Experimental Study Of The Inhibition Of PESV By Promoting Autophagy On H₂₂ Hepatoma

Objective: Observe the protein expression difference of angiogenesis and autophagy pathways,and the change of microvessel density after the therapy of polypeptide extract from scorpion venom(PESV) combined with Rapamycin on the H22 hepatoma growth. Make the effect of autophagy and anti-angiogenesis clearly of PESV and PESV combined with chemotherapy for tumor. Then explore the mechanism on autophagy and anti-angiogenesis, and provide theoretical basis for its anti-cancer clinical application.Methods: 1. Establish subcutaneous H22 hepatoma tumor model: the tumor-bearing mice were divided into four groups randomly:the control group,the PESV group,the Rapamycin group,and the combination group( PESV+ Rapamycin). The tumor volume was measured once every other day and then the tumor volume growth curve was drawn,and the tumor inhibitory rate was calculated.2. Observed H22 hepatoma tissue pathological changes under light microscopy after the routine HE staining was done. 3. Signing microvessel by factor VIII to detect the change of MVD, and observed the effect of PESV 、Rapamycin and combined therapy to angiogensis. 4. Immunofluorescence and Immunohistochemistry were performed to detect the expression level of mammal target of rapamycin(m TOR), hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor-A(VEGF-A),UNC-51-likekinase-1(ULK1)and microtubule-associated protein1 light chain 3(LC3A). 5. The expression of m TOR,HIF-1α,VEGF-A,ULK1 and LC3 A were confirmed and quantified by western blotting.Results: 1. The effect of PESV on the growth of H22 hepatoma transplantation tumor Compared with the model group, the growth speed and weight of H22 hepatoma transplantation tumor were inhibited obviously in the PESV group, Rapamycin group and the combination group after 14 days’ intervention(P<0.05,P<0.01), and the tumor inhibitory rates are the 17.7%,29.2%, 44.8%. And the tumor volume and weight of the Rapamycin group and the combined group were more obviously reduced(P<0.01). Compared with the Rapamycin group, the tumor volume and weight in the combination group were also obviously reduced(P<0.05). 2 The tumor tissue’s pathological changes of each group The results of HE staining showed that in the combined group, there were some large necrotic areas, and the tumor cells showed the morphologic changes of apoptosis and necrotic, such as nuclear fragmentation, nuclear pyknosis and so on. In the PESV group and the Rapamycin group there were also obvious necrosis areas and cells, but the levels of necrosis were all lower than the combination group. In the control group, there were fewer necrotic areas, and the necrotic areas were smaller, what’s more, tumor cells’ growth were exuberant, necrotic and apoptotic cells were fewer. 3The difference of MVD in each tumor group Immunohistochemical staining showed that factor VIII positioned in vascular endothelial cells.The positive cells’ cytoplasm was stained tan,and microvascular distributted dispersedly. Compared with control group, the MVD of Rapamycin group, PESV group and combined group were lower, and there was significant difference statistically for each comparison(P < 0.05, P < 0.01). Compared with the Rapamycin group,the combined treatment group was decreased more significantly(P<0.05). 4 The expression of m TOR, HIF-1α, VEGF-A, ULK1 and LC3 A detected by Immunohistochemical and Immunofluorescence staining.According to the fluorescence microscopy of frozen section, the green fluorescence of m TOR, HIF-1 α and VEGF-A was more evident respectively in the control group, intervention groups had lower fluorescence. The green fluorescence of ULK1 and LC3 A in combination group was more obvious respectively while other groups were lower. Immunohistochemical staining showed that m TOR, VEGF- A, ULK1 and LC3 A mainly expressed in tumor cell cytoplasm. The positive cells displayed brownish yellow granules on the cytoplasm. HIF-1α expressed in the nucleus and cytoplasm where the positive cells displayed brownish yellow granules. The semi-quantitative grey scale value analysis, the expression of m TOR, HIF-1α, VEGF- A in three intervention groups were lower than the control group(P<0.05,P<0.01), the expression of ULK1 and LC3 A were higher than control group(P<0.05). Compared with the Rapamycin group, the expressions of m TOR,HIF-1α and VEGF-A in the combination group were also obviously reduced(P<0.05), the expressions of ULK1 and LC3 A were enhanced(P<0.05). 5. The expression of m TOR, HIF-1α, VEGF- A, ULK1 and LC3 A detected by Western blotting. Western blotting showed that compared with control group, the expression bands of m TOR, HIF-1α, VEGF- A in the intervention groups were reduced(P<0.05), and the ULK1 and LC3 A were enhanced(P<0.05). Compared with the Rapamycin group, the tumor volume and weight in the combination group were also obviously reduced(P < 0.05). Compared with the Rapamycin group, the expressions of m TOR,HIF-1α,VEGF-A in the combination group were also obviously reduced(P<0.05), and the expressions of ULK1 and LC3 A were enhanced(P<0.05).Conclusion: 1. PESV and PESV combined with Rapamycin can inhibit the growth of H22 hepatoma tumor,and the combination group is more effective. 2. PESV and PESV combined with Rapamycin can inhibit angiogenesis in H22 hepatoma tumor, that may be related to inhibiting m TOR, HIF-1α and VEGF- A protein expression, and the combination group is more effective. 3. PESV and PESV combined with Rapamycin can promote autophagy of H22 hepatoma tumor cells, by enhancing the protein expression of ULK1 and LC3 A, and the combination group is more effective.