| Vagina atrophy(VA)is a common disease in postmenopausal women.It is due to the decrease of estrogen level and the degradation of vaginal tissue.It is mainly manifested as vaginal dryness,itching,and pain during intercourse.With the global aging,the number of postmenopausal women continues to increase.About 40% of postmenopausal women will develop symptoms related to vaginal atrophy,and only about 20% of them will seek for help.The current treatments are estrogen supplements and vaginal lubricants.Many women refuse to use estrogen because of the concerns about the side effects of estrogen,and vaginal lubricants are less effective in some patients with severe symptoms.As one of the main components of extracellular matrix(ECM),collagen plays an important role in the formation of tissues and organs,and participates in the expression of cell functions and signal transmission.The reason why collagen is widely used in biomedical applications is that it can form fibers with a certain strength and stability through its own aggregation and cross-linking,and has the characteristic of biocompatibility and safety.At present,21 types of collagen have been found,of which type Ⅰ,Ⅱ,and Ⅲ proteins are the most abundant in the human body,accounting for about 80% to 90% of the total collagen.Among them,vaginal tissue contains a large amount of type Ⅲ collagen.As the level of estrogen decreases,the collagen content in the vaginal tissues decreases as well.This topic aims to discuss the effect of type Ⅲ collagen on vagina atrophy in rats and the related mechanisms that may be involved,and to provide a theoretical basis for clinical treatment of vagina atrophy.PART Ⅰ ESTABLISHMENT OF RAT VAGINA ATROPHY MODELOBJECTIVE: To establish the vagina atrophic rat model and investigate the histological changes of vagina tissue.METHODS:(1)Vagina atrophic rat model was established by ovariectomy.Ten normal female SD rats of 6-8 weeks were selected,and 10 rats were randomly divided into two groups:(1)sham group(n = 5): After anesthesia and disinfection,the abdominal was exposed for 30 minutes,and the abdomen was closed.(2)Model group(n = 5): After anesthesia and disinfection,the abdomen was excised,the bilateral ovaries were exposed with toothless forceps,the bilateral uterine arteries were ligated,removed the bilateral ovaries,and sutured the incision.(2)H&E and Masson stain were used to investigate the histological change of vagina epithelial and collagen content in rat vagina tissue.RESULTS: The weight before and after operation was measured.After ovariectomizing,the rat vagina epithelial became thinner,collagen disarranged through H&E and Masson stain.CONCLUSION: Vagina atrophy rat model was established by ovariectomy.PART Ⅱ EFFECT AND MECHANISM OF TYPE Ⅲ COLLAGEN ON VAGINA ATROPHY RATSOBJECTIVE: To investigate the effect of type Ⅲ collagen on vagina atrophy,and provide supplement for clinical use.METHODS:(1)Forty-five female SD rats of 6-8 weeks were selected and randomly divided into 5 groups:(1)Sham group(n=10);(2)Model group(n=10): only ovariectomized;(3)Collagen group(n=10): 28 days after ovariectomy,vaginal administration of type Ⅲ collagen 0.5ml daily for 14 consecutive days;(4)Estrogen group(n=10): 28 days after ovariectomy,vaginal administration of 0.5ml estrogen daily,given continuously for 14 days;(5)Carbomer group(n = 5): 28 days after ovariectomy,0.5 ml of carbomer was given daily for 14 days.(2)The location of biotin-labeled collagen in vagina tissue was traced.(3)Vagina epithelial thickness was measured by H&E stain,the arrangement of collagen was assessed by Masson stain.The p H value and microorganism were observed.(4)The expression level of COL-Ⅰ、COL-Ⅲ、TIMP-1、MMP-1、VEGF、Ki-67、IL-6 was examined by IHC and q PCR,the protein level of AQP-2 was examined by Western blot.(5)The ultrastructure change was observed by scan microscopy.(6)Endometrium thickness was measure by H&E stain.(7)Cells were inducted to age cells to observe cell proliferation and apoptosis.RESULTS:(1)The immunofluorescence result showed that biotin-labeled collagen was visible in the vagina epithelium and muscle layer.(2)The results of H&E stain showed that the thickness of the vaginal epithelium after estrogen and collagen treatment was thicker than that of model group and closed to the sham group.Masson stain results showed that the arrangement of connective tissue in vagina of the model group and the carbomer group was disordered,and that of the vagina tissues treated with estrogen and collagen was closed,which was both similar with sham group.(3)Immunohistochemistry and qPCR results showed that the expressions of COL-I,COL-Ⅲ,TIMP-1,VEGF,and Ki-67 in the vaginal tissue after collagen and estrogen treatment were higher than those in the model group,which were close to the sham group;the expressions of MMP-1 and IL-6 in model group were higher than those in collagen group,estrogen group and sham group.Western blot results showed that the expression level of AQP-2 protein in vaginal tissue after collagen and estrogen treatment was higher than that of the model group and the carbomer group,and similar to sham group.(4)Electron microscopy results showed that after ovariectomy,the wrinkle wall of the vaginal epithelium disappeared,microvilli disappeared,and the intercellular space became larger.After type Ⅲ collagen treatment,the microvilli appeared in the vaginal epithelium,and the space between the cells decreased which was similar with sham group.(5)The endometrium of rats observed by H&E stain showed that the thickness of endometrium after collagen treatment was closed to the model group,and the thickness of endometrium after estrogen treatment was thicker,which was closed to that of the sham group.(6)Flow cytometry and CCK-8 results showed that type Ⅲ collagen can promote D-gal induced proliferation of senescent cells and inhibit apoptosis.CONCLUSION: Collagen treatment can be seen in the vagina epithelium and muscle layer,and promote the expression of COL-Ⅰ,COL-Ⅲ,TIMP-1,VEGF,Ki-67,and AQP-2 in vagina tissue,and reduce the expression of MMP-1 and IL-6,thereby promoting extracellular formation and reducing the degradation of extracellular matrix,and reduces the expression of inflammatory factors,increases the water content in tissues,and promotes the proliferation of epithelial cells.At the same time,it can improve the vaginal ultrastructure,promote the growth of microvilli,reduce the distance between cells.Compared with estrogen,it has not caused abnormal proliferation of the endometrium.At the same time,it could promote the proliferation of age cells and inhibit apoptosis. |
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