatment of osteoporosis was screened out.(2)To verify the biological effects of more optimized PEMF parameters on osteoblast and osteoclast precursors in vitro level.Specific methods are as follows:BMSCs were extracted from mice and determined by flow cytometry and alizarin red staining.BMSCs were divided into four groups:Osteogenesis group,Osteogenesis+PEMF(50Hz,1.6mT),Osteogenesis+PEMF(75Hz,1.6mT),Osteogenesis+PEMF(15Hz,2mT).ALP staining,activity analysis and the expression of osteogenic genes ALP,OCN and RUNX2 were detected 7 days after induction.After 14 days of induction,alizarin red staining and quantitative analysis were performed to evaluate the osteogenic ability of the cells.Osteoclast precursor cells RAW264.7 were given PEMF intervention during the process of osteoclast differentiation induced by RANKL,namely:cells were divided into RANKL group,RANKL+PEMF(1.6mT,50Hz),RANKL+PEMF(1.6mT,75Hz),RANKL+PEMF(0.5mT,15Hz).Seven days after induction,TRAP staining,TRAP quantitative analysis,and osteoclast-related genes CTSK,NFATcl,TRAP expression were detected.(3)The biological effects of more optimized PEMF parameters in the treatment of ovx-induced osteoporosis mice were verified in vivo.The three-month-old mice were given PEMF intervention after their ovaries were removed.They were divided into control group,OVX group,OVX+PEMF(8Hz,1.6mT),OVX+PEMF(50Hz,1.6mT),OVX+PEMF(75Hz,1.6mT).After one month of intervention,Micro-CT scan of femur,pathological section and staining,detection of bone transformation markers CTX,P1NP,and tibial metaphyseal osteogenesis and osteoclast related markers ALP,OCN,RUNX2,CTSK,NFATc1,TRAP were detected.Results:(1)PEMF(50Hz,1.6mT),PEMF(75Hz,1.6mT)and PEMF(15Hz,2mT)were the potential optimal intervention parameters for BMSC in vitro.The potential optimal intervention parameters for RAW264.7 cells in vitro were:PEMF(50Hz,1.6mT),PEMF(75Hz,1.6mT),PEMF(15Hz,0.5mT).and the potential optimal were used to treat OVX-induced osteoporosis mice were:PEMF(8Hz,1.6mT),PEMF(50Hz,0.5mT).1.6MT),PEMF(75Hz,1.6MT).(2)Flow cytometric phenotype analysis showed that the extracted cells were BMSCs.ALP staining and activity analysis showed that PEMF(15Hz,2mT)significantly increased the expression of ALP compared with the untreated group(P<0.05).Alizarin red staining and quantitative analysis showed that the absorbance value of Alizarin red in PEMF(50Hz,1.6mT)and PEMF(15Hz,2mT)groups was significantly increased compared with the untreated group,but the difference between the two groups was not statistically significant(P>0.05),while the absorbance value of alizarin red was not increased in PEMF(75Hz,1.6MT)(P>0.05).QRT-PCR showed that after osteogenic induction differentiation,the expression of osteogenic related genes ALP and OCN were significantly increased after PEMF(15Hz,2mT)intervention(P<0.05)compared with the untreated group,The expression of the above genes was decreased in PEMF(50Hz,1.6mT)and PEMF(75Hz,1.6MT)(P<0.05).For osteoclast precursor cells,compared with RANKL group,the number of TRAP positive cells(P<0.05)and cell volume were decreased in PEMF(15Hz,0.5mT)group,and the expression of osteoclast related genes(NFATcl and TRAP)were inhibited.However,after PEMF(50Hz,1.6mT)and PEMF(75Hz,1.6mT)treatment,the number and volume of TRAP staining positive cells did not decrease compared with the RANKL group.The expression of osteoclast related genes(CTSK,NFATc1,TRAP)was increased,and the expression difference of NFATc1,TRAP was statistically significant(P<0.05).(3)Micro-CT analysis showed that PEMF(50Hz,1.6mT)and PEMF(75Hz,1.6mT)intervention could significantly inhibit the decrease of Tb.N and BV/TV and the increase of Tb.SP(P<0.05)induced by OVX.However,PEMF(8Hz,1.6mT)group had no significant effect on the decrease of Tb.N,BV/TV and increase of Tb.SP induced by OVX(P>0.05).He staining and TRAP staining showed that the intervention of PEMF had an inhibitory effect on the morphological destruction of bone tissue after OVX,especially PEMF(50Hz,1.6mT)and PEMF(75Hz,1.6mT)could significantly improve the bone microstructure of bone trabeculae and reduce the number and size of osteoclasts(P<0.05).ELISA and qRTPCR analysis showed that compared with OVX mice,PEMF(50Hz,1.6mT)and PEMF(75Hz,1.6mT)significantly promoted the expression of osteogenic genes(OCN,Runx2)(P<0.05)and serum P1NP(P<0.05).PEMF(75Hz,1.6mT)1.6MT)was more obvious.In addition,PEMF(50Hz,1.6mT)and PEMF(75Hz,1.6mT)inhibited the secretion of bone resorption markers CTX-I and the expression of osteoclast related genes(CTSK,NFATc1,TRAP)(P<0.05),and PEMF(75Hz,1.6mT)had a more significant inhibitory effect.Conclusions:The more optimized treatment parameter for intervention of osteoblast precursor cells in vitro is PEMF(15Hz,2mT),and the more optimized treatment parameter for osteoclast precursor cells is PEMF(15Hz,0.5mT).PEMF(1.6mT,75Hz)is the optimal treatment parameter for osteoporotic mice in vivo.The third chapter:to clarify the biological mechanism of more optimized PEMF which were used to treat osteoporosis in vitro and in vivoObjective:To clarify whether more optimized PEMF parameters can inhibit the biological mechanism of NLRP3 inflammasome activation in the treatment of osteoporosis.Materials and Methods:The more optimized PEMF intervention parameters can be used to inhibit the activation of NLRP3 inflammasome to treat osteoblast precursor cells and the osteoclast precursor cells.(1)The specific method for osteoblast precursor cells is:BMSC cells were treated with H2O2 and divided into Control group,H2O2 group,H2O2+NAC group,H2O2+NLRP3 inhibitor group,and H2O2+PEMF(15Hz,2mT)group.ALP staining and activity analysis were performed after 7 days of osteogenic differentiation.After 14 days of osteogenic differentiation,alizarin red staining and quantitative analysis were performed,and the expression levels of ROS,antioxidant related genes SOD1,SOD2,GPX1 and CAT,and the expression levels of osteogenic related genes ALP,OCN,Runx2 and NLRP3 inflammasome related genes and proteins NLRP3,caspasel and ASC in different treatment groups were detected.(2)The specific method of osteoclast precursor cells is:Osteoclast precursor RAW264.7 cells were divided into RANKL induction group,RANKL+NAC group,RANKL+NLRP3 inhibitor group,and RANKL+PEMF(15Hz,0.5mT)group.TRAP staining and quantitative analysis were carried out after 7 days of induction,and the expression of ROS and antioxidant related genes SOD1,SOD2,GPX1 and CAT in different treatment groups were detected.The osteoclast related genes TRAP,CTSK and NFATcl,NLRP3 inflammasome related NLRP3,caspasel,ASC expression were also detected.(3)The biological mechanism of PEMF intervention parameters with optimized levels in vivo to treat osteoporotic mice by inhibiting NLRP3 inflammasome activation.The specific method is:After ovarian removal,the mice at 3 months were divided into Control group,OVX group,OVX+NLRP3 inhibitor group,and OVX+PEMF(75Hz,1.6mT).After one month of intervention,femoral micro-CT scan,pathological sections and staining were performed.The secretion of bone transformation markers CTX,P1NP and IL-B was detected by ELISA.The expression of NLRP3,caspasel and ASC protein in femoral epiphysis was detected by immunohistochemistry.qRT-PCR were used to detect osteogenesis and osteoclastrelated markers ALP,OCN,RUNX2,CTSK,NFATc1,TRAP,NLRP3 inflammasome NLRP3,caspasel,ASC expression.Results:(1)CCK8 experiment shows that 500uM H2O2 can be used as the best concentration of mouse BMSC injury model.After H2O2 treatment of BMSCs,the number of ALP active cells(p<0.05),the expression of mineralized nodules and osteogenic factors(OCN)were significantly down-regulated(p<0.05).NLRP3 inflammasome-related factors(NLRP3,ASC,Caspase-1)expression was significantly up-regulated(p<0.05),while pretreatment with MCC950(NLRP3 inhibitor)reversed the above trend,suggesting that H2O2 may cause osteogenic differentiation and mineralization of BMSCs through the activation of NLRP3 inflammasomes.NAC and PEMF(15Hz,2mT)can achieve results similar to MCC950 pretreatment.After NAC and PEMF(15Hz,2mT)pretreated the cells,the number of ALP active cells and the absorbance value increased,the alizarin red stained mineralized nodules and the absorbance value increased(p<0.05),and osteogenic genes(ALP,OCN)in PEMF(15Hz,2mT),the expression of RUNX2 in the NAC group was significantly upregulated(p<0.05).Compared with the H2O2 group,the expression of antioxidant genes(SOD1,SOD2,GPX1,CAT)in the PEMF(15Hz,2mT)group was significantly up-regulated(p<0.05),the intracellular reactive oxygen species(DCFH-DA fluorescence intensity)decreased significantly,and there was no statistical difference between NAC and H2O2 group,but the 4 antioxidant genes showed an up-regulation trend.In addition,compared with the H2O2 group,the expressions of NLRP3,ASC,and Caspase-1 in the PEMF(15Hz,2mT)and NAC groups were significantly downregulated(p<0.05).It is suggested that PEMF can alleviate the decline of BMSC osteogenic differentiation induced by H2O2 by inhibiting the ROS/NLRP3 axis.(2)RAW264.7 were induced by RANKL can produces more osteoclasts,larger osteoclasts,and osteoclast-related genes(CTSK,NFATc1,TRAP)and NLRP3 inflammasome-related genes(NLRP3,ASC,Caspase-1)expression is up-regulated;MCC950 treatment can inhibit the differentiation of RAW264.7 into osteoclasts,the expression of osteoclast genes and NLRP3 inflammasomes,suggesting that NLRP3 is involved in the formation of RANKL-induced osteoclasts.After the intervention of RANKL-induced osteoclasts with NAC and PEMF(15Hz,0.5mT),the number,volume and activity of osteoclasts decreased(P<0.05).The number and area of Factin rings in the cells and the number of bone resorbed lacunae showed a decreasing trend.The expression of osteoclast related genes(CTSK,TRAP and NFATcl)decreased,and the difference of expression of TRAP and NFATcl in the NAC group and PEMF(15Hz,0.5mT)group was statistically significant.In addition,the expression of NLRP3 inflammasome related genes(NLRP3,ASC,Caspase-1)in PEMF(15Hz,0.5mT)group was down-regulated,and the difference of ASC expression was statistically significant.The expression of antioxidant genes(SOD1,SOD2,GPX1,CAT)was up-regulated,and the difference in expression of SOD1 and SOD2 in NAC and PEMF(15Hz,0.5mT)group was statistically significant,and the intracellular reactive oxygen species(DCFH-DA fluorescence intensity)was decreased.These results suggest that PEMF can alleviate the osteoclast formation induced by RANKL by inhibiting the ROS/NLRP3 axis.(3)Micro-CT analysis showed that compared with the OVX group,NLRP3 inhibitor treatment and PEMF(75Hz,1.6mT)intervention can significantly inhibit the decrease of Tb.N,BV/TV and the increase of Tb.Sp induced by OVX(p<0.05)),but has no effect on Tb.Th(p>0.05).It can be observed by HE and TRAP staining that NLRP3 inhibitor and PEMF intervention can inhibit the destruction of bone tissue after OVX,can significantly improve the bone microstructure of trabecular bone,and reduce the number and size of osteoclasts(p<0.05).ELISA analysis found that compared with the OVX group of mice,NLRP3 inhibitor treatment and PEMF(75Hz,1.6mT)intervention significantly inhibited the secretion of bone resorption markers CTX-I and IL-1β(p<0.05),and increased serum P1NP levels(p>0.05).The qRT-PCR test results showed that compared with the OVX group,the expression of osteogenic genes(ALP,OCN,Runx2)mRNA was up-regulated after PEMF(75 Hz,1.6mT)and NLRP3 inhibitor treatment,and the up-regulation of OCN was statistically significant(P<0.05).After PEMF(75 Hz,1.6mT)and NLRP3 inhibitor intervention,the expression of osteoclast-related genes(CTSK,NFATc1,TRAP)decreased.Among them,PEMF(75Hz,1.6 mT)intervention group(CTSK,NFATc1,TRAP),NLRP3 inhibitor group(NFATc1)was statistically significant(p<0.05).The above studies suggest that NLRP3 inhibitors and PEMF intervention can improve the degeneration of bone microstructure in OVX models and increase bone mass.In addition,compared with the OVX group,the PEMF(75Hz,1.6mT)and NLRP3 inhibitor groups increased the expression of antioxidant genes(SOD1,SOD2,GPX1,CAT)after intervention,and the difference in the expression of GPX1 and CAT was statistically significant(P<0.05).It is suggested that PEMF(75Hz,1.6mT)group and NLRP3 inhibitor treatment can reduce the increase of ROS induced by OVX.However,compared with the OVX group,after the intervention of NLRP3 inhibitor and PEMF(75Hz,1.6mT),the expression of NLRP3,ASC,and caspasel genes and proteins in the femur showed a downward trend,but the difference was not statistically significant(P>0.05).Conclusions:PEMF can improve osteogenic differentiation,inhibit osteoclast formation in vitro,and reverse bone loss after OVX in vivo.Mechanism studies have shown that this is related to the decrease in the expression of ROS and NLRP3 inflammasomes after PEMF intervention,suggesting that PEMF can treat osteoporosis may be closely related to the inhibition of the ROS/NLRP3 axis signaling pathway.