Study On The Antibacterial Effect, Anti-inflammatory And Antioxidant Mechanism Of Tibetan Medicine Sanchensan On Bacterial Pneumoni

Objective:On the basis of previous studies,the antibacterial effect of Sanchen Powder(SCP)alone and combined with vancomycin(VAN)on methicillin resistant Staphylococcus aureus(MRS A)and its mechanism were further observed in vitro;We explored the therapeutic effect of SCP on pneumonia and its mechanism of anti inflammation and anti-oxidation via in vivo,providing experimental basis for clinical treatment of children’s pneumonia with SCP.Method:(1)In vitro antibacterial experiment:SCP was extracted with 50%ethanol to obtain the ethanol extraction of Sanchen Powder(ESCP).① The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of ESCP and the MIC value of VAN were measured by standard micro broth dilution method;② The fractional inhibitory concentration index(FICI)of ESCP combined with VAN was observed by the checkerboard method;③ The effect of ESCP and combined with VAN on MRSA in mature biofilm was observed by using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium5carboxanilide reduction test(microplate method)combined with scanning electron microscope to evaluate the effect of ESCP combined with VAN on MRSA biofilm;④ The effect of ESCP combined with VAN on MRSA virulence factors was observed by plasma coagulase test and DNA enzyme test.(2)In vivo experiment:First,the young rats were divided into blank group and moldmaking group.The young rats in the model-making group were induced pulmonary inflammation by oral and tracheal instillation of lipopolysaccharide(LPS),and were divided into high dose SCP group(HSCP group),low dose SCP group(LSCP group),dexamethasone group(DEX group)and model group(Model group).After 2 h of LPS interference,SCP,dexamethasone and the control solvent were administered for 2 and 4 days,and the materials were taken 24 h after the last administration.① Observe the general condition and body weight of young rats;② Peripheral blood was taken to test the four items of blood coagulation and blood routine test;③ HE staining was used to observe the pathology of lung tissue;④Enzyme linked immunosorbent assay(ELISA)was used to detect the content of hypothalamic fever central mediator and the inflammatory factors of bronchoalveolar lavage fluid(BALF);⑤The activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)in lung tissue were detected by biochemical method;⑥The content of TLR4,IκB α,p65,Nrf2,p62 protein in TLR4/NF-κB and Nrf2/p62 pathway in lung tissue was detected by western blot to study the therapeutic effect of SCP on pneumonia and its mechanism of anti inflammation and anti-oxidation.Result:(1)In vitro antibacterial experiment:① MICs of ESCP and VAN were 25.6 mg/mL and 2 μg/mL respectively by standard micro broth dilution method.The MBC of ESCP is 51.2 mg/mL;② The checkboard method showed that FICI=2,ESCP and VAN had no relation;③ Microwell plate experiment and coagulase experiment showed that the inhibition of bacteria growth in mature biofilm and coagulase by ESCP and VAN combined application was better than that by single application;④ESCP and VAN alone or in combination have no significant effect on DNA hydrolase.(2)In vivo experiment:① The coagulation function of the model animals was normal,and SCP had no significant effect on the normal coagulation function of the animals;② At the 4th day,HSCP,LSCP and DEX groups could significantly reduce the number of neutrophils(p<0.001),but no significant effects on peripheral blood leukocytes,platelet counts and eosinophils were observed;③The pathological score of lung tissue in HSCP group was significantly lower than that in model group(p<0.01),while LSCP group and DEX group had no significant improvement on lung injury condition;④ The HSCP group can significantly increase cyclic adenosine monophosphate(cAMP)(p<0.001)and reduce prostaglandin E(PGE)(p<0.001),and its inhibitory effect on PGE is significantly better than LSCP group(p<0.01)and DEX group(p<0.01).LSCP group and DEX group had effects on cAMP and PGE only after 4 days of administration.HSCP group,LSCP group and DEX group had no significant effect on hypothalamic arginine vasopressin(AVP);⑤ In this experiment,we observed that HSCP and LSCP had significant effects on the cytokines downstream of TLR4/NF-κB pathway,but no significant effects on the expression of TLR4,p65,IκBα and other proteins in the pathway.HSCP group and DEX group significantly inhibited the levels of IL-6,TNF-α and IL-4 at 2 and 4 days,and promoted IL-10 and TGF-β.LSCP group inhibited IL-6 at 2 days(p<0.05),and significantly inhibited IL-6,TNF-α and IL-4 at 4 days,and increased IL-10 and TGF-β;⑥In this study,it was observed that HSCP and LSCP had an effect on the expression of Nrf2,p62 proteins in Nrf2/p62 signal pathway,but there was no significant difference.However,HSCP group had a significant inhibitory effect on the MDA level(p<0.01),and significant increase on the SOD activity(p<0.05)at 4 days,LSCP group could significantly reduce the MDA level(p<0.05)at 4 days,and DEX group could reduce the MDA level at two time points.The HSCP group was significantly better than LSCP group and DEX group in all the above indicators.The HSCP group had significant effect after 2 days of administration,while the LSCP group had significant effect after 4 days of administration.The HSCP group and LSCP group had better effect after 4 days of administration than 2 days.Conclusion:(1)In vitro antibacterial experiment:① ESCP has antibacterial effect on MRS A,mainly manifested in the inhibition of planktonic bacteria,coagulase and the destruction of biofilm integrity;② The combination of ESCP and VAN has a significant inhibitory effect on MRS A coagulase and the amount of viable bacteria in mature biofilm.(2)In vivo experiment:① SCP does not affect the coagulation function of normal blood coagulation system;② SCP can inhibit inflammatory reaction by reducing the levels of L-6 and TNF-α,while SCP can increase IL-10,TGF-β and reducing the level of IL-4 to regulate the inflammatory response to achieve anti-inflammatory effect;③SCP can play an antioxidant role by significantly increasing SOD activity and reducing MDA level.This study only observed the trend of the influence of SCP on the expression of upstream proteins TLR4,p65,IκBα,Nrf2,p62 and other proteins of inflammatory reaction,but there was no significant statistical significance,which needs further study.