| Objective To observe the expression of TNF-?,IL-1 as well as IL-6 in the serum of patients with mild and moderate intrauterine adhesions,and to investigate the role of inflammation in pathogenesis of IUA.To determine whether Zuogui pills combined with human umbilical cord mesenchymal stem cells transplantation can improve the uterine fibrosis in IUA rats on the basis of the successful IUA model rats,and to explore the possible mechanism of it.Methods Clinical research: selecting 30 cases of IUA and 30 patients with normal endometrium.After diagnosed,the blood samples were collected,and the supernatant was collected after centrifugation.TNF-?,IL-1 and IL-6 levels in serum of two groups were measured by ELISA.Animal model: Forty-eight female rats were randomly divided into four groups;(A)control group: without thermal damage;(B)Thermal damage sustained 10s;(C)Thermal damage sustained 15s;(D)Thermal damage sustained 20s;each group has 12 rats.At post-operative day 3,7,14 and 21,three rats from each group were sacrificed to remove the bilateral uteri.The sections of the uteri were examined by HE and Masson staining to observe the morphology and fibrosis of endometrium.Experimental study: Thirty female rats were randomly divided into five groups,each group has 6 rats.(A)control group: without thermal damage and other treatment;(B)Model group: no treatment after establishment of model;(C)Chinese medicine group:Zuogui pills treatment after establishment of model;(D)h UC-MSC transplantation group: intravenous transplantation of P3 h UC-MSCs after establishment of model;(E)combination group: Zuogui pills treatment and intravenous transplantation of P3 h UC-MSCs after establishment of model.After 28 days of treatment,the rats were sacrificed to remove the bilateral uteri and collect the cardiac blood.The sections of the uteri were examined by HE and Masson staining to observe the morphology,gland and fibrosis of endometrium.TNF-?,IL-1 and IL-6 in serum of each group was measured by ELISA.The localization and expression of CD29 and P38 MAPK in endometrium were detected by immunofluorescence double staining.The expression of TGF-?1,P38 MAPK and P65NF-k B in endometrium were detected by Western blot.Results Clinical research: The expression of TNF-?,IL-1 and IL-6 in patients with IUA were significantly higher than the normal control group(P<0.05).Animal model:Compared with the endometrial morphology of each group,the A group showed that the uterine cavity was star type,the endometrium was complete,the glands were rich,and the structure was regular.B,C,D three groups of endometrium showed varying degrees of damage.At the 14,21 days after operation,the endometrial morphology of group B has been basically restored.There was a small number of newborn glands,no significant fibrosis changes.The C group showed that the uterine cavity was narrow,the muscular layer was complete,the glands were few,the endometrial stroma was filled with collagen fibers at the 14,21 days after operation.At the 7,14,21 days after operation.The group D showed that the uterine cavity was completely closed,the endometrium was damaged excessively,and the structure of muscular layer was blurred.Experimental study:(1)Morphological observation: Compared with group B,the number of endometrial glands was increased,the collagen fibers in the endometrial stroma was decreased in the three treatment groups,and the changes of group D and E were significantly higher than those of group C(P<0.05).However,compared with the group A,the morphological recovery of the uterine cavity was worse,and the difference was statistically significant(P<0.05);(2)Inflammatory factor expression:Compared with group B,the expression of TNF-?,IL-1 and IL-6 in the serum of the three treatment groups were significantly decreased(P<0.05),and the changes of group D and E were significantly more than those of group C(P<0.05).However,the expression of TNF-?,IL-1 and IL-6 in group D and E was still higher than group A(P<0.05);(3)the expression of CD29:There were no significant difference in the former three groups(P>0.05).The mean optical density of CD29 in group D and E was significantly stronger than the other three groups(P<0.05).The expression of CD29 in group D was significant higher than group E(P<0.05).The expression of P38MAPK: the group B had a significant higher mean optical density than the other four groups(P<0.05).Compared with group B,The mean optical density of P38MAPK in the three treatment groups were significantly decreased(P<0.05),and the changes of group D and E were significantly more than those of group C(P<0.05).However,the expression of P38 MAPK in group D and E were still higher than group A(P<0.05).There were no significant difference in group D and E(P>0.05);(4)The result of Western blot: The expression of TGF-?1,P38 MAPK and P65NF-k B in group B was stronger than the other 4 groups(P<0.05).The grey level of TGF-?1,P38 MAPK and P65NF-k B in the three treatment groups were significantly decreased(P<0.05),and the changes of group D and E were significantly more than group C(P<0.05).However,the expression of TGF-?1,P38 MAPKand P65NF-k B in group D and E were still higher than group A(P<0.05).The grey level of TGF-?1 in group D was significantly lower than group E(P < 0.05).The expression of P38 MAPK and P65NF-k B in group D and E had no significant difference(P>0.05).Conclusions ?The high expression of TNF-?,IL-1 and IL-6 may play an important role in the pathogenesis of IUA.?Using the catheter filled with boiling water to injury the endometrium for 15 seconds,can cause damage to the basal layer of the endometrium,fibrosis adhesions after 14 days,so as to establish a stable rat IUA model.?Zuogui pills combined with human umbilical cord mesenchymal stem cell transplantation can effectively improve the degree of endometrial fibrosis of IUA rats.The mechanism might be achieved through inhibiting the P38MAPK/NF-k B signal transduction pathway and down-regulating the expression of proinflammatory cytokines and fibrosis factors such as TNF-?,TGF-?1,IL-1 and IL-6. |
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