| Objective: This study aims to clarify the relationship between antioxidant and SHP2-mediated cytokines produced by Lactobacillus plantarum LP45 and osteoblast/osteoclast differentiation and apoptosis,and to elucidate the mechanism by which LP45 antagonizes glucocorticoid-induced changes in osteoblast and osteoclast functions and oxidative stress,so as to elucidate the mechanism of Lactobacillus plantarum LP45 intervention in osteoporosis at the molecular level and provide a new scientific basis and new ways for This will provide a new scientific basis and a new approach for the early prevention of osteoporosis.Methods:1.Macrophages RAW264.7 were divided into LPS group,LP45 group,LPS+LP45 group and blank control group,and the levels of intracellular oxidative stress response indicators were detected by oxidative stress probe staining,and the expression of NOX4,P22,P47 oxidative stress-related proteins and IL-1β,NLRP3,IRF3,INF-β inflammatory factor-related by ELISA protein expression as well as MDA and SOD concentrations.2.The macrophage osteoblast and macrophage osteoblast culture systems were completed in truss well chambers and divided into LPS,LP45,LPS+LP45 and empty control groups,and the expression of RANKL was detected by immunofluorescence staining RANKL expression was detected by immunofluorescence staining.and the expression of OPG,RANKL,RUNX3,β-catenin,osteoblast-associated proteins and NF-κB,CREB,AP-1 and osteoblast-associated proteins were determined by protein immunoblotting,while osteoblast proliferative potential was determined by CCK-8.Results:1.The results of RAW264.7 macrophage oxidative stress levels in each group showed that fluorescence microscopy revealed that ROS levels were significantly lower in the LPS group compared to the blank control group(P<0.01);ELISA results showed significantly lower MDA values and higher SOD values in the LP45 group compared to the control group.(P<0.01);MDA values were significantly increased and SOD values were significantly decreased in the LPS and LPS+LP45 groups compared to the LP45 group(P<0.01);MDA values were significantly decreased and SOD values were significantly increased in the LPS group compared to the control group.(P<0.01);2.The expression of RAW264.7 oxidative stress-related macrophage protein in each group showed NOX4 and P47 expression in the LP45 group compared to the empty control group(P<0.01);P47 expression in the LPS group and LPS +LP45 group significantly increased NOX4,P22 and P47expression(P<0.01);LPS+LP45 group significantly decreased NOX4,P22 and P47 expression(P<0.01);LPS+LP45 group significantly decreased NOX4,P22 and P47 expression(P<0.01);expression of NOX4,P22 and P47 was further reduced in the LPS+LP45 group compared with the LP45 group;3.In terms of SHP2 expression in macrophages in each group,SHP2 expression was significantly reduced in the LPS and LP45+LPS groups compared with the LP45 group(P<0.01),while SHP2 expression was significantly increased in the LP45+LPS group compared with the LPS group(P<0.01);in the expression levels of inflammatory factor-related proteins in each group,SHP2 expression was significantly increased in the LPS group compared with the LP45 group(P<0.01);compared with the LP45 group,the LPS group and The expression levels of IL-1β,NLRP3,IRF3 and INF-β were significantly increased in the LP45+LPS group(P<0.01),while the expression levels of L-1β,NLRP3,IRF3 and INF-β were suppressed and significantly decreased in the LP45+LPS group compared to the LPS group(P<0.01).4.All groups Results of osteoblast activity and RANKL immunofluorescence staining showed that osteoblast activity after 48 hours of culture using the CCK assay was significantly different between the empty control,LPS and LP45 groups(P<0.01);immunofluorescence staining showed that RANKL expression in the LP45 group osteoblasts was significantly lower than in the control group(P<0.01).The immunofluorescence results of RANKL expression also showed that RANKL expression was significantly lower in the LP45 group compared to the empty control group(P<0.01);expression was significantly higher in the LPS and LPS+LP45 groups compared to the LP45 group(P<0.01);in the LPS+LP45group compared to the LPS group5.The expression of osteoblast-associated proteins in each group showed that the expression of RANKL was significantly higher in the LPS and LP45+LPS groups compared to the LP45 group(P<0.01);the expression of RANKL was significantly lower in the LP45+LPS group compared to the LPS group(P<0.01);the expression of RANKL was significantly higher in the LPS group compared to the LPS group(P<0.01);the expression of RANKL was significantly higher in the LPS group compared to the LP45 group(P<0.01);the expression of RANKL was significantly higher in the LPS group compared to the LP45 group(P<0.01).LPS group was significantly lower(P<0.01);OPG,RANKL and RUNX3 expression levels were significantly increased in the LP45 group compared to the empty control group(P<0.01);OPG,RANKL and RUNX3 expression levels were significantly increased in the LPS and LP45+LPS groups compared to the LP45 group(P<0.01);significantly decreased OPG,RANKL and RUNX3 expression levels in the LP45+LPS group compared with the LP45 group(P<0.01);significantly decreased OPG and RANKL expression levels in the LP45+LPS group compared with the LPS group(P<0.01).6.In the LP45+LPS group,the LPS group decreased expression levels of OPG,RANKL and RUNX3 compared to the LPS group(P<0.01).The expression levels of osteoclast-associated proteins in each group were examined: the expression levels of NF-κB,CREB and AP-1 were significantly lower in the LP45 group compared to the empty control group(P<0.01);compared to the LP45 group,after LPS addition and LP45 +LPS group significantly increased the expression levels of NF-κB,CREB and AP-1(P<0.01);NF-κB and CREB expression levels were significantly decreased in the LP45+LPS group compared with the LPS group(P<0.01)and AP-1expression level was decreased(P<0.05);Conclusions:LPS mediates the generation of oxidative stress,degrades SHP2 and related kinase activation to disrupt osteoblast differentiation and promote osteoclastogenesis.LP45 can play an antioxidant and anti-inflammatory role by inhibiting the production of oxidative stress and inflammatory factors to activate the SHP2 signaling pathway,promote osteoblast differentiation and inhibit osteoclastogenesis to maintain bone homeostasis and improve bone metabolism. |
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