The Effects Of Ligustrazine On Angiogenesis And Regulation Of KLF4/VEGF/ADAMTS1 Pathway In H22 Hepatocellular Carcinoma Mice

Objective:To study the effects of ligustrazine（tetramethylpyrazine,TMP）on angiogenesis and angiogenesis-related protein expression in H22 liver cancer mice.Methods:H22 liver cancer mice were divided into four groups randomly:model group（normal saline）,dox control group(DOX,2mg·kg^-1),ligustrazine low and high dose group(TMP 25,50mg·kg^-1).The mice were treated with drugs by intraperitoneal injection.Blood was collected at the end of the experiment to detect serum transaminase（ALT and AST）levels.Subaxillary tumors of H22 liver cancer mice were weighed and the tumor inhibitory rate was calculated.HE staining was used to observe the histological changes of underarm tumors in H22 liver cancer mice.The angiogenesis of tumor tissue was observed by immunohistochemistry.The expression changes of vascular endothelial growth factor（VEGF）,Kruppel-like factor 4（KLF4）and human metallopeptidase containing platelet-reactive protein 1（ADAMTS1）in the subaxillary tumor tissues of H22 liver cancer mice were detected by Western Blot.Results:1.Surface view of underarm tumor in H22 liver cancer mice.Compared with the model group,the low-dose and high-dose ligustrazine groups had smaller axillary tumors significantly,while the difference between the dox control group and the high-dose ligustrazine group was not significant statistically.2.Compared with the model group,the tumor weight and tumor inhibition rate of the low-dose and high-dose ligustrazine groups were reduced significantly,and there was no significant statistically difference between the high-dose ligustrazine group and the dox control group.The tumor inhibition rates of low and high dose TMP were31.6%and 53.53%,respectively.3.The results of serum ALT and AST showed that,compared with the model group,serum ALT and AST significantly decreased,while serum ALT and AST increased in the adriamycin control group.It showed that TMP had a significant inhibitory effect on the elevation of serum ALT and AST in liver cancer mice,and could improve the liver function of liver cancer mice.4.HE staining results showed that the tumor tissues in the model group presented a typical columnar and glandular-like nests,and the liver cancer cells were arranged into cords and clumps and infiltrated into the surrounding liver tissues.There was severe necrosis and bleeding in some areas.The low and high dose ligustrazine group could reduce the number of nests,and large areas of vacuolar necrosis appeared in the tumor tissue.There was no significant difference between ligustrazine high dose group and dox control group.5.Immunohistochemical results show that the model group,CD34 positive expression rate is high,a number of capillaries,ligustrazine low and high dose group with the increase of dose,angiogenesis gradually reduce,ligustrazine high-dose group and adriamycin control group there was no statistically significant difference effect of inhibiting vascular,showed that different concentrations of TMP can restrain varying degrees of liver cancer in mice tumor microvascular generation.6.Western Blot results showed that,compared with the model group,the low-dose and high-dose ligustrazine groups significantly inhibited the protein expression of VEGF and increased the protein expression of KLF4 and ADAMTS1.Conclusion:Ligustrazine has a certain inhibitory effect on angiogenesis in H22 liver cancer mice,and its mechanism is related to inhibition of angiogenesis by down-regulating VEGF protein expression and increasing of KLF4 and ADAMTS1 protein expression.