Studies On Oxidative Stress In Vaginal Discharge Of Patiants With Vaginitis And Its Induced Vaginal Epithelial Cell Apoptosis

BackgoundDiseases of the genitourinary system is a common and frequently encountered disease, which including vulvitis, vaginitis, cervicitis, endometritis, salpingitis and pelvic inflammation. Vaginitis is one of the most common genital system disease of women. The infected vaginitis are divided into three types:Bacterial vaginosis, Vulvovaginal candidiasis and trichomonas vaginitis. At present, the specific pathogenic mechanism of viginitis is also elusive, and the more received mechanism is that vaginitis is a discruption or drift of normal microbiota of bacteria in the vagina where high concentration of aerobia and anaerobia take the place of lactobacillus and become the dominant bacteria. So the microbial population balance is destroyed. Discuss the relation between microbial population and various vaginitis has become a critical problem in the vaginitis prevention and treatment. There are also no related studies about antioxidants activity in the vaginal discharge and vaginitis pathogenic mechanism. We utilized the case resources and the patients’vaginal discharge of the second hospital of Lanzhou University to investigate the activities of antioxidants in viginal discharge and viginal mucosa epithelial cell associated apoptosis. This research could provide an important rationale foundation for the treatment and prevention of women viginitis.ObjectivesThe antioxidants, lipid peroxidation and vaginal lactobacillus in the vaginal discharge were observed for the first time. At the meantime, we investigate the specific mechanism of vaginitis induced apoptosis in bacterial vaginitis, monilial vaginitis and trichomonas vaginitis using the vaginal discharge of patients. Comparing the different mechanism of vaginitis for the apoptosis, and discussing the relationship between apoptosis and antioxidants and lipid peroxidation.Contents and Methods1.Vaginal discharge antioxidants activity study method in vaginitis patientsA speculum was used to collect vaginal discharge from posterior fornix of the vaginal canal. All samples were collected used the same test system which contained two swabs. One cotton swab was placed in a tube containing2ml of saline and sent to be evaluated microscopically for the presence of bacterial, candida, trichomoniasis vaginitis and evaluated for the lactobacillus accounts. The another cotton swabs were placed in a tube containing lml of PBS and stored at-70℃for further MDA, SOD, CAT, H2O2, pH and Vitamin C detection.2. Oxidative stress induced vaginal mucosa epithelial cell apoptosisThe mucosa epithelial cells were obtained clinically from the bacterial vaginitis, candida vaginitis and trichomoniasis vaginitis patients. The cells viability and apoptosis were detected in the three above vaginal mucosa epithelial cells. Indirect immunofluorescence (IFA)and western blot analysis were used to identify the activated caspase3.The DNA ladder experiment was employed to detect the apoptosis in the vaginitis and normal women vagina.The apoptosis related mitochondrial pathway proteins, such as Bcl-2family proteins and apoptosis family proteins, and ER stress associated proteins, such as Grp78, CHOP, caspase12, were estimated by western blot method. Thus we combinated the first part and this part study to analyze aggregately, and predict the correlation between antioxidants activity and lipid peroxidation level and the apoptosis event in the vaginal mucosa epithelial cells of the patients.Results1.Vaginal discharge pathogenic bacteria results1.1Vaginis kinds and patients age distruibution A total of197female patients (aging20～49years, average age34.5years) with vaginitis were involved in this study. Meanwhile, a total of60healthy females aged24～47years (average age35.1years) as the control group. Moreover, there was no significant difference in ages among the four groups (P>0.05). We observed the distribution of the bacterial vaginitis, candida vaginitis and trichomoniasis vaginitis in all of the197patients, and discovered that bacterial vaginitis66cases (account for33.5%), candida vaginitis73cases (account for37.05%) and trichomoniasis vaginitis58cases (account for29.44%).1.2pH analysis in the vaginal dischargeIn all of the vaginal discharge in197vaginitis cases, the pH were exclusively greater than4.5. Comparing with the normal acid environment in vagina, the environment in vaginitis patients took on a basicity. But all of the pH in the normal health women vagina were lower than4.5. We also found that the candida vaginitis share the highest level in the three vaginitis.1.3The detected rate of the lactobacillu in three vaginitisThe detected rate of lactobacillus was declined significantly in the bacterial vaginitis, candida vaginitis and trichomoniasis vaginitis compared with the normal health women vaginal discharge. The decreased detected rate of lactobacillus was proportional to the risk factor of various vaginitis. The results showed that the more decreased of lactobacillus, the higher risk factor was discovered in the vaginitis. Among the three vaginitis, the highest detected rate was in the candida vaginitis (12.07%), and the risk factor was increased to16.564.2. Vaginal discharge lipid peroxidase and antioxidants activity results2.1Analysis of the lipid peroxidation in the vaginal discharge of vaginitis patients:Through detecting the lipid peroxide MDA level in all197patients’ and60normal health women’s vaginal discharge, we found that the MDA level in all of the bacterial vaginitis, candida vaginitis and trichomoniasis vaginitis enhanced significantly compared with the normal control group (health women, P<0.05), but there are no differences among the three kinds of vaginitis. After treating with drugs to the three vaginitis, MDA level was decreased significantly compared with the control group. The analysis of H2O2in the three vaginitis patients’ vaginal discharge showed that the average level of the three vaginitis ascended to20.05±24.22, and higher than the level (2.78±2.34) in the health women vaginal discharge. In addition, the H2O2level in the trichomoniasis vaginitis was higher significantly than the H2O2level in the bacterial vaginitis and candida vaginitis groups (P<0.01).2.2Analysis of the antioxidants and antiperoxidase in the vaginal discharge of vaginitis patientsThe CAT activity of vaginal discharge of bacterial vaginitis, candida vaginitis and trichomoniasis vaginitis decreased significantly compared with the activity of the normal vaginal discharge (P<0.01). Interestingly, the CAT activity of vaginal discharge in candida vaginitis was lower significantly than the CAT activity in bacterial vaginitis (P<0.05). Comparing with the SOD activity of vaginal discharge in the normal vaginal discharge, the SOD activity of the vaginal discharge in bacterial vaginitis, candida vaginitis and trichomoniasis vaginitis decreased significantly. But there were no significant difference of SOD in vaginal discharge among bacterial vaginitis, candida vaginitis and trichomoniasis. Except for the CAT and SOD detection, the vitamin C amount was also detected in the various vaginitis. The vitamin C amount in the vaginal discharge of the three vaginitis was higher significantly than the control group (P<0.001), but the difference among three groups is not significant. Through the activity (amount) of CAT, SOD and vitamin C in all of the three vaginal discharge decreased obviously, they could recover to the normal level after drug treatment.3. Oxidative stress induced vaginal mucosa epithelial cell apoptosis results3.1Indirect immunofluorescence (IFA) and western blot analysis were used to identify the activated caspase3. The results indicated that the cleaved activated caspase3level increased significantly in the bacterial vaginitis, candida vaginitis and trichomoniasis vaginitis. The DNA ladder experiment was also employed to detect the apoptosis in the vaginitis and normal women vagina. We found that there were some obvious DNA fragments forming the DNA ladder in the mucosa epithelial cells of three kinds of vaginitis.3.2The mitochondria pathway triggered apoptosis in the bacterial vaginitisWe detected the apoptosis associated proteins, such as the classical biomarker protein Bcl-2and Bax, in the mucosa epithelial cells of vaginal patients. The results showed that the Bcl-2protein level decreased significantly, and the Bax protein increased significantly in the three vaginitis mucosa epithelial cells compared with the normal women.3.3The endoplasmic reticulum stress induced apoptosis in the candida vaginitis and trichomoniasis vaginitisWestern blot results confirmed that the level of Grp78, CHOP and activated casapse12were all increased compared with the control group.Conclusion1. In the vaginal discharge of bacterial vaginitis, candida vaginitis and trichomoniasis vaginitis, the lactobacillus detection rate was decreased, pH was enhanced, which indicated that the flora environment was destroyed.2. The activity of lipid peroxidase MDA, amount of oxidants H2O2were both increasing significantly, and antiperoxidase SOD, CAT, antioxidants vitamin C were enhancing compared with the normal vagninal discharge of vaginitis patients, which showed that the three kinds of vaginitis patients were affected by the endoplasmic reticulum stress induced apoptosis.3. The vaginal mucosa epithelial cells of bacterial vaginitis patients were affected by the mitochondrial pathway induced apoptosis, and the patients with candida vaginitis and trichomonasis vaginitis were affected by the endoplasmic reticulum stress triggered apoptosis.