| Objective: The expression of HIF-1a and its regulatory factors PHD2,VHL in the eutopic and ectopic endometrium in endometriosis by clinical study,and to investigate the relationship of PHD2, VHL and endometriosis; To observe the effect of Bushenwenyanghuayu on kidney yang deficiency and blood stasis Endometriosis Rat Models of HIF-1a, PHD2, VHL expression,research the mechanism Bushenwenyanghuayu in treatment of endometriosis.Methods:1 The first part: the expression of HIF-1a and its regulatory factors PHD2,VHL in endometriosis.Experimental group: in September 2013 to August 2014 selected 20 cases of endometriosis patients with surgical treatment of laparoscopy or laparotomy from Hebei Province Hospital of traditional Chinese medicine, The Second Hospital of Hebei Medical University and The sixth hospital of shijiazhuang Intraoperative to return the same in ectopic and eutopic endometrium in patients with fresh tissue signed informed consent(patients) with postoperative confirmed by pathological diagnosis of endometrial tissue.Preoperative 3 months not used hormone therapy, age 26 to 39 years old,Control group:collect in the same period for uterine leiomyoma whole hysterectomy patients with uterine fibroids and normal endometrium tissues as control group, and confirmed by pathology.Patients age 30 to 40 years old.Two groups of patients age distribution are balanced,there was no statistically significant difference.Using immunohistochemistry,Western-blotting detection of ectopic endometrium, eutopic endometrium,uterine leiomyoma, normal tissues of HIF-1a, PHD2, VHL protein expression,RT-PCR was used to detect the expression of HIF-1a m RNA, PHD2 m RNA,VHL m RNA.2 The second part: the establishment of kidney yang deficiency and blood stasis Endometriosis Rat ModelSimulation of department of gynaecology "wading in the rain, feel the cause of cold evil", "the theory of traditional Chinese medicine on the basis of pathogenic cold impairing Yang", and the traditional "transplantation" endometriosis animal model with kidney yang deficiency blood stasis to establish model of combination of disease and syndrome.On the basis of frozen time and sequence for 3 different models, as follows: frozen 1 month,make endometriosis models, frozen again 1 month is set to "pre-and post-frozen" group; frozen 1 months,endometriosis after building not frozen "pre-frozen" group;first not frozen, lines of endometriosis after building,continue to freeze a month, is set to "post- frozen" group, and blank control group.With macro performance,physical and chemical inspection index to evaluate the clinical conforms to the degree of the model.3 The third part: Bushenwenyanghuayu prescription impact on HIF-1a,PHD2, VHL expression of kidney yang deficiency and blood stasis endometriosis RatWill only 50 SD rats were divided into blank control group,sham operated, model group, traditional chinese medicine, gestrinone, 10 in each group, Which the conventional breeding were control group and sham operated;model group, traditional chinese medicine, gestrinone uses the "extension of the refrigerator with ice water immersion" for 30 days,combined with "autologous transplantation of law" to create the kindey yang deficiency blood stasis model,After giving chinese medicine group "the kidney yang and removing blood stasis",gestrinone "pregnant three ketene" gastric perfusion, model group, control group, sham operated group was given the same volume of ectopic focus, to observe the morphology and size of the rats after 4 weeks, light microscope to observe the morphological changes of eutopic and ectopic endometrium tissue HE staining,immunohistochemical SABC method, Western blotting detection of HIF-1a, PHD2, VHL protein,RT-PCR was used to detect the expression of m RNA, HIF-1a PHD2 m RNA,VHL m RNA.Result:1 Part 1: the expression of HIF-1a, PHD2, VHL in endometriosis1.1 The expression of HIF-1a, PHD2,VHL in ectopic endometrium, eutopic endometrium, uterine leiomyoma, normal endometrium tissue by Immunohistochemical method.1.1.1 The expression of HIF-1aThe positive expression of HIF-1a showed brown granular. In the ectopic tissues, the cells arranged in disorder, epithelial cells were lightly stained,glandular epithelium turned shallow; eutopic glandular relatively clear,positive cells were heavier, cells arranged in disorder in fibroids tissues, and had no complete glands, the structure was in fuzzy, cells shrinked and necrosis,the expression of positive cells shallowed. In the normal endometrium glandular integrity, the positive cells were lightly stained. The average optical density value of the expression, the higher of the quantity showed the greater of the content. The order from high to low of HIF-1a expression was:ectopic endometrium group, eutopic endometrium group, uterine leiomyoma group, normal endometrium group.The expression of HIF-1a in ectopic endometrium enhanced. Compared with eutopic endometrium,uterine leiomyoma and normal endometrium group, the differences were statistically significant(P<0.05); Compared with the normal endometrium group and uterine leiomyoma group, the eutopic endometrium group was statistically significant(P<0.05).Compared with the normal endometrium group,the uterine leiomyoma group was statistically significant(P<0.05).1.1.2 The expression of PHD2The positive expression of PHD2 showed brown granular. Ectopic endometrium gland was integrity, the expression of positive cells were lightly stained;eutopic endometrium glandular was integrity, the epithelium is thin,the positive cells were lightly stained; there was no integrity glands in uterine fibroids group, the positive cells were stained heavier; normal endometrium glands were intact, cells arranged closely, the positive cells were stained heavier. The expression of PHD2 from high to low in each group was: normal endometrium group, uterine leiomyoma group,eutopic endometrium group and ectopic endometrium group. Compared with uterine leiomyoma group,eutopic endometrium and ectopic endometrium group, the expression of PHD2 in normal endometrium group was statistically significant(P<0.05);Compared with eutopic and ectopic endometrium group, the uterine leiomyoma group was statistically significant(P<0.05); Compared with eutopic endometrium group, the ectopic endometrium group was statistically significant(P<0.05).1.1.3 The expression of VHLThe positive expression of VHL showed brown granules. The ectopic endometrial glands showed incomplete, the positive cellsstained shallow;eutopic endometrium glands were intact, cells stained shallow; uterine leiomyoma group glandular epithelial cellswere not integrated,the epithelium was thin and flat, positive cells were stained heavier; the normal endometrium glands were integrated,the epithelial cells were columnar, there was a small amount expression in the connective tissue, the positive cells in the free-end were highly. The expression of VHL from high to low was : normal endometrium group, uterine leiomyoma group, eutopic endometrium group and ectopic endometrium group. Compared with uterine leiomyoma group,eutopic endometrium group and ectopic endometrium group, the expression of VHL in normal endometrium group was statistically significant(P<0.05); the uterine leiomyoma group compared with eutopic endometrium group and the ectopic endometrium group was statistically significant(P<0.05).1.2 The expression of HIF-1a, PHD2, VHL in ectopic endometrium, eutopic endometrium, uterine leiomyoma, normal endometrium tissues by Western-blot.1.2.1 The expression of HIF-1a proteinAccording to the statistical results, The expression of HIF-1a protein from high to low in each group was : ectopic endometrium group,eutopic endometrium group,uterine leiomyoma group,normal endometrium group, ectopic endometrium group was statistically significant compared with other groups(P<0.05); Compared with the uterine leiomyoma group, the expression of eutopic endometrium was no significant difference(P>0.05).Compared with normal endometrium group, there was statistically significant(P<0.05),there was statistical significance between uterine leiomyoma and normal endometrium group(P<0.05).1.2.2 The expression of PHD2 proteinAccording to the statistical results, the expression of PHD2 protein from high to low in each group was as follows, normal endometrium group, uterine leiomyoma group,eutopic endometrium group and ectopic endometrium group. Compared with ectopic endometrium group, normal endometrium group and eutopic endometrium group had statistical significance(P<0.05),Compared with eutopic endometrium group and ectopic endometrium group,the expression in uterine leiomyoma group was significant difference(P<0.05).There was significant difference in eutopic endometrium group and ectopic endometrium group(P<0.05).1.2.3 The expression of VHL proteinAccording to the statistical results, the expression of VHL protein from high to low in each group: normal endometrium group, uterine leiomyoma group,eutopic endometrium group and ectopic endometrium group,Compared with the normal endometrium group, there was statistically significant in uterine leiomyoma group, eutopic endometrium group and ectopic endometrium group(P<0.05); Compared with the eutopic endometrium group and ectopic endometrium group,there was statistically significant in the uterine leiomyoma group(P<0.05); Compared with eutopic endometrium group, there was no significant difference in ectopic endometrium group(P>0.05)PHD2 m RNA, VHL m RNA expression1.3 RT-PCR detection of eutopic and ectopic endometrium HIF-1a m RNA,1.3.1 The expression of HIF-1a mRNAAccording to the statistical results, the expression of HIF-1a mRNA order from high to low was as follows: ectopic endometrium, eutopic endometrium group, uterine leiomyoma group, normal endometrium group. Enhanced expression of HIF-1a in ectopic endometrium, compared with that in eutopic endometrium, uterine leiomyoma and normal endometrium group the differences were statistically significant(P<0.05); Compared with eutopic endometrium, there was statistically significant in uterine and normal endometrium groups(P<0.05), Compared with the normal endometrium group,there was statistically significant in uterine leiomyoma group(P<0.05).1.3.2 The expression of PHD2 m RNAAccording to the statistical results, the expression of PHD2 mRNA order from high to low is normal endometrium group,uterine leiomyoma group,eutopic endometrium,ectopic endometrium group.there was statistically significant difference between ectopic endometrium group and the normal endometrium group(P<0.05), there was no significant difference between the expression of eutopic endometrium,uterine leiomyoma group(P>0.05); there were statistically significant between eutopic endometrium and normal endometrium group(P<0.05), no statistical significance compared with uterine leiomyoma group(P>0.05);there were statistically significant between uterine leiomyoma group and the normal endometrium group(P<0.05).1.3.3 The expression of VHL mRNAAccording to the statistical results, the expression of VHLmRNA order from high to low was as follows, normal endometrium group,uterine leiomyoma group,eutopic endometrium group,ectopic endometrium group.Ectopic endometrium group showed significant differences compared with eutopic endometrium, uterine leiomyoma and normal endometrium group(P<0.05); Compared with eutopic endometrium and uterine leiomyoma group,there was statistically significant in normal endometrium group(P<0.05),there was statistical significance between the uterine leiomyoma group and the normal endometrium group(P<0.05).1.4 The correlation between HIF-1a and PHD2, VHL expressionAccording to the statistical results,HIF-1a was negatively correlated with PHD2, the correlation coefficient was-0.908; HIF-1a was negatively correlated with VHL, the correlation coefficient was-0.875.2 Part 2: the establishment of Rat Model of Endometriosis due to kidney yang deficiency and blood stasis2.1 During the experiment, no significant difference was observed in blank control group rats appearance, white coat color was luster, spirit was fine, lively, and responsive, their diet, urination and defecation were normal.the rats appeared dirty hair, unresponsive, curled up, lazy, hind legs move slowly, chills, cluster, afraid of cold, claws and tails were pale, the tail subcutaneous were purple, the ears’ color was pale, breathing lightly,lackluster, little drinking, and loose stool in other three groups. In the rats of Pre-and post-frozen group, their performance was worse apparently.2.2 Outlooks of ectopic endometrium by the naked eyesIn pre-and post-frozen group, grafts grew well, volume was apparently increased, the uplift of dark red or bright utricle, internal full of effusion, visible surface vessels,blood vessels surrounding in ectopic tissues was rich, some of the ectopic tissue with the surrounding tissue adhesion; in pre-frozen group, post-frozen group, graft volume was small, fine surface vessels, tissue blood flow around the ectopic was poor.2.3 The body temperature of rats in each groupFrozen after 1 month, in pre-and post-frozen group and pre-frozen group rats,the body temperature were lower than post-frozen group and blank control group, after statisticsal processing, pre-and post-frozen group of rats body temperature and post-frozen and blank control group were statistically significant(P<0.05),there was no significant difference compared with pre-frozen(P>0.05; post-frozen group and normal group had no significant difference(P>0.05). After 2 months, pre-and post-frozen and pre-frozen group, blank control group had statistical significance(P<0.05), there was no statistical significance compared with post-frozen group(P>0.05), pre-frozen group and normal group had no significant difference(P>0.05).2.4 The weight of rats in each groupFrozen 1 month later, the body weight of all rats increased, but compared with the pre-and post-frozen and pre-frozenrats, the post-frozen group and control group body weight increased slowly, after statistics processing, pre-and post-frozen, compared with model pre-frozen, blank control group rats weight difference was statistically significant(P<0.05), post-frozen compared with pre-frozen group was statistically significant(P<0.05), but compared with normal group it had no significant difference(P>0.05).Frozen 2 months later pre-and post-frozen rats compared with other groups were statistically significant(P<0.05),post- frozen compared with pre- frozen group had no statistical significance(P>0.05).2.5 Comparison of serum sex hormone levels in all groupsEach rats of estradiol, progesterone and testosterone levels from low to high order is: pre-and post-frozen,post-frozen,pre-frozen, blank control group,after statistical processing, pre-and post-frozen estradiol levels compared to the rest of the groups differences were statistically significant(P<0.05),post- frozen compared with pre-frozen group was not statistically significant(P>0.05).The expression of testosterone, progesterone level was comparatively different,which was statistically significant between any two groups(P<0.05).2.6 Comparison of whole blood viscosity and reduced viscosity by HemorheologyThe results showed that the levels of whole blood viscosity from high to low order was: pre-and post-frozen,post-frozen,pre-frozen,blank control group.After statistics processing, The differences in the whole blood viscosity of low cut, cut and high cut were statistically significant between these groups(P<0.05).The levels of reduction viscosity from high to low order was: pre-and post-frozen,post-frozen, pre-frozen,blank control group.There was significant difference between every group low shear reduction viscosity(P<0.05); pre-and post-frozen group,post-frozen group and blank control group in cut all were statistical significant(P<0.05),compared with pre-frozen group was not statistically significant(P>0.05); the expression of high cut between pre-and post-frozen group, pre-frozen and blank control group were statistical significant(P<0.05).compared with post-frozen group was not statistically significant(P>0.05),post-frozen group compared with pre-frozen group,normal group were statistical significant(P<0.05).2.7 Comparison of rat Viscera indexThe Viscera index of spleen, thymus,uterus from low to high order was: pre-and post-frozen,post-frozen,pre-frozen, blank control group, pre-and post-frozen rats spleen index comparing with other groups were statistically significant(P<0.05),post-frozen compared with normal group about spleen index(P<0.05); pre-frozen rats thymus index compare with other groups were statistically significant(P<0.05),pre-frozen group difference in the rats uterus index frozen model,blank control group was statistically significant(P<0.05).2.8 The levels of serum CA125, c AMP and c GMPResults showed that the pre-and post-frozen group rats serum CA125 and c GMP was higher than other groups,compared with others pre-and post-frozen group was statistically significant(P<0.05), the levels of CA125 and c GMP in pre-frozen and post-frozen were higher than normal group(P<0.05); rats serum c AMP was lower than the rest of the group,compared with the other groups was statistically significant(P<0.05), the levels of c AMP in pre-frozen and post- frozen was lower than normal group(P<0.05).3 Part 3 :the effect of Bushenwenyanghuayu prescription on the HIF-1a,PHD2, VHL expression in the rat models of endometriosis due to kidney yang deficiency and blood stasis.3.1 The general condition of ratsControl group rats showed white coat color luster, spirit was well and lively,sensitive reaction, normal diet and discharge.Compared with control group, the hair color were darker, more lively, spirit was better, more movement, the reaction was sensitive, eating normally in the sham-operated group. The rats in the model group showed the body dirty, unresponsive,curled up and lazy,less eating, loose stool. In the traditional Chinese Medicine group, gestrinone group rats showed that dirty hair color were dark,sensitive reaction, activity, less eating, normal waste.3.2 Observation of ectopic foci in the morphologyIn the Traditional Chinese medicine, gestrinone group and model group, grafts grew well, volume increased obviously, the uplift of the dark red or bright utricle, in which full of fluid, the surface blood vessels were clearly visible, blood vessels surrounding ectopic tissues were rich, some abdominal viscera adhered to ectopic tissue.3.3 HE staining under light microscopeAccording to the results of control group, the membrane with lumens,complete, neat rows. Sham-operated group endometrium was slightly thickened, the film inside the lumens, disordered arrangement of cells.Endometrial lining model group compared with sham-operated group more thick, glandular, tube cavity disappeared, disordered arrangement of cells and inflammatory cells increased, traditional chinese medicine group lumen was complete, but the shape was irregular, lining order, membrane cells arranged in neat;gestrinone group of endometrial was incomplete,lumens was irregular, the membrane cell shrinked, cells arrangement were not neat.3.4 The protein expression of PHD2, VHL, HIF-1a in the eutopic endometrium, ectopic tissue by SABC methodThe results showed that HIF-1a positive particles appeared, mainly expressed in the endometrium, membrane, glandular cavity in the nucleus and cytoplasm;PHD2 mainly expressed on the cell membrane and small amounts of expression;VHL positive staining was mainly in the nucleus and cytoplasm.The average optical density reflected the content of index, the larger of the quantity reflected the higher of the content of index.Results showed that the expression of HIF-1a order from high to low model group was gestrinone group,Chinese Medicine,sham-operated group,control group.The model group rats HIF-1a expression were higher than that in sham-operated group, control group(P<0.05); the expression of HIF- 1a in Chinese Medicine and gestrinone group were lesser than model group(P<0.05); the expression of VHL PHD2 order from high to low control group was sham-operated group, Chinese Medicine,gestrinone group,model group.The expression of VHL PHD2 in model group was lower than the control group and sham-operated group, the expression of Chinese Medicine and gestrinone group were higher than that in model group(P<0.05).3.5 Results the protein expression of HIF-1a, PHD2, VHL by western blotting method in ectopic tissueThe results showed that the expression of HIF-1a order from high to low was model group, gestrinone group,chinese medicine group,sham-operated group,control group. The model group was higher than that in the control group, sham-operated group, compared with the two groups were statistically significant(P<0.05); the expression of HIF-1a was higher in Chinese Medicine and gestrinone group compared with the model group, there was statistically significant(P<0.05), Chinese Medicine and gestrinone group had no statistical significance(P>0.05). The expression of PHD2 order from high to low was control group, sham-operated group, gestrinone group,Chinese Medicine group,model group.The expression of PHD2 in the control and sham-operated group, were higher than model group,there was statistically significant difference between the two groups(P<0.05). The expression of PHD2 Traditional Chinese Medicine group and gestrinone group was higher than that of model group, was statistically significant(P<0.05), gestrinone group was higher than Chinese Medicine group(P<0.05). The expression of VHL order from high to low was control group, chinese medicine group,gestrinone group,sham-operated group,model group.The expression of VHL of model group was lower than that of the control group, sham-operated group,there was statistically significant difference between the two groups(P<0.05);traditional chinese medicine group and gestrinone group the expression of VHL was higher than that of model group, two groups compared with the model group were statistically significant(P<0.05), there was no significant difference between the chinese medicine group and the gestrinone group(P>0.05).3.6 mRNA Expression of HIF-1a, PHD2, VHL of eutopic endometrium and ectopic tissue in each groupThe expression of HIF-1a mRNA order from high to low was model group, Chinese Medicine group, gestrinone group,sham-operated group,control group.According to the results of model group HIF-1a m RNA expression was higher than the control and sham-operated group, compared to the groups were statistically significance(P<0.05), compared with model group, Traditional Chinese Medicine and gestrinone group were lower,there was statistically significant(P<0.05), traditional chinese medicine was higher than gestrinone group, there was no statistical significance(P>0.05); the expression of PHD2 m RNA order from high to low was control group,sham- operated group, gestrinone group,chinese medicine group, model group. Model group rats PHD2 m RNA expression were lower than the control group and sham-operated group, compared with the two groups were statistically significant(P<0.05); Model group rats PHD2 m RNA expression were lower than gestrinone group,Chinese Medicine group(P<0.05). The expression of VHL m RNA order from high to low was control group,sham-operated group, gestrinone group,Chinese Medicine group,model group.The expression of VHL m RNA in model group were lower than control and sham-operated group, compared with the two groups were statistically significant(P<0.05).The expression VHL m RNA in model group were lower than gestrinone group and chinese medicine group(P<0.05).3.7 The correlation of HIF-1a and PHD2, VHL expressionThe results showed that HIF-1a negatively correlated with PHD2(correlation coefficient of 0.799, p=0.799), HIF- 1 a and negatively correlated with VHL(correlation coefficient of 0.625, p = 0.625).Conclusions:1. The clinical studies showed that: the expression of HIF-1a in ecpotic tissues increased, and the expression of PHD2 and VHL proteins in ectopic endometrium reduced, which confirmed hypoxia exist in the state of endometriosis. The correlation study shows that HIF-1a and PHD2, VHL were negatively correlated,which confirms that in the condition of hypoxic,PHD2 can not be activated. Meanwhile, VHL does not recognize the HIF-1a subunit, thus enhance the expression of HIF-1a, which confirms that HIF-1a, VHL,PHD2 play important roles in the pathogenesis of endometriosis.2.We evaluate from the changes of the general conditions of rats, the view of pelvic ectopic lesions, serum progesterone, testosterone, estradiol,blood rheology bindexes, body temperature and weight of rats, and find that the experimental rats of “pre-and post-frozen group”, which followed the way “frozen one month for endometriosis model and then frozen another month”, matches the kidney yang deficiency and blood stasis syndrome clinical features of endometriosis most, which can be an animal model as kidney yang deficiency and blood stasis.3 The therapeutic method of tonifying kidney, warming yang and dissolving stasis can reduce the expression of HIF-1a in the model rats of endometriosis due to kidney yang deficiency and blood stasis improve the state of endometriosis hypoxic, thus increase the activity of PHD2 and mediate the degradation of HIF-1a. At the same time, VHL combined HIF-1a subunit, the latter protein was degraded and thus the expression of HIF-1a was decreased, and the expression of PHD2 and VHL was increased, which take effects in the treatment of kidney yang deficiency and blood stasis endometriosis. |
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