Based On The PI3K/AKT/mTOR Signaling Pathway, The Molecular Mechanism Of Repairing The Follicular Developmental Disorder Caused By Tripterygium Glycosides By The Method Of Invigorating The Kidney And Promoting Blood Circulation Was Studied

Part one The isolation, primary culture and identification of rat ovarian granulosa cellOBJECTIVES:To isolate the rat ovarian granulosa cell for primary culture and to search the best time point of the granulosa cell for the detection.METHODS:Sacrifice the female mature SD rat in 48h after subcutaneous injection of PMSG 40IU. Put the ovary into PBS fluid and wash, release the granulosa cells from the follicles with empty entry needle. Collect the granulosa cells after filter and centrifugation. Put the granulosa cells into the culture dish with DME/F12/mycillin/ fetal bovine serum medium and cultured in the environment of 37℃ and 5% CO2 incubator. In 24h after incubation, the cells were divided into 5 groups:blank controlled group, GTW group, west medicine group (GTW+estradiol valerate), TCM high dosage group (GTW+XJGSW high dosage) and TCM low dosage group (GTW+XJGSW low dosage). The blank control group were cultured with the serum from the rat intragastrically administrated with physiological saline, other groups were cultured with the serum from the rat intragastrically administrated with 80mg/kg·d GTW. After 48h, all groups were cultured with corresponding serums respectively. The tests except MTT were proceeded in 48h after cultivation with corresponding serums. The HE stain and FSHR immunocytochemical testing were proceeded in 120h after cell culture, while the MTT testings were proceeded in the 48h,72h,96h,120h and 144h after cell culture to observe the growth, proliferation and apoptosis of the granulosa cells so as to determine the best time for the coming experimental test.RESULTS:The HE stain showed that the cells were with complete cellular morphology and clear edge with irregular polygon shape. The nucleus was stained with deep blue color and the cytoplasm with carnation, which presented to be granulosa cells. The FSHR immunocytochemical test showed that the positive cells presented yellow-brown color in the cytoplasm and the purple-blue color in the nucleus while the negative cells presented purple-blue color in the nucleus and clear color in the cytoplasm. The FSHR positive rate was higher than 90%. The MTT test presented fastest proliferation speed of the granulosa cells during 96h-120h of culture with the peak OD value.CONCLUSIONS:The isolation and primary culture of the rat ovarian granulosa was successful. The granulosa cells growth fastest in 96h-120h after isolation and the test could be proceeded with the cells collected at 120h after isolation in the following experimentPart two Experimental study of the molecular mechanism of GTW induced follicle development disorder and the recovery by reinforcing kidney and activating blood based on PI3K/AKT/mTOR signal pathwayOBJECTIVES:Based on the PI3K/AKT/mTOR signal pathway to study the molecular mechanism of the follicle development disorder caused by GTW and the mechanism of its recovery treated with kidney reinforcement and blood activation TCM formula Xinjiaguishenwan.METHODS:To isolate and culture the rat ovarian granulosa cell as methods mentioned in part one. In 24h after the normal culture, the cells were divided into 5 groups:blank controlled group, GTW group, west medicine group (GTW+ estradiol valerate), TCM high dosage group (GTW+XJGSW high dosage) and TCM low dosage group (GTW+XJGSW low dosage). The blank control group were cultured with the serum from the rat intragastrically administrated with physiological saline, other groups were cultured with the serum from the rat intragastrically administrated with 80mg/kg·d GTW. After 48h, all groups were cultured with corresponding serums respectively. The tests were proceeded in 48h after cultivation with corresponding serums. The granulosa cells proliferation rate was tested with MTT methods. Western-blotting was proceeded to test the PI3K, p-AKT, p-mTOR, IGF-land IRS-1 relative protein expressions and real time PCR were proceeded to test the PI3KmRNA, p-AKTmRNA, p-mTORmRNA, IGF-1mRNA and IRS-1 mRNA relative expressions.RESULTS:MTT results showed that comparing to the blank control group, all other groups presented lower OD value and the GTW group presented lowest OD value. The different was significant (P<0.01). Comparing to the GTW group, the OD values of western medicine group and TCM groups were increased significantly(P< 0.01). Comparing to the western medicine group, the OD value difference of TCM high dosage group was not significant (P>0.05), but that of TCM low dosage group decreased significantly (P< 0.05). The western-blotting and RT-PCR results showed that comparing to the blank control group, GTW group presented significantly low expressions of PI3K, p-AKT, p-mTOR, IGF-1, IRS-1 proteins and mRNAs (P< 0.01). In 48h after treatment of estradiol valerate and different dosages of XJGSW, The PI3K, p-AKT, p-mTOR, IGF-1, IRS-1 proteins and mRNA expressions of western medicine group and XJGSW high dosage group increased significantly (P< 0.01) and the PI3KmRNA, p-mTOR protein and IRS-1 protein expressions presented similarly (P< 0.01 or P< 0.05). Yet the PI3K protein, p-AKT protein and mRNA, p-mTOR mRNA, IGF-1 protein and mRNA, IRS-1 protein and mRNA differences of XJGSW low dosage group presented no significant difference (P>0.05) comparing to the GTW group, but were with the tendency of increase. Comparing to the western medicine group, the PI3K protein and mRNA of XJGSW high and low dosage groups, the p-AKT protein and mRNA of XJGSW high dosage group, the p-mTOR protein and mRNA of XJGSW high dosage group, the IGF-1 protein and mRNA of XJGSW high dosage group, IGF-1 protein of XJGSW low dosage group, IRS-1 protein and mRNA of XJGSW high dosage group, IRS-1 mRNA of XJGSW low dosage group presented no significant difference (P>0.05), but the p-AKT protein and mRNA, p-mTOR protein and mRNA, IGF-1 mRNA and IRS-1 protein of XJGSW low dosage group presented significant difference(P< 0.01 or P< 0.05).CONCLUSIONS:GTW may deregulate the PI3K, p-AKT, p-mTOR, IGF-1 and IRS-1 protein and mRNA expressions of ovarian granulosa cells to inhibit the activity of PI3K/Akt/mTOR signal pathway. The mitosis signal of ovarian granulosa cells was blocked and the proliferation of granulosa cell decreased accordingly. Thus the growth of follicle slowed and the ovarian function was defected. While XJGSW, the kidney reinforcement and blood activation TCM formula, may upregulate the PI3K, p-AKT, p-mTOR, IGF-1 and IRS-1 protein and mRNA expressions of ovarian granulosa cells to promote the activity of PI3K/Akt/mTOR signal pathway. The mitosis signal of ovarian granulosa cells was accelerated and the proliferation of granulosa cell increased accordingly. Thus the growth of follicle accelerated and the defected ovarian function was recovered.