The Research On The Mechanism Of Guishenwan Decotion Treatment To Kidney Deficiency-thin Endometrium Of Rats Based On The Regulation Of MiR-135a

Objective:1.To establish the rat model of kidney deficiency-thin endometrium, according to the current kidney deficiency rat model and thin endometrium rat model, simulating thin endometrium patient with kidney deficiency in clinical. To clarify the modeling steps, the use of drugs and medication time.Providing more in line with the actual clinical model of combination of diseases and syndromes to study of the thin endometrium, in-depth understanding of the pathological characteristics of kidney deficiency-thin endometrium and provide experimental basis to explore effective treatment method.2. To analysis the regulatory effect of miR-135a on JAK2/STAT3 signal pathway and expression of HOXA10 gene under the Guishenwan decotion effect, in order to clarify target and pharmacodynamic mechanisms of Guishenwan decotion in the treatment to thin endometrium, so as to enrich the ancient clinical application, but also provide a scientific basis for the application of traditional Chinese medicine in assisted reproductive technology.Methods:1. To establish the rat model of kidney deficiency-thin endometrium by injection of 95% ethanol through the SD rat uterine cavity and 450mg/kg/days *8 days hydroxyurea intragastric administration. HE staining was used to detect endometrial tissue structure and to measure the thickness of endometrium; immunohisto chemical method to detect endometrial cell markers include keratin protein and vimentin protein, and endometrial receptivity marker protein such as VEGF protein.2. After the establishment of rat model of kidney deficiency-thin endometrium, intervention by Guishenwan decotion administration low and high dose for two estrous cycles. HE staining detection of endometrial tissue structure and to measure the thickness of endometrium; using immunohistochemical method to detect the proteins of the endometrium such as vimentin, keratin, VEGF, COX-2 and so on; using Western blot method to detect the rat endometrium JAK2, p-jak2, STAT3, p-STAT3 expression; using QRT PCR method for detection of miR-135a and HOXA10 gene, ITG beta 3 gene and Emx2 of rat endometrial.Results:1.Kidney deficiency-thin endometrium modeling success, morphology, the structure of endometrium of the control group was integrity, the model group endometrial thinning intimal due to the continuous, reduce wrinkles and parts glandular epithelial, glandular epithelial cells and endometrial epithelial cells showed low columnar, even flat; interstitial fibrous tissue hyperplasia, gland rarefaction, visible part of glandular epithelial atrophy. The endometrial thickness of the model group (453.95+60.46um) was less than that of the control group(709.13+114.05um) thin (P<0.01). The weight of spleen and thymus of the model group was milder than the control group (P<0.01).2. The spleen weight of the control group was heavier than that of the rest of the groups (P<0.05). the spleen weight of the low dose group of Guishenwan decotion was heavier than that of the estrogen group and the high dose group of Guishenwan decotion (P<0.05). The thymus weight of the control group was heavier than that of the rest of the groups (P<0.05). The thymus weight of the low dose group of Guishenwan decotion and the high dose group of Guishenwan decotion was heavier than that of the model group and the estrogen group (P<0.05).3. The endometrium of the model group was thinner than that of the other groups (P<0.05). The endometrium of the control group was thicker than that of the other groups (P<0.05); The thickness of the endometrium of the low dose group of Guishenwan decotion compare with the high dose group of Guishenwan decotion and estrogen group were no significant difference (P>0.05).4. The expression of VEGF protein of each group were higher than those in the model group(P<0.05). Compared to the control group, the expression of VEGF of the estrogen group, the low dose group of Guishenwan decotion and the high dose group of Guishenwan decotion were higher(P<0.05). The expression of VEGF of the low dose group of Guishenwan decotion was higher than the estrogen group (P<0.05). The expression of COX-2 of the control group and the high dose group of Guishenwan decotion was higher than that of the other groups (P<0.05). The expression of COX-2 of the estrogen group and the low dose group of Guishenwan decotion were no significant difference(P>0.05). The expression of COX-2 of the control group and the high dose group of Guishenwan decotion were no significant difference(P>0.05)5. The expression of ITG β3 protein of the estrogen group and the model group was lower than that of the other groups (P<0.05). The expression of ITG β 3 proteinthe of the and the control group had no statistical difference (P>0.05).6. The expression of JAK2 protein of the model group and the control group showed no significant difference (P>0.05). The expression of JAK2 protein of the groups of Guishenwan decotion was lower than the the model group and the control group (P<0.05). The expression of JAK2 protein of the high dose group of Guishenwan decotion was higher than the estrogen group(P<0.05). The expression of P-JAK2 protein of the model group was higher than that of the other groups(P<0.05). The expression of STAT3 protein of the control group and the estrogen group was higher than that of the groups of Guishenwan decotion(P<0.05). The expression of P-STAT3 protein of the model group was higher than that of the other groups(P<0.05). The expression of P-STAT3 protein of the groups of Guishenwan decotion was lower than that of the control group and the estrogen group(P<0.05).7. The relative expression of ITG β 3 gene of the model group was higher than that of the other groups(P<0.05). The relative expression of ITG β 3 gene of the estrogen group and the high dose group of Guishenwan decotion was higher than that of the control group (P<0.05). The relative expression of ITG β 3 gene of the control group and the low dose group of Guishenwan decotion had no statistical difference(P>0.05).8. The relative expression of HOXA10 gene of the low dose group of Guishenwan decotion was higher than that of the other groups(P<0.05). The relative expression of HOXA10 gene of the model group was lower than that of the other groups(P<0.05).9. The relative expression of miR-135a of the model group was lower than that of the other groups(P<0.05). The relative expression of miR-135a of the high dose group of Guishenwan decotion was higher than that of the control group and the low dose group of Guishenwan decotion(P<0.05).10. The expression of miR-135a and JAK2 was highly negatively correlated, r=-0.814, P<0.01.The expression of miR-135a and P-JAK2 was lowly negatively correlated, r=-0.351, P<0.01. miR-135a and P-STAT3 was moderately negatively correlated, r=-0.734, P<0.01; miR-135a and STAT3 was not related.11. The expression of JAK2 and P-STAT3 was positively high correlated, r=0.84, P<0.01. P-JAK2 and STAT3 was not related.12. The protein expression of JAK2 and VEGF was moderately negatively correlated, r=-0.75, P<0.01. P-STAT3 and VEGF was moderately negatively correlated, r=-0.762, P<0.01. P-STAT3 and COX-2 was lowly negatively correlated, r=-0.478, P<0.01. VEGF and COX-2 was lowly negatively correlated, r=0.304, P<0.05.13. miR-135a and HOXA10 was not related, r=0.273, P>0.05. miR-135a and ITG β3 was lowly negatively correlated, r=-0,307, P<0.05. miR-135a and EMX2was moderately negatively correlated, r=-0.726,P<0.01.14. HOXA10 and ITG β3 was moderately negatively correlated, r=-0.565, P<0.01.EMX2 and ITG β3 was lowly positively correlated, r=0.389, P<0.01. The expression of HOXA10 and ITG β 3 protein was positively correlated, r=0.772, P<0.01.Conclusions:1. To establish the rat model of kidney deficiency-thin endometrium, according to the current kidney deficiency rat model and thin endometrium rat model, simulating thin endometrium patient with kidney deficiency in clinical. The model can be the actual situation of clinical response of thin endometrium of kidney deficiency syndrome.2. Guishenwan Decotion can effectively improve the symptoms of the kidney deficiency, and effectively increase the endometrial thickness, enhanced the protein expression of VEGF, COX-2and ITG β 3 in thin endometrium, effectively promote the proliferation of endometrial repair. Therefore, using Guishenwan decotion in assisted reproductive technology for thin endometrium with kidney deficiency,in order to improve the success rate of fertilization.3. The protein expression of JAK2, STAT3, P-JAK2 and P-STAT3 was inhibited by Guishenwan Decotion. Low and high dose group of Guishenwan decotion can enhance the expression of miR-135a;The expression of miR-135a was enhanced in low and high dose group, especially in high-dose group;The expression of EMX2mRNA was inhibited in low and high dose group, especially in high-dose group;The expression of the ITG β 3mRNA was inhibited by the Guishenwan decotion. Guishenwan decotion as a multi-target compound decotion, the complex mechanism of action of multiple gene and protein expression were investigated. The results of the research provide a theoretical foundation and scientific basis for further development of drug research and discover of Guishenwan decotion.4. The mechanism of Guishenwan decotion treatment to Kidney deficiency-thin endometrium of rats based on the regulation of miR-135a, is through the upregulation of miR-135a expression in the endometrium, which has negative moderating effect to the expression of EMX2mRNA, so as to improve the endometrial receptivity. miR-135a and HOXA10 gene is not related.The intervention of the Guishenwan decotion can up regulation of the expression of HOXAlOmRNA.which can enhance the protein expression of ITG β 3 of the endometrium, so as to improve the endometrial receptivity.