A Preliminary Study On The Relationship Between Autophagy And Collagen Expression In Vaginal Fibroblasts In Patients With Pelvic Organ Prolapse

ObjectivePelvic organ prolapse(POP)is characterized by abnormal position and dysfunction of pelvic organs(including the bladder,uterus,vagina,rectum and etc.),and has severe impact on the quality of life of patients severely in different degrees.The cause of POP is vaginal delivery,getting older,or aging and pathological repair of the connective tissue,which lead the pelvic floor muscle and tissue to become weakened.Numerous studies have found that the total collagen content in pelvic floor support tissue from POP patients decreased significantly.The collagen,mainly type-? and type-?collagen,expressed in pelvic floor support tissue is synthesized and secreted by fibroblasts.The effect of the type-?/? collagen ratio change on POP patients has also been widely concerned.Type-?collagen(collagen ?,Col ?)is related to tissue toughness and has larger diameter.Type-? collagen(collagen ?,Col ?),with less content compared to type-?collagen,is related to tissue elasticity and has smaller diameter.The increase of type-?/? collagen ratio can enlarge the diameter of collagen fiber and improve toughness,while the decrease of type-?/? collagen proportion lead to smaller diameter of collagen fiber,reduced toughness and improved elasticity.Autophagy is a kind of "self-phagocytosis" phenomenon in cells,which is an important process of the turnover of substance in cells,and can digest and degrade organelles and proteins in cells that are damaged,aged or denatured through lysosome pathway.Plentiful literatures have showed that the number of type-?and type-? collagen in POP patients decreased,but the mechanism of collagen reduction is still unclear.So is there a correlation between the decrease of collagen and the clearance function of autophagy? Whether can increase collagen number through regulating cells' autophagy activity,and eventually delay the occurrence of POP?In order to explore the above hypothesis,we took the vaginal wall fibroblasts from POP patients or non-POP patients as research objects in this paper.Firstly we detected the expressions of intracellular autophagy marker protein(LC3,Beclin1)and collagen.Then we measured the changes of autophagy activity and collagen expression in cells after we used starvation to induce or autophagy inhibitor 6-amino-3-methyl-purine(3-Methyladenine,3-MA)to suppress autophagy,so as to verify their correlation preliminarily.Method 1.Vaginal wall tissues of 10 cases of POP patients and 10 cases of non-pop patients who received surgical treatment in The Third Affiliated Hospital of Guangzhou Medical University from June 2017 to December 2018 were selected for primary culture and identification of vaginal wall fibroblasts by collagenase digestion.2.Fibroblasts from the vaginal wall of POP patients and non-POP patients were collected from the 4th to 10 th generation in good growth condition.Each group was cultured for 5 days in DMEM/F12 culture medium supplemented with 10% fetal bovine serum,1% penicillin and streptomycin.Then the expressions of autophagy marker proteins(LC3 and beclin1)and collagen(Col ? and Col ?)were detected by western blotting.3.The number of autophagy in the vaginal wall fibroblasts from POP patient was observed by transmission electron microscopy.4.Well-grown vaginal wall fibroblasts from POP patients and non-POP patients were randomly divided into control group(DMEM/F12 culture medium containing sugar and serum)and starvation treatment group(DMEM/F12 culture medium without sugar and serum).After culture for 4 hours,the expressions of LC3 and Beclin1 were detected by western blotting.5.Well-grown vaginal wall fibroblasts from POP patients and non-POP patients were randomly divided into control group(DMEM/F12 culture medium containing sugar and serum),starvation treatment group(DMEM/F12 culture medium without sugar and serum)and 3-MA treatment group(DMEM/F12 culture medium containing sugar and serum +10mmol/L 3-MA).After culture for 4 hours,the mRNA expression levels of LC3,Beclin1,Col?and Col ? were detected by real-time fluorescent quantitative polymerase chain reaction(RT q-PCR),and the protein expression levels of LC3,Beclin1,Col? and Col ? were detected by western blotting.Result 1.On the 3rd to 5th day after culture and inoculation,cells isolated from vaginal wall tissue showed adherent growth,and individual adherent cell with uneven distribution exhibited spindle shape and clear boundary.On the 10 th to 12 th day after culture,the spindle adherent cells grew to 80%-90% confluence and were arranged in a fish-school or whirlpool pattern.The second generation of fibroblasts were detected by immunocytochemical staining and proved to be the vaginal wall fibroblasts,because of their positive staining of vimentin,negative staining of cytokeratin,and sampling sites.2.Compared with non-POP patients,the protein expression levels of LC3,Beclin1,Col I and Col ? in vaginal wall fibroblasts from POP patients showed a decreasing trend when being cultured in DMEM/F12 culture medium containing sugar and serum,the difference was statistically significant(P < 0.05).3.The results of transmission electron microscopy showed that compared with the control group,the number of autophagosomes in vaginal wall fibroblasts from POP patients increased in starvation treatment group,while decreased in 3-MA treatment group.4.After starvation treatment,the expression levels of autophagy-related protein LC3 II and Beclin1 in vaginal fibroblasts of POP patients and non-POP patients were(0.808±0.015),(0.901±0.059)and(0.750±0.049),(0.980±0.044)respectively,indicating that the level of autophagy increased.5.Compared with the control group,the expression of LC3 and Beclin1 in the vaginal wall fibroblasts of POP patients and non-POP patients increased,while the expression of LC3 and Beclin1 in the 3-MA treatment group decreased.6.In the fibroblasts of vaginal wall of POP patients and non-POP patients,compared with the control group,the expression of LC3 protein in the 3-MA treatment group was(0.349±0.015)and(0.464±0.055),and the expression of Beclin1 protein was(0.140±0.049)and(0.340±0.036),which indicated that the autophagy level decreased under the action of 3-MA.7.Compared with the control group,the expression of Col I and Col ? in the vaginal wall fibroblasts of POP patients in starvation group and 3-MA group decreased,and the quantitative expression of Col I and Col ? was shown in Table 5.There was no significant difference(P > 0.05).However,the expression of Col I and Col ? in the vaginal wall fibroblasts of non-POP patients after induced autophagy was lower than that of the control group.The difference was statistically significant(P < 0.05).Conclusion 1.The primary culture of vaginal wall fibroblasts isolated from POP patients and non-POP patients was successful.2.Compared with non-POP patients,the expressions of autophagy marker proteins and collagen in vaginal wall fibroblasts from the POP patients showed lower trends;3.Both starvation induction and 3-MA drugs regulate autophagy through macroautophagy pathway.The decrease of collagen in POP patients may be related to other autophagy pathways.4.Collagen expression in vaginal wall fibroblasts of non-pop patients decreased after enhanced autophagy,suggesting that the risk of POP increased in patients without prolapse under the influence of enhanced autophagy.