| Objective:To explore the inhibitory effect of Hydroxysafflor yellow（HSYA）on H22 liver cancer cells and the influence of PI3K/Akt/mTOR signaling pathway related proteins.Methods:1.The H22 liver cancer model was established and grouped:Forty-eight Kunming male mices,SPF grade,eight mices were selected as the blank group,and the mices that be left were injected with H22 cells under the skin to establish the H22 liver cancer mouse model.After 24hours of inoculation,they were divided into model group,HSYA low dose,medium,and high dose groups,and positive group random Ly,every group has 8 mices.HSYA low,medium and high dose groups(20,40,60 mg·kg-1·d-1)were administered by intraperitoneal injection,0.2m L each one day at a time.The mices of positive group were injected with 25 mg·kg-1·d-1dose cyclophosphamide by intraperitoneal injection,0.2 m L each,once every other day;the mices of model group and blank group were injected with 0.2m L normal saline each one day at a time.Keeping continuous medication,after 14 days,the mices were sacrificed and weighed.2.Using the Immersion method to measure the volume of the tumor tissue.3.The tumor tissue and spleen were weighed to calculate the tumor inhibition rate and spleen index.4.After the tumor tissue was fixed and embedded,the HE staining method was used to observe the morphology change of tumor tissue.5.TUNEL method to detect apoptosis of liver cancer cells.6.Immunohistochemical method and Western blot method were used to detect LC3 protein level which belong to autophagosome membrane marker protein.7.Immunohistochemistry and Western blot was used method to detect the protein expression which related PI3K/Akt/mTOR pathway.Results:1.The effect of HSYA on the general state and body weight of H22 liver cancer mice:The mices in the model group have poor mental state,their fur is smoother,their amount of food,water and activity were reduced significantly,and the weight of the mices increases rapidly;the mices in the administration group have better mental states.The mices of HSYA medication groups the fur were shiny,the amount of food,water and activity were increased significantly,and the weight was lower（P<0.05）.2.The effect of HSYA on the volume of H22 liver cancer mice:The tumor volume of the mices in model group is larger than the medicinal groups（P<0.05）.3.The effect of HSYA on the mass,spleen index and tumor inhibition rate of H22 liver cancer mice:The tumor mass of the mices in the model group was heavier than the medicinal groups.Compared with the model group,the tumor mass of the mices in the medicinal groups was reduced（P<0.05）.And the HSYA high-dose group was different obviously（P<0.01）.Compared with the blank control group,the spleen index of medicinal groups decreased（P<0.05）,and compared with the model group,the medication group decreased（P<0.05）.And the tumor inhibition rates of HSYA low,medium and high dose groups and positive group were32.01%,39.82%,44.51%,and 73.64%,respectively.4.The effect of HSYA on the pathological morphology of H22 liver cancer mice:Tumor morphology changes by HE staining,the model group has densely packed tumor cells,the number of cells is large,disorderly arrangement,large and deep stained nuclei,high nucleoplasm ratio,multiple mitosis,significant cell atypia,unclear boundary with surrounding tissues,and tumor cells have infiltrated peripheral muscle tissue;the tumor cells in the HSYA low-dose group are aggregated,the number of cells is large and the tumor cells have a disordered arrangement,the cells are mainly round and oval,the nucleoplasm is relatively large,the nuclear staining is deep and the atypia is significant;the HSYA medium-dose group has fewer tumor cells,The cell arrangement is slightly sparse,some tumor areas have necrosis,and the tumor cells are of different sizes;the tumor cells in the HSYA high-dose group are significantly reduced,megakaryocytes and multinucleated tumor cells are rare,and the nuclear staining is lighter.The tumor tissue shows large areas of necrosis,and the boundary with surrounding tissues is obvious;In the positive group,tumor cells were significantly reduced,tumor cell atypia was not obvious,tumor cell nucleus staining was lighter,cell volume was significantly reduced,and the boundary with surrounding tissues was clear,not infiltrating the surrounding tissues,and tumor tissue necrosis was seen in most locations.5.The effect of HSYA on the apoptosis of H22 liver cancer cells:Apoptotic cells can show nuclear pyknosis,round,crescent and irregular shapes.The microscopic results showed that apoptotic cells were rare in the model group,and the apoptotic cells in medicinal groups were more,and a clear distribution of apoptotic cells was visible,with a significant difference statistically（P<0.05）.6.The effect of HSYA on the expression of LC3 protein of H22 liver cancer cells:The expression of LC3 protein in tumor cells of the model group was less,and the expression of LC3protein in tumor cells of the medicinal groups increased.Among them,the HSYA high-dose group was different obviously from the model group（P<0.01）,suggesting that HSYA can induce hepatocarcinoma cells to spontaneously bite.7.The effect of HSYA on the expression of PI3K/Akt/mTOR pathway:Compared with the model control,the results of immunohistochemistry and Western-bloting indicated that the expression of PI3K,Akt and mTOR protein in tumor cells of the HSYA each dose group and the positive group was significantly reduced（P<0.01）.Western-bloting results suggest that,with the gradual increase in the dose of the medicinal group,the protein expression of P-PI3K/PI3K,P-Akt/Akt,and P-mTOR/mTOR gradually decreased,compared with the model group,with a statistically significant difference（P<0.05）.Conclusion:HSYA has inhibitory effects on H22 liver cancer cells;its inhibitory effects on H22 liver cancer cells may be related to the induction of tumor cell apoptosis and autophagy;its mechanism may be through inhibiting PI3K/Akt/mTOR signaling pathway to induce apoptosis and autophagy bite. |
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