The Mechanism Of Shengjiang Powder In The Treatment Of Osteomyelitis Based On The Intervention Of CGAS/STING Pathway On Immune Escape Of Staphylococcus Aureus Biofilm

Objective1.A retrospective study on the clinical efficacy of Shengjiang Powder in the treatment of tibia fracture related infection.This study provides reference for the clinical treatment of tibia fracture related infection.2.To investigate the role of cGAS/STEING pathway in the intervention of osteomyelitis caused by Staphylococcus aureus biofilm and the mechanism of biofilmmediated immune escape.3.To explore the feasibility and mechanism of Shengjiang Powder in the treatment of osteomyelitis under the guidance of the theory of " Huo Yu Fa Zhi ".Methods1.Between January 2010 and January 2022,a total of 43 patients were included in the study,with 22 patients in the observation group and 21 patients in the control group.The observation group was treated with Shengjiang Powder combined with debridement and antibiotic bone cement,while the control group was treated with debridement and antibiotic bone cement.The basic data of the two groups were recorded,and the changes of inflammatory indicators were dynamically observed and recorded on the first day,the seventh day,the fourteenth day and the twenty-eighth day after treatment,as well as the infection control after one month of treatment and the recurrence rate of infection after six months.2.Animal experimentsStaphylococcus aureus biofilm steel plate was cultured,and the plate with biofilm was implanted into the femur of rats to establish osteomyelitis(BIO-OM)rat model.After modeling,the experiment is divided into two parts.(1)In the study of the role of cGAS/STING pathway in the intervention of osteomyelitis caused by Staphylococcus aureus biofilm and its mechanism,the animals were divided into six groups:①Sham operation group,②Ctrl group,③Staphylococcus aureus group:implanted with Staphylococcus aureus plate,④Biofilm group:implanted with biofilm plate.⑤STING activation(ADU-S100)group,⑥STING inhibitor(C-178)group.Each group had 12 rats.(2)In the study of the feasibility and mechanism of Shengjiang Powder in the treatment of osteomyelitis,the animals were divided into five groups:①Sham group,②Model group,③Levaquin group,④SJS-High-dose group and⑤SJS-Low-dose group.Each group had 12 rats.After modeling,the growth and quantity of biofilm on the surface of steel plate were analyzed by scanning electron microscope(SEM).The differences between BIO-OM rats and control rats were observed by X-ray,micro-CT and pathological tissue sections.The expression levels of cGAS,STING,TBK1,IRF3,NF-kB and mRNA were compared between BIO-OM rats and control rats by quantitative PCR and Western Blot.3.Cell experiment(1)Establish a Sting-activated and Sting-inhibited macrophage cell line and a macrophage-biomembrane co-culture model,and detecting cell proliferation and apoptosis by flow cytometry;The expressions of cGAS,STING,TBK1,IRF3,NF-kB and mRNA were detected by quantitative PCR and Western Blot in macrophages infected with biofilm and control cells.The expression levels of pro-inflammatory cytokines were detected by ELISA.Bacterial phagocytosis assay was used to observe the survival ability of biofilm in cells and the bactericidal ability of macrophages,and to evaluate the effect of Staphylococcus aureus biofilm on the immune response of macrophages.(2)Macrophage-biofilm co-culture model was established,and cell proliferation and apoptosis were detected by flow cytometry.The protein and mRNA expression levels of cGAS/STING signaling pathway related molecules in each group were compared by quantitative PCR and Western Blot,and the protein expression levels of TNF-α,IL-6,IL-1 β,IL-17 and IL-23 immune response related proteins in each group were also compared.Bacterial phagocytosis test was used to observe the effect of different concentrations of Shengjiang Powder on the immune response of macrophages.Results1.Shengjiang Powder had a good short-term effect on patients with tibial fracture related infection.There was no significant difference in age,sex,injury site,injury mechanism,fracture type and open fracture between the two groups.WBC,CRP and ESR had no significant difference on the first day of treatment(P>0.05).There was statistical significance on the 7th day,14th day and 28th day after treatment(P<0.05),and the observation group was better than the control group.One month after treatment,in the observation group,18 cases were cured,3 cases were improved,and 1 case was not cured.The cure rate was 81.82%and the effective rate was 95.45%.In the control group,15 cases were cured,3 cases were improved and 3 cases were not cured.The cure rate was 71.43%and the effective rate was 85.71%.There was no significant difference in infection control between the two groups one month after treatment(P>0.05).After 6 months of treatment,in the observation group,there were 3 cases of recurrence and 19 cases of no recurrence,the recurrence rate was 13.64%.In the control group,there were 9 cases with recurrence and 12 cases without recurrence,the recurrence rate was 42.86%.The recurrence rate of the two groups after 6 months of treatment was statistically significant(P<0.05),and the observation group was better than the control group.There were no safety issues in either group.2.Inhibition of the expression of cGAS/STING pathway can significantly improve the periosteal reaction and inflammatory reaction in rats with Staphylococcus aureus osteomyelitis.In rat osteomyelitis model,the protein and mRNA expression levels of cGAS/STING signaling pathway in each group were significantly different.The expression levels of cGAS and STING in S.Aureus and Biofilm groups were higher than those in Sham group,and were significantly different from those in S.Aureus and Biofilm groups,the protein expression of cGAS and STING in ADU-S100 group was significantly increased(P<0.05),and the protein expression of cGAS and STING in C-178 group was significantly decreased(P<0.05)compared with S.Aureus and Biofilm groups.The expression levels of TBK1,IRF3 and NF-κB in ADU-S100 group were significantly higher than those in Sham group(P<0.05),and the protein expression levels in S.Aureus and Biofilm group were also significantly higher than those in Sham group(P<0.05).The expression levels of TBK1,IRF3 and NF-κB in C-178 group were lower than those in S.Aureus and Biofilm group(P<0.05).The expression levels of TNF-α,IL-6,IL-β,IL-17 and IL-23 immune response related proteins were detected by ELISA.Compared with the blank group,the immune response levels of TNF-α,IL-6,IL-1β,IL-17 and IL-23 in the macrophage-Staphylococcus aureus group,the macrophage-Staphylococcus aureus biofilm group and the Stingactivated macrophages-Staphylococcus aureus biofilm group increased(P<0.05).Compared with the macrophage-staphylococcus aureus biofilm group,the immune response level of the Sting activated macrophage-staphylococcus aureus biofilm group was increased,and the immune response level of the Sting inhibited macrophagestaphylococcal biofilm group was significantly decreased(P<0.05).The results of CCK8 cell viability test in co-cultured cells of each group showed that compared with the macrophage-S.aureus biofilm group,the cell proliferation activity of the Sting activated macrophage-S.aureus biofilm group was increased(P<0.05),and the cell viability of the Sting inhibited macrophage-S.aureus biofilm group was significantly decreased(P<0.05).The protein and mRNA expression levels of cGAS,STING,TBK1,IRF3 and NF-κB were detected by Western Blot and RT-PCR.Compared with the blank group,the protein and mRNA expression levels of cGAS,STING,TBK1,IRF3 and NF-κB in the macrophage-Staphylococcus aureus group,the macrophage-Staphylococcus aureus biofilm group and the Sting-activated macrophage-S.aureus biofilm group were increased(P<0.05).Compared with the macrophage-S.aureus biofilm group,the expression level of the above factors was increased in the Sting-activated macrophage-S.aureus biofilm group,and was decreased in the Sting inhibited macrophage-S.aureus biofilm group(P<0.05).Compared with the blank group,the immune response levels of TNF-α,IL-6,IL-1β,IL-17 and IL-23 were increased in the macrophage-Staphylococcus aureus group,the macrophage-Staphylococcus aureus biofilm group and the Sting-activated macrophage-S.aureus biofilm group(P<0.05).Compared with the macrophage-S.aureus biofilm group,the immune response level of the Sting activated macrophage-S.aureus biofilm group was increased,and the immune response level of the Sting inhibited macrophage-S aureus biofilm was significantly decreased(P<0.05).The phagocytic efficiency of the macrophage-staphylococcus aureus biofilm group,the macrophage-staphylococcus aureus biofilm group,the Sting-activated macrophages-staphylococcus aureus biofim group and the Sting-inhibited macrophages-staphylococcus aureaus biofim group decreased to different degrees,and the phagocytic function of the Sting-activated macrophages-saureus biofim was significantly decreased(P<0.05).3.Shengjiangsan regulates immune escape mediated by Staphylococcus aureus biofilm in osteomyelitis infection through cGAS-STING pathway.The rats with osteomyelitis were treated with Shengjiang Powder,and the protein expression levels of cGAS/STING signaling pathway in each group were detected,including cGAS,STING and their signaling pathway related molecules TBK1,IRF3 and NF-κB.The protein expression levels of cGAS and STING in the model group were significantly higher than those in the sham operation group(P<0.05).Compared with the model group,the expression levels of cGAS and STING in SJS-High-dose group,SJSLow-dose group and the positive drug Levaquin group were significantly decreased(P<0.05).Compared with the sham operation group,the mRNA expression levels of cGAS,STING,TBK1,IRF3 and NF-κB in the model group were significantly increased(P<0.05).Compared with the model group,the expression levels of cGAS,STING,TBK1,IRF3 and NF-κB mRNA in SJS-High-dose group,SJS-Low-dose group and the positive drug Levaquin group were significantly decreased(P<0.05).CCK8 method was used to detect cell viability and proliferation,and cell proliferation activity was observed.The results showed that compared with the blank group,the cell proliferation ability of the model group was significantly enhanced(P<0.05).After each group was cultured for 120 minutes after the intervention of drugcontaining serum,the protein expression levels of signal molecules related to cGAS/STING signaling pathway were detected,including cGAS,STING,TBK1,IRF3 and NF-κB.The expression levels of cGAS and STING in the model group were higher than those in the blank group(P<0.05).Compared with the model group,the expression levels of cGAS and STING in the Levaquin group,SJS-High-dose group,SJS-Low-dose group were significantly decreased(P<0.05),while the expression levels of TBK1,IRF3 and NFκB in the model group were increased(P<0.05).Compared with the model group,the levels of TBK1,IRF3 and NF-κB were down-regulated in the Levaquin group,SJS-Highdose group,SJS-Low-dose group.Compared with the blank group,the expression levels of cGAS,STING,TBK1,IRF3 and NF-κB mRNA in the model group were increased(P<0.05).Compared with the model group,the expression levels of cGAS,STING,TBK1,IRF3 and NF-κB mRNA in the Levaquin group,SJS-High-dose group,SJS-Low-dose group were decreased(P<0.05).The expression of TNF-α,IL-6,IL-1β,IL-17 and IL-23 immune response related proteins in the model group was significantly increased after the intervention of Shengjiang Powder medicated serum(P<0.05).Compared with the model group,the expressions of TNF-α,IL-6,IL-1β,IL-17 and IL-23 in the Levaquin group,SJS-High-dose group,SJSLow-dose group were significantly decreased(P<0.05).Compared with the blank group,the phagocytic efficiency of the model group,the Levaquin group,SJS-High-dose group,SJS-Low-dose group decreased in different degrees.Compared with the model group,the phagocytic efficiency of the Levaquin group,SJSHigh-dose group,SJS-Low-dose group increased in different degrees.Conclusion1.For patients with tibial fracture related infection,Shengjiang Powder combined with debridement and antibiotic bone cement has a good short-term effect.Sheng-jiang Powder is helpful for the decrease of blood inflammatory indexes in the acute stage of tibial fracture related infection,and can reduce the recurrence rate of tibial fracture-related infection after treatment.2.Staphylococcus aureus biofilm mediates immune escape through the cGAS/STING signaling pathway in osteomyelitis.3." Shengjiang Powder " regulates the body’s immune response by targeting the cGAS/STING pathway,inhibits the immune escape of the biofilm of Staphylococcus aureus,and creates favorable conditions for the cure of osteomyelitis infected by Staphylococcus aureus.