Effect Of Different Drugs On Leptin,State3 And Caspase3 In Rat Model Of Endometriosis

Endometriosis (EMS), is an estrogen-dependent gynecological disease. And the biological features change with the periodic female hormone. Clinical symptoms include:pelvic pain, pelvic mass, infertility etc. EMS has a series of characteristics which are similar with other similar malignant tumor, such as metastasis, invasion, adhesion, angiogenesis, reduced apoptosis and so on, also known as "benign tumor". In our previous studies,we had found that leptin, leptin receptor (OB-R) and stat3 would be expressed in ectopic endometrium of EMS. These suggested that could activate the JAK2/stat3 pathway through the combination of OB-R and leptin, promoting the proliferation of EMS lesions, and at the same time accompanied with disorder of local energy metabolism. In addition, caspase3 plays an important role in the apoptosis of ectopic endometrial cells.Simvastatin, a kind of statins, could down-regulate cholesterol, by suppressing cholesterol synthesis, reducing low density lipoprotein, and increasing high density lipoprotein. It also can regulate metabolism, anti-tumor, anti-inflammation, anti-apoptosis and prevent ischemia reperfusion injury through multiple signal pathways in different organs. Metformin is an effective medicine for the treatment of type 2 diabetes in the past. In recent years, metformin may play an important role in the anti-tumor effect through different mechanisms. Since EMS has abnormal energy metabolism, and decreased apoptosis, Whether simvastatin and metformin, which could improve the energy metabolism of the body and promote apoptosis through a variety of ways, can be used in the treatment of EMS! In this experiment, we try to study the pathogenesis of EMS and provide a new target for the treatment of EMS by exploring the abnormal energy metabolism and cell apoptosis of EMS lesions.Objevtive1. To establish the EMS model of Lewis rats by intra-peritoneal transplantation.2. To observe the effects of puerarin. simvastatin. metformin and mifepristone on EMS lesion in rats.3. To investigate whether the four drugs could have any effect of leptin and stat3 in EMS lesions.4. To investigate the effect of the four medicine on apoptosis of the EMS lesions by observing the expression of caspase3.Method1. To choice 70 healthy female Lewis rats, divided into two groups:donor group (n= 9) and experimental group (n= 61). Then to establish EMS allograft model of rats, estradiol benzoate 0.05mg/100g, intra-muscular injection,4 weeks.2. To observe the volume of EMS lesions in rats, and calculate the success rate of the EMS model.3.50 EMS rats were divided into five groups randomly:1) in the control group; 2) puerarin group (50mg/kg); 3) simvastatin group (5mg/kg); 4) metformin group (126mg/kg); 5) mifepristone group (6.33mg/kg). Each group were given corresponding drugs. Continue daily estradiol benzoate 0.05mg/100g, for 4 weeks.4. The lesion volume was observed again, removing of lesions for the next step.5. to detect Leptin, Stat3, Caspase3 by immunohistochemistry (IHC), real-time fluorescence quantitative polymerase chain reaction (RT-PCR) and Western blotting (Western-blot).Result1.86.9% of rats (n=53) were successfully modeled, and the average volume of each lesion was 27.01+3.72 mm3.2. Metformin group and mifepristone group was smaller than that control group; puerarin group was greater than control group; simvastatin group shows no difference with control group.3. Frozen section IIIC showed:1). leptin expression of puerarin group compared with control group, shows no significant difference; the other three groups were lower than control group; 2).the expression of Stat3 in simvastatin group was lower than that in control group; puerarin group was not significantly different from that in control group; metformin group and mifepristone group was higher than control group; 3). There was no significant difference in the expression of Caspase3 between the puerarin group and control group; simvastatin group was lower than control group; metformin group and mifepristone group was higher than that in control group.4. RT-PCR showed:the expression of leptin mRNA in four experimental groups were lower than control group; 2). expression of stat3 mRNA in puerarin group and simvastatin group were lower than control group; metformin group and mifepristone group was higher than control group.3). the expression of mRNA Caspase3 in puerarin group and control group had no significant difference; another three experimental groups were lower than that of control group.5. Western blot showed:1). leptin of simvastatin group was lower than control group; metformin group and mifepristone group was higher than control group; puerarin group and control group show no significant difference; 2). stat3 of four experimental group were lower than the control group; 3). caspase3 in puerarin group was lower than control group; another three groups was higher than control group.Conclusion1. EMS model of intra-peritoneal transplantation in Lewis rats has many advantages, such as high survival rate, easy to observe, strong vitality, ability to receive multiple operations, complete immune system and similar the organization and biochemical characteristics with human.2. Puerarin can reduce the mRNA expression of leptin, Stat3, caspase and the amount of caspase3 protein in the EMS, while increasing the amount of leptin and Stat3 protein through signal pathway of stat3 and inhibition of apoptosis of EMS cells. Puerarin is not suitable for the treatment of endometriosis.3. Though lesions were not inhibited, Simvastatin down-regulated the mRNA expression and protein of leptin, stat3 and caspase3. Therefore, further experiments are needed to investigate whether simvastatin can be used in the treatment of EMS.4. Metformin could induce apoptosis through caspase3 rather than stat3 pathway. Metformin is expected to become a new drug for the treatment of EMS.5. Mifepristone can inhibit the growth of EMS lesions through caspase3-induced apoptosis.6. Leptin can be used for the diagnosis of EMS, but not become a reference of the efficacy for EMS.7. Through the study of the energy metabolism and apoptosis of EMS, it is helpful to clarify the proliferation and death of EMS cells, identify new therapeutic concepts, and explore new therapeutic drugs.