Effects Of Disintegrin Of Gloydius Brevicaudus Venom On Endometriosis Of Rat And Its Mechanism

OBJECTIVE:Endothelial cells could transplant and grow in ectopic organization,as we know Endometriosis(EMT),which similar to the biological behavior of malignant tumor. The key pathological progresses of EMT are heterotrophic adhesion, cultivation and growth of the endometrial cells and vascular endothelial cells. Thus blocking the processes effectively has become an important treatment strategy of EMT. Integrin plays an important role in the processes of adhesion, migration and growth of cell. MMP- 9 is another key bio-marker in these pathological progresses. First of all, MMP- 9 activate the work of integrin by interacting with it to enhance cell adhesion; secondly MMP-9 improve the budding and angiogenesis of vascular endothelial cells.While researches show that disintegrin from snake venom containing RGD sequence could inhibit the effect of integrin in adhesion, migration and growth of cell. So we try to apply the disintegrin to block the progresses of adhesion,migration and growth of EMT in rats and investigate the mechanism of it by assaying the expression of MMP-9 in endometrial cells of rat in vivo and in vitro. Our aim is to investigate the effect and mechanism of the disintegrin for treating EMT and provide experimental data for a new therapy of EMT.METHODS:1.Separation, purification and active identification of Gloydius brevicaudus Venom(GBV) : 1)Superdex 75 gel filtration chromatography: separate the venom according to the proteins molecular weight. 2) DEAE Sepharose Fast Flow anion exchange chromatography: separate the fraction according to different isoelectric points. 3) SDS-PAGE: identify the purity of the protein. 4) Born method to assay the ability of antiplatelet aggregation of the protein.2. Therapeutic effects of the disintegrin on the different development stages in rat EMT model:1) The protocol of EMT modeling surgery on rats: peel the endometrial falling off, and transplant it into the abdominal wall autonomously where is rich in blood vessels. Then open abdominal cavity to observe the growth of ectopic foci after 28 days. Dosing before the surgery, after the surgery, and after the model successful built.2) Effects of disintegrin dosing before EMT modeling: soaking the peeled endometrial in1.5μg/ml disintegrin solution for 30 minutes before ectopic transplantation. In compared group, the endometrial is soaked in normal saline. Observe the growth of ectopic foci 28 days later.3) Effects of disintegrin dosing after EMT modeling: dosing 1.5 mg/kg disintegrin through the sublingual vein injection after EMT surgery. Administrated 1.5 mg/kg disintegrin by tail intravenous after one week and three weeks, respectively. The compared group gives a same dose normal saline. And then observe the growth of ectopic foci 28 days after the surgery.4) Effects of disintegrin dosing after EMT model successful built: observe the growth of ectopic foci 28 days after the EMT surgery and then divided the modeled rats into three groups: NS group, low dose group(0.75 mg/kg) and high dose group(1.5 mg/kg) randomly.The rats were treated by disintegrin tail intravenous administrated.They were dosed rightly, in the second week, and in the third week, respectively.The compared group give a same dose normal saline.Open abdominal cavity to observe the the growth of ectopic foci three weeks after the surgery.3. Effect of disintegrin on the expression level of MMP-9 of rat EMT model in vitro and in vivo :1) Assay the effects of disintegrin on the expression of MMP-9m RNA of rat endometrial cells cultured by the disintegrin by RT-PCR in vitro:The cells was treated by disintegrin 1.5μg/ml for 24 hours. The cells were harvested and their total RNA were extracted. The m RNA of MMP-9 were tested by RT-PCR. The control group were treated by normal saline.2) Assay the expression of MMP-9 of endometrial cells of EMT modeled rats in vivo effected by the disintegrin by Western blot:Extract the total protein of ectopic foci of modeled rats collected from every group of three dosage strategies. Assay the expression level of MMP-9 each group.RESULTS:1.Separation, purification and active identification of Gloydius brevicaudus Venom(GBV) :Integrin of GBV was purified into a single protein band, whose molecular weight is about 7700 D. It has the activity of antiplatelet aggregation induced by ADP. The IC50 is 0.594μg / ml.2. Effects of the disintegrin on the different development stages of rat EMT model:1) The EMT modeled surgery on rats: We find transparent or translucent cysts in the abdominal cavity in the successes model rats 28 days after the transplantation grafts surgery.The blood vessels could be seem clearly, some of the ectopic foci cysts connect or adhere to the tissue or fat around, some other cysts even attach to the bowel. The average size of ectopic foci is 37.10±14.77 cm3.2) Effects of disintegrin dosing before modeling surgery on the forming of EMT lesion of rats:the size of ectopic endometrial of NS group(n=6) and disintegrin group(n=11) are 33.41±28.85 cm3 and 8.93±11.34 cm3 respectively, and the weights are 29.01±12.99 mg and 11.62±11.42 mg respectively. So the size and weight of ectopic foci both inhibited by disintegrin of GBV(*P<0.05).3) Effects of disintegrin dosing after modeling on the forming of EMT lesion of rats: The sizes of ectopic endometrial of NS group(n = 6) and disintegrin dosing group(n = 7) were58.50±37.71 cm3 and 5.33±5.48 cm3 respectively, and the weights are 101.98±75.19 mg and19.34±9.71 mg respectively. So the size(**P<0.01) and weight(*P<0.05) of ectopic foci treated by 1.5mg/kg·d-1 disintegrin after modeled group have reduced significantly compared to the NS group.4) Effects of disintegrin dosing after model successful built on the forming of EMT lesion of rat(n=6):The sizes of ectopic foci before the dosing of NS group, disintegrin low dose treatment group and disintegrin high dose treatment group are 36.19 ±19.09 cm3,35.15±15.88 cm3 and 39.95 ± 9.33 cm3 respectively, and the sizes of ectopic foci after the dosing are 31.20 ± 15.57 cm3, 24.44±14.29 cm3 and 22.01±10.47 cm3 respectively. We find the size of the endometrial lesion of disintegrin dosing groups administrated by 0.75mg/kg·d-1and 1.5mg/kg·d-1 were both reduced(*P<0.05) obviously compared to the control group.3. Effect of disintegrin on the expression of MMP-9 of rat EMT model in vitro and in vivo :1) Effect of disintegrin on the expression level of MMP-9m RNA of the rat ectopic endometrial cell in vitro:Assay the MMP-9m RNA in cultured of rat endometriosis cells after dosing in vitro by RT-PCR.We find out that the disintegrin(1.5 μg /ml) can inhibit the expression level of MMP-9m RNA of ectopic lesions obviously. The results show that: the MMP-9m RNA expression level of disintegrin group have reduced significantly compared with control group(*P<0.05).2) Effect of disintegrin on the expression of MMP-9 protein of EMT rats in vivo: Assay the expression of MMP-9 of rats with EMT by Western blot. The results show that the expression of MMP-9 of the three dosage strategies, namely dosing before modeling, dosing after modeling, and dosing after successes modeled, have decreased obviously compare to the control groups(*P<0.05).CONCLUSIONS:The disintegrin from GBV could reduce the size and weight of ectopic foci in EMT rats. The therapeutic effect might be associated with down-regulation of MMP-9 by blocking the binding of the legends such as extracellular matrix(ECM) to integrin.