| ObjectiveA mouse model of Premature Ovarian Insufficiency(POI)induced by 4-vinylcyclonhexene diepoxide(VCD)was used to explore the mechanisms of traditional Chinese medicine in promoting transplanted embryonic embryonic stem cells' ovarian-restoring function by improving the ovarian microenvironment through regulating the TAK1-TGF-? signal pathway.MethodsSixteen C57B/L6 mice were randomly divided into control group(CON)and model group(MOD),with 8 rats in each group.The model group was intraperitoneally injected with VCD sesame oil solution for 15 days at a dose of 160 mg · kg-1 to establish a mouse POI model.The control group was given an equal amount of sesame oil intraperitoneally.On the 50th to 55th day,based on the estrous cycle of the mice,harvest mice' s organs at the estrus period.The estrus cycle smears were collected to monitor the changes of estrous cycle,and the number of follicles at each developmental stage was counted after HE staining of ovarian tissue sections,and comparative analysis was performed to determine whether the modeling was successful.A total of 100 female C57B/L6 mice were randomly divided into control group(CON),model group(MOD),Chinese medicine group(HY),stem cell group(SC)and stem cell combined with Chinese medicine group(SCHY),20 rats in each group..The control group(CON)was given intraperitoneal injection of the same volume of sesame oil for 15 days from the first day(D1)of the experiment,once a day;from the first day of the experiment(Dl),the mice was gavaged with the equal volume of saline until the organs is taken.The model group(MOD)intraperitoneal injected with VCD sesame oil solution for 15 days from the first day(D1)of the experiment,once a day;from the first day of the experiment(D1),the mice was gavaged with the same volume of saline until they were sacrificed.From the first day(D1)of the experiment,the Chinese medicine group(HY)intraperitoneal injected with VCD sesame oil solution for 15 days,once a day;from the first day of the experiment(Dl),the mice was gavaged with the Huyang-Yangkun decoction continuously until they were sacrificed.From the first day(D1)of the experiment,the stem cell group(SC)intraperitoneal injected with VCD sesame oil solution for 15 days,once a day;from the first day of the experiment(D1),the mice was gavaged with the same volume of saline until they were sacrificed;10 days after the modeling(D25),the mouse embryonic stem cell suspension was injected once through the tail vein.From the first day(Dl)of the experiment,the stem cell combined with Chinese medicine group(SCHY)intraperitoneal injected with VCD sesame oil solution for 15 days,once a day;ofrom the first day of the experiment(D1),the mice was gavaged with the Huyang-Yangkun decoction continuously until they were sacrificed;Ten days after modeling(D25),mouse embryonic stem cell suspension was injected once through the tail vein.The es trous cycle of mice was monitored by vaginal exfoliated cell smears.The body weight of the mice and the weight of the ovaries and uterus were weighed to calculate the organ index.The pathological sections of the liver and kidney tissues were prepared for HE staining,and the effects of interventions on liver and kidney were observed.The number of follicles in the ovarian tissue was counted after HE staining,and the levels of serum sex hormones FSH,AMH and E2 were detected by ELISA.In the second experiment,the ovarian tissues of each group were obtained.Pathological sections and immunohistochemical staining were performed on some ovarian tissues,and the protein expressions of TNF-?,IL6,FSHR,PR,ER,TGF-?,BMP4,TAK1,Smad4 and JNK were observed under microscope.Pictures were taken for quantitative analysis after.miRNA and mRNA were extracted from some ovarian tissues,and primers of miR143,miR145,miR135,miR429 and TAK1,TARF6,TAB1,TAB2,Smad4,Smad2,Smad3,MKK6,P38 and JNK genes were designed.The miRNA and mRNA expressions were quantitatively detected by fluorescence quantitative PCR.Total protein was extracted from some ovarian tissues,and the protein expression of TGF-?,Smad4 and FSHR in ovaries were detected by Western-Blot.The results of various tests above were used to investigate the therapeutic mechanism of Traditional Chinese medicine combined with stem cells therapy for treating VCD-induced Premature Ovarian Insufficiency.ResultsEstrous cycle:The control group showed regular estrous cycle with 100%normal rate,and the model group' s normal rate was 12.5%.There was a significant difference between the two groups(P<0.05).Ovary morphology:Follicles of various developmental stages were observed in the ovary of the control group,and there were more antral follicles.The blood vessels in the ovaries were abundant too.In the model group,the ovarian volume was reduced,and there were few follicles in the ovaries of model group,and more atretic follicles remained,and the blood supply was reduced compared with the control group.Numbers of follicles at different developmental stages were counted:Compared with the control group,the number of follicles in all developmental stages in the model group were decreased(P<0.05).Organ index:The organ index of mice ovary and uterus decreased after VCD model(P<0.05);while in traditional Chinese medicine group,stem cell group and stem cell combined with traditional Chinese medicine group,the organ index increased compared with the model group(P<0.05).There was no statistical difference between the three treatment interventions on the ovarian and uterine organ index.Estrous cycle:There was a statistically significant difference in estrous cycle among all groups(P<0.01).Except for the control group,the estrous cycle of each group changed,and the abnormal rate in the combined treatment group was the lowest and the normal rate highest(5.26%).Changes in liver and kidney tissue structure:the pathological morphology of liver and kidney tissues of each group was normal.Follicle counts:Compared with the control group,the number of follicles in the model group was decreased(P<0.01).However,compared with the model group,all types of follicles,except antral follicles,in the traditional Chinese medicine group,the stem cell group and the combine group were increased(P<0.05);the number of primordial follicles was statistically different between the stem cell group and the control group(P<0.05);compared with the modelgroup,the number of antral follicles in the combined treatment group Elevated(P<0.01).Changes in serum sex hormone levels:Compared with the control group,the FSH level was higher in the model group(P<0.01),and the AMH level was lower(P<0.01).Compared with the model group,The FSH level in Chinese medicine group,stem cell group and stem cell combined with Chinese medicine group decreased(P<0.Ol),while the AMH level was relatively increased(P<0.05).The AMH levels of Chinese medicine,stem cell and stem cell combined with Chinese medicine treatment group and control group were statistically deferent(P<0.05).There was no significant difference in E.levels among all the groups(P>0.05).miRNA expression:4 microRNAs'(miR143,miR145,miR135,miR429)expression were not statistically different among all groups(P>0.05).Immunohistochemical expression of inflammatory factors in mouse ovary:Compared with the control group,the immune-related inflammatory factors TNF-?(P<0.05)and IL-6(P<0.01)were increased in the model group.Compared with the model group,the TNF-a level in the stem cell group and stem cell combined Chinese medicine group was relatively lower(P<0.05),and the IL-6 in the Chinese medicine group,stem cell group and stem cell combined Chinese medicine group was relatively lower(P<0.01).Immunohistochemical expression of mouse ovarian hormone receptor:Compared with the control group,the expression of FSHR(P<0.05)and PR(P<0.01)was decreased in the model group;compared with the model group,The FSHR in stem cell group and stem cell combined Chinese medicine group was relatively higher(P<0.01),while the PR in the Chinese medicine group,stem cell group and stem cell combined with Chinese medicine group was relatively higher(P<0.05).There was no significant difference in the expression of ER among all the groups(P>0.05).Immunohistochemical expression of TGF-?-TAK1 pathway protein in mouse ovary:Compared with the control group,the expressions of TGF-?,BMP4,TAK1,JNK(P<0.01)and Smad4(P<0.05)proteins in model group was significantly lower.Compared with the model group,TGF-? expressions in the traditional Chinese medicine group and the stem cell combined with traditional Chinese medicine group were lower(P<0.05),while the expressions of BMP4 in the stem cell group and stem cell combined Chinese medicine group were relatively lower(P<0.05),the expression of Smad4 and TAKlin stem cells group and Chinese medicine group were reduced(P<0.05 or P<0.01).Mouse protein expression:Compared with the control group,the expression of TGF-? in the model group was increased(P<0.05).Compared with the model group,the expression of TGF-? in the Chinese medicine group,stem cell group and combine group was relatively Lower(P<0.05).Compared with the control group,the expression of Smad4 in the model group was increased(P<0.05);compared with the model group,the expression of Smad4 was decreased in the three treatment groups(P<0.05);while in the traditional Chinese medicine group and stem cells group,Smad4 expression was different from that in the control group(P<0.01).Compared with the control group,the expression of FSHR in the model group was decreased(P<0.05);compared with the model group,the expression of FSHR was increased in the three treatment groups(P<0.01).The relative expression of mRNA in each group of mice:Compared with the control group,the mRNA expressions of TAK1,TRAF6,TAB1,TAB2,Smad4,Smad2,Smad3,MKK6,P38 and JNK increased in the model group(P<0.05).Conclusion1.VCD was intraperitoneally injected for 15 days at a dose of 160 mg·kg-1.The VCD-induced C57B/L6 mouse model of Premature Ovarian Insufficiency was successfully established.2.Chinese medicine,embryonic stem cell transplantation,and stem cell combined with traditional Chinese medicine treatments can improve serum AMH level and decrease FSH level in POI mice,increase the number of follicles,and repair the ovarian function.3.The mechanism of Chinese medicine improving the therapeutic effect of embryonic stem cell therapy is based on the TGF-?-TAK1 pathway. |
PDF Full Text Request