Study On The Therapeutic Effects And Mechanism Of Tibetan Medicine Ershiwuwei Guijiu Pills In Rats With Cervicitis

Objective:Taking the Tibetan medicine Ershiwuwei Guijiu pills（EGP）as the research object,by throughing the experimental study of antibacterial activity in vitro to explore the inhibitory effects of EGP on the common pathogenic bacterium Staphylococcus aureus（SA）and by establishing two models of cervicitis in rats to explore the potential mechanism of EGP effects on cervicitis from the perspective of oxidative stress and cell apoptosis,so as to provide experimental basis for the clinical promotion and secondary development of EGP.Methods:1.Study on the antibacterial activity of EGP in vitroThe size of the inhibition zone was determined by agar plate drilling method;The minimum inhibitory concentration（MIC）was determined by micro broth dilution method;The growth curve of bacteria,and the relative amount of extracellular nucleic acid and soluble protein content were determined by ultraviolet spectrophotometer（UV）;Alkaline phosphatase（AKP）activity was determined by microplate reader;The total protein content of bacteria was determined by SDS-PAGE gel electrophoresis.2.Study on the therapeutic effects and mechanism of EGP on rats model of cervicitis induced by phenol mucilageSD female rats were randomly divided into blank group,model group,low,medium,high(0.2,0.4,0.8 g·kg^-1)dose group of EGP,and anti-uterine tablet group(0.4725 g·kg^-1).Except for the blank group,the rat model of cervicitis caused by phenol mucilage was established by injecting 25%phenol mucilage deep into the vagina.After12 days of intragastric administration,the general condition and uterine index of rats were observed;Hematoxylin and eosin（HE）staining was used to observe the histopathological damage in uterine tissue;Td T-mediated d UTP nick-end labeling（TUNEL）staining was used to observe cell apoptosis in uterine tissue;Enzyme-linked immunosorbent assay（ELISA）method was used to detect the contents of superoxide dismutase（SOD）and malondialdehyde（MDA）in uterine tissue;Quantitaive real-time polymerase chain reaction（q RT-PCR）method was used to detect the expression of nuclear factor erythroid 2-related factor（Nrf2）,heme oxygenase-1（HO-1）,and NADPH oxidase 4（Nox4）m RNA in uterine tissue;Western blot method was used to detect the expression of Nrf2,HO-1,and Nox4 protein in uterine tissue.3.Study on the therapeutic effects and mechanism of EGP on rats model of cervicitis induced by phenol mucilage mixed with Staphylococcus aureus infectionSD female rats were randomly divided into blank group,model group,medium dose(0.4g·kg^-1)group of EGP,and anti-uterine tablet group.Except for the blank group,the rats model of cervicitis induced by phenol mucilage mixed with SA was established by successively injecting 25%phenol mucilage and SA deep into the vagina.After 12days of intragastric administration,the general condition and uterine index of rats were observed;HE staining was used to observe the histopathological damage in uterine tissue;TUNEL staining was used to observe cell apoptosis in uterine tissue;ELISA method was used to detect the contents of SOD and MDA in uterine tissue;q RT-PCR method was used to detect the expression of Nrf2,HO-1,Nox4,B lymphocytoma-2（Bcl-2）,Bcl2 associated X protein（Bax）,Cysteine protease 3（caspase-3）,Cysteine protease 9（caspase-9）m RNA in uterine tissue;Western blot method was used to detect the expression of oxidative stress protein Nrf2,HO-1,Nox4,and apoptotic damage-related protein Bcl-2,Bax,cleaved caspase-3,and cleaved caspase-9 in uterine tissue.Results:1.Study on the antibacterial activity of EGP in vitroThe inhibition zone diameters of EGP at concentrations of 50,100 and 200mg·m L^-1against SA were（12.33±0.75）mm,（16.33±0.41）mm and（19.17±0.68）mm,respectively;The MIC against SA was 100 mg·m L^-1;Compared with control group,the growth of SA was significantly slowed down（P<0.05）,the extracellular AKP activity was significantly increased（P<0.05）,the relative amount of extracellular nucleic acid was significantly increased（P<0.01）,the content of extracellular soluble protein was significantly increased（P<0.01）,and the expression of total bacterial protein was significantly decreased.2.Study on the therapeutic effects and mechanism of EGP on rats model of cervicitis induced by phenol mucilageCompared with blank group,the weight gain of the model group was significantly slowed down（P<0.01）,the score of vulvar inflammation was significantly increased（P<0.01）,the uterine index was significantly increased（P<0.01）,the activity of SOD was significantly decreased（P<0.01）,and the expression of Nrf2,HO-1 m RNA and protein were significantly decreased（P<0.01）,the content of MDA was significantly increased（P<0.01）,the expression of Nox4 m RNA and protein were significantly increased（P<0.01）;Compared with model group,the body weight of rats in each administration group of EGP was significantly increased（P<0.01）,the score of vulvar inflammation was significantly decreased（P<0.01）,the uterine index was significantly decreased（P<0.05）,the activity of SOD was significantly increased（P<0.05）,the expression of Nrf2,HO-1 m RNA and protein were significantly increased（P<0.01）,the content of MDA was significantly decreased（P<0.05）,the expression of Nox4m RNA and protein were significantly decreased（P<0.01）;The results of HE staining showed that EGP could significantly reduce the degree of inflammation in rats;The results of TUNEL staining showed that EGP could significantly reduce cell apoptosis.3.Study on the therapeutic effects and mechanism of EGP on rats model of cervicitis induced by phenol mucilage mixed with SA infectionCompared with blank group,the weight gain of the model group was significantly slowed down（P<0.01）,the score of vulvar inflammation was significantly increased（P<0.01）,the uterine index was significantly increased（P<0.01）,the activity of SOD was significantly decreased（P<0.01）,the expression of Nrf2,HO-1,Bax,caspase-3,caspase-9 m RNA and protein were significantly decreased（P<0.01）,the content of MDA was significantly increased（P<0.01）,the expression of Nox4,Bcl-2 m RNA and protein were significantly increased（P<0.01）;Compared with model group,the body weight of rats in medium dose group of EGP was significantly increased（P<0.01）,the score of vulvar inflammation was significantly decreased（P<0.01）,the uterine index was significantly decreased（P<0.05）,the activity of SOD was significantly increased（P<0.05）,the expression of Nrf2,HO-1,Bax,caspase-3,caspase-9 m RNA and protein were significantly increased（P<0.01）,the content of MDA was significantly decreased（P<0.05）,the expression of Nox4,Bcl-2 m RNA and protein were significantly decreased（P<0.01）;The results of HE staining showed that EGP could significantly reduce the degree of inflammation in rats;The results of TUNEL staining showed that EGP could significantly reduce cell apoptosis.Conclusion:In this study,two models of cervicitis in rats were established to investigate the potential mechanism of action of EGP in the treatment of cervicitis from the perspective of oxidative stress,and it was found that EGP could decrease the expression of oxidative stress protein,increase the expression of antioxidative stress protein.By inhibiting the expression of Nox4,activating the Nrf2/HO-1 signaling pathway,it can exert antioxidant effect,reduce inflammatory reaction and cell apoptosis,thereby inhibit the development of cervicitis.