Therapeutic Effect Of Ginsenoside Rh2on Experimental Model With Endometriosis And Its Mechanism Of Action

AIMThe study is to establish models of endometriosis in vitro and in vivo, and to investigate the therapeutic effect of Ginsenoside Rh2on models of endomtrisosis and explore its molecular mechanisms of action.METHODS1. After an adaptive feeding for a week, estrus cycle phase of rat was checked by means of vaginal smear. With surgical autotransplantation, rats with regular estrus cycles were selected to establish the rat model of endometriosis. After the recovering period of3weeks, the model rats were laparotomized to investigate the growth condition of the transplant.2. The rat models randomly divided into five groups:model control group(0.5%CMC-Na), Gestrinone group(1.5mg/kg) and Rh2low-,medium-and high-dose group(5、10、40mg/kg).After oral administrations for21days, rats with endometriosis were laparotomized again, and the volumes of peritoneal implants were measured.3. When rat models were sacrificed, blood samples in abdominal aortas were collected to measure levels of estrogen(E2), progesterone(P) and antiendometrium antibody (EMAb) using enzyme-linked immunosorbent assay (ELISA). Histopathologic changes of ectopic endometrium were tested by HE staining and the expressions of apoptosis factors, such as Bcl-2、Bax、Caspase-3and Caspase-9, were determined by western blotting.4. In order to establish cell model of endometriosis, endometrial cells of ectopic endometrium from patients with endometriosis were cultured in vitro. With the treatment of digestion in collagenase II, ectopic-endometrial tissues were isolated and purified.5. Endometrial cells were incubated in Phenol Red-free DMEM/F12media supplemented with10%charcoal stripped FBS, and cultured continuously for24h、48h、72h with the different concentration of Rh2(0.0001-100μmol/L), to analyse the effect of Rh2on the growth of endometrial cells.6. In order to investigate the alteration of phosphorylase kinase in endometrial cells, we screened the differential expression of phosphorylase kinase between the control group(DMSO) and Rh2group(30μmol/L) using high-throughput protein chip and Real-time RT-PCR technology.7. By Western blot technology, we examined the protein expression, such as pp38n pSrc、Bcl-2、Bax etc, between the control group(DMSO) and Rh2group(30μmol/L) with or without specific antagonists.RESULTS1. After oral administration of0.5%CMC-Na, Gestrinone(1.5mg/kg) and various doses of Rh2for21days, the volume of peritoneal implants in model control group obviously increased, while the volume of peritoneal implants in Gestrinone group, and Rh2low-,medium-and high-dose group(5%10、40mg/kg) obviously decreased. Rh2(40mg/kg) significantly inhibited the growth of ectopic endometrium(.P<0.05).2. The levels of E2, P, EMAb in serum were measured by ELISA after different treatments of Rh2. Rh2could dose-dependently reduce the level of E2, P and EMAb, and Rh2(40mg/kg) obviously decreased the level of E2, P and EMAb suggesting that its mechanism of action may relate to reducing the level of E2、P and EMAb.3. Ginsenoside Rh2(5、10、40mg/kg) could increase the expression of Bax/Bcl-2、 Caspase-9、Caspase-3in ectopic endometrium, suggesting that Rh2induces the apoptosis of ectopic endometrium by upregulating the expression of Bax/Bcl-2、 Caspase-9、Caspase-3. 4. Ginsenoside Rh2could serve a dual function in endometrial cells:it can promote proliferation at low concentration (0.0001～1μmol/L), and inhibit growth at high concentration (10μmol/L～100μmol/L).5. Bispenol A(BPA) can promote the proliferation of endometrial cells at the concentration of0.01μmol/L and0.0001μmol/L, with the proliferation rate reaching to16%and12%, respectively. Ginsenoside Rh2can enhance the inhibitory effect on endometrial cells in the presence of BPA(0.01μmol/L).(P<0.05).6. Ginsenoside Rh2can upregulate the expression of phosphorylated-Src(Y419), downregulate the the expression of phosphorylated protein, such as JNK pan (T183/Y185,T221/Y223、GSK-3α/β(S21/S9)、β-catenin, ERK1/2(T202/Y204,T185/Y187) and MSK1/2(S376/S360). Futher analysis shows the difference of protein kinase mainly involved Src pathway and p38MAPK pathway.7. When Src/p38MAPK pathway was inhibited, Ginsenoside Rh2can enhance the expression of Bax/Bcl-2, suggesting that inhibition of endometrial cell by Rh2may be related to inhibition of Src/p38MAPK pathway.CONCLUSION1. Ginsenoside Rh2, a natural product monomer, can obviously inhibit the growth of ectopic endometrial at the dose of40mg/kg, and it could have a promising role to treat endometriosis.2. Ginsenoside Rh2inhibits the growth of ectopic endometrium in the established rat model of endometriosis, and its mechanism of action may be related to reducing the level of E2, P and EMAb, and increasing the protein expression of Caspase-9, Caspase-3and Bax/Bcl-2.3. Ginsenoside Rh2may have double effects on endometrial cells:it can promote proliferation at low concentration (0.0001～1μmol/L), and inhibit growth at high concentration (10μmol/L～100μmol/L).4. Bispenol A(BPA) can promote the proliferation of endometrial cells, Ginsenoside Rh2can enhance the inhibitory effects on endometrial cells in the presence of BPA.5. Ginsenoside Rh2can upregulate the expression of phosphorylated-Src(Y419), downregulate the the expression of phosphorylated protein, such as JNK pan (T183/Y185, T221/Y223)、GSK-3α/β(S21/S9)、β-catenin、ERK1/2(T202/Y204, T185/Y187) and MSKl/2(S376/S360).Ginsenoside Rh2can enhance the expression of Bax/Bcl-2to induce apoptosis of endometrial cells When Src/p38MAPK pathway was inhibited.