Study On The Anti-tumor Effect Of Leeches Based On Angiogenesi

"Anti-angiogenic" therapy is currently a hot direction of anti-tumor research,and its purpose is to cut off the key aspects of tumor progression.Vascular endothelial growth factor(VEGF)is the strongest angiogenic factor.Modern medical research has confirmed that there is a certain correlation between the content of thrombin in blood and the expression of VEGF in patients with malignant tumors.Leech is a common anticoagulant traditional Chinese medicine.Its main anticoagulant active ingredients are thrombin inhibitors.We speculate that leech can inhibit the formation of new blood vessels and play an anti-tumor role through its anticoagulant action.Therefore,this project aims to study the relationship between leech anticoagulation and anti-tumor.Firstly,the anti-tumor effect of leeches was determined by human hepatocellular carcinoma Hep G2 as tumor cell model.Secondly,the human umbilical vein endothelial cells(HUVEC)as the in vitro model,the chick chorioallantoic membrane(CAM)as the in vivo model and the zebrafish as the overall model were used to observe the effects of leeches on angiogenesis.Finally,the model of tumor-bearing mice was established to observe the effect of leeches on the density of tumor angiogenesis in mice,the content of thrombin and VEGF were measured to elucidate the anti-tumor mechanism of leeches in inhibiting angiogenesis.This project was expected to reveal the reasons why leech is both anticoagulant and anti-tumor,provide a basis for Hirudo as a low toxic and anti-tumor Chinese medicine,and provide a theoretical basis for the further development of Guangxi characteristic animal drug.The main experimental methods and results are described below.Ⅰ.Preparation and in vitro activity assay of leech extractLow-temperature aqueous extraction method was used to extract the Whitmania pigra Whitman and Hirudinaria manillensis,and the anticoagulant activities were respectively applied to the samples by thrombin titration method and a coagulation method.It aimed to determine the effectiveness and rationality of the extraction method.Results:Both the Whitmania pigra Whitman and Hirudinaria manillensis had good anticoagulant activity in vitro.The activity of Hirudinaria manillensis was far greater than that of Whitmania pigra Whitman.Conclusion:The method of low-temperature water extraction can ensure the anticoagulant activity of Whitmania pigra Whitman and Hirudinaria manillensis.Ⅱ.Study on Effect of Leech Extract on Tumor CellsTo determine the anti-tumor effect of Whitmania pigra Whitman and Hirudinaria manillensis,taking human hepatocellular Hep G2 as the representative,the inhibition rate of Hep G2 proliferation was determined by MTT assay at the concentrations of 2,4,and 10 mg/mL of them at 24,48,and 72 h.Results:After 72 hours of administration,the proliferation of Hep G2 was significantly inhibited at doses of 2,4 and 10 mg/mL(p<0.01).The inhibition of the Hirudinaria manillensis was stronger than that of Whitmania pigra Whitman,and there was a significant difference(p<0.01).Conclusion:The Whitmania pigra Whitman and have significant anti-tumor effects,and the Whitmania pigra Whitman has a weaker inhibition on the proliferation of Hep G2 than Hirudinaria manillensis.Ⅲ.Study on the effect of leech extract on angiogenesis1.Study on the proliferation of HUVECs by extracts of leech1.1 Study on the proliferation of normal HUVECs by leech extractThe MTT assay was used to determine the proliferation inhibition effects of the Whitmania pigra Whitman and Hirudinaria manillensis on normal cultured HUVECs to determine the direct effect of the two kind of leeches on endothelial cells.Results:When the concentration of water extracts of Whitmania pigra Whitman and Hirudinaria manillensis was 1 mg/mL,the inhibition rate of HUVECs proliferation reached 25.2%.When the extracts of Whitmania pigra Whitman were increased to 4,12 mg/mL,there was no significant increase in the inhibition of HUVEC proliferation.When the extract of Hirudinaria manillensis increased to 12 mg/mL,the inhibition of HUVECs proliferation increased.At the same time,there was no significant difference in the inhibition of HUVECs proliferation between Whitmania pigra Whitman and Hirudinaria manillensis water extract at the same concentration.Conclusion:Both the Whitmania pigra Whiman and Hirudinaria manillensis have an inhibition effect on the proliferation of HUVECs.There is no significant difference between the two kind of leeches.1.2 Study on the proliferation of pathological HUVECs by leech extractThe MTT assay was used to determine the proliferation inhibition of HUVECs cultured in media containing 12.5%Hep G2 culture supernatants.RESULTS:When the concentration of water extracts of Whitmania pigra Whitman and Hirudinaria manillensis was 1 mg/mL,the inhibition rate of HUVECs proliferation reached above 24%,and the difference was significant(p<0.01).As the concentration of the two kind of leeches increased to 12 mg/mL,the proliferation inhibition rates of HUVECs increased.At the same concentration,there was no significant difference in the inhibition rate of HUVECs proliferation between Whitmania pigra Whitman and Hirudinaria manillensis.Conclusion:Both the Whitmania pigra Whitman and Hirudinaria manillensis have the effect of inhibiting the proliferation of pathological HUVEC,and the effect is equivalent.2.Angiogenesis effect of leech extract on CAMThe CAM model was used to directly observe the effects of the Whitmania pigra Whitman and Hirudinaria manillensis on angiogenesis.Results:Compared with the blank group,the inhibition rates of CAM new vessels were 76.04%,67.36%,and 63.15%when the Whitmania pigra Whitman were at concentrations of 80,40,and 20 mg/mL,respectively.The inhibition rates of CAM neovascularization were 74.97%,57.56%,and 52.01%,when the Hirudinaria manillensis were at concentrations of 60,40,and 20 mg/mL,respectively.When the concentration of the two kind of leeches were at 20 mg/mL,the number of new vessels had a significant difference compared with the blank group(p<0.01).At the same time,when the concentrations of both were at 20 and 40 mg/mL,the number of new vessels in the Whitmania pigra Whitman group was slightly less than that of the Hirudinaria manillensis group,and the difference was statistically significant(p<0.05).Conclusions:The Whitmania pigra Whitman and Hirudinaria manillensis have a strong inhibition effect on the angiogenesis of CAM,and the Whitmania pigra Whitman inhibits it more strongly than Hirudinaria manillensis.3.Angiogenesis effect of leech extract on CAMHep G2 were inoculated on CAM to generate a tumor angiogenesis model.Direct observations of the effects of Whitmania pigra Whitman and Hirudinaria manillensis on tumor angiogenesis were performed.Results:Compared with the model group,the inhibition rates of CAM neovascularization were 80.03%.66.79%and 65.17%when the concentrations of the extracts of Whitmania pigra Whitman were at 80,40.and 20 mg/mL,respectively.When the concentrations of the extracts of Hirudinaria manillensis were at 60.40 and 20 mg/mL,the inhibition rates of CAM neovascularization were 76.11%,65.56%,and 64.63%,respectively.When the two kind of leeches were at a concentration of 20 mg/mL,compared with the model group,the number of new vessels has a significant difference(p<0.01).At the same time,when the concentrations were at 20 and 40 mg/mL,there was no significant difference in the number of angiogenesis between Whitmania pigra Whitman group and Hirudinaria manillensis group.Conclusion:This experiment directly reflects the inhibition of angiogenesis in CAM tumors by Whitmania pigra Whitman and Hirudinaria manillensis.4.Angiogenesis of zebrafish in leech extractsThe zebrafish was used as an in vivo model to observe the effects of Whitmania pigra Whitman and Hirudinaria manillensis on the number of angiogenesis.Results:Compared with the blank group,when the concentration was above 30 mg/mL in the Whitmania pigra Whitman,and above 20 mg/mL in the Hirudinaria manillensis group,the vascular number in the zebrafish group was significantly reduced(p<0.01).At the same concentration of the Whitmania pigra Whitman and Hirudinaria manillensis,the inhibition effect of Whitmania pigra Whitman on the intersegmental angiogenesis of zebrafish was weaker than that of Philippine burdock.The difference was especially significant when the concentration was 80μg/mL.Conclusion:The concentration of Whitmania pigra Whitman and Hirudinaria manillensis can inhibit the intersegmental angiogenesis in zebrafish within a certain range,and the inhibition effects of phenytoin on intersegmental angiogenesis in zebrafish.The effect is slightly stronger than that of the Whitmania pigra Whitman.Ⅳ.Antitumor and anti-angiogenic effects of leech in tumor-bearing miceThe Whitmania pigra Whitman and Hirudinaria manillensis were exposed to H22 tumor-bearing mice and their anti-tumor effect was observed.Plasma thrombin and VEGF levels were measured.Immunohistochemistry was used to evaluate the microvessel density(MVD)of tumor tissues.It aimed to determine whether leeches can act through antithrombin,thereby inhibiting VEGF expression and affecting angiogenesis,ultimately plays an role in anti-tumor effect.1.Anti-tumor effect of Whitmania pigra Whitman and Hirudinaria manillensisThe corresponding H22 tumor-bearing mice were continuously intragastrically administered(0.16 mL/10 g)with high-dose(1 g/mL)and low-dose(0.25 g/mL)by Whitmania pigra Whitman and Hirudinaria manillensis.At the same time,the positive drug group was injected intraperitoneally with cyclophosphamide(30 mg/kg).Results:By the cyclophosphamide group during the administration period,the mice had low activity and decreased food intake.The activities and diets of the other mice group were normal and no death was observed.Compared with the model group,each drug group could significantly inhibit tumor growth;when the high-dose group of the Whitmania pigra Whitman and Hirudinaria manillensis,and the dose of cyclophosphamide was 30 mg/kg,the H22 tumor-bearing mice had similar anti-tumor effects and fewer side effects on tumor-bearing mice in the Whitmania pigra Whitman and Hirudinaria manillensis group.The Results showed that the Whitmania pigra Whitman and Hirudinaria manillensis has a strong anti-tumor effect.2.Determination of thrombin and VEGF in plasma of H22 tumor-bearing miceThe content of thrombin in plasma of H22 tumor-bearing mice was measured by chromogenic substrate method,and the plasma VEGF level in H22 tumor-bearing mice was measured by enzyme-linked immunosorbent assay(ELISA).Results:Compared with the model group,the thrombin level was lower in H22 tumor-bearing mice in each administration group,and the difference was significant(p<0.01).At high doses,the thrombin content in H22 tumor-bearing mice was not significantly different from Whitmania pigra Whitman and Hirudinaria manillensis.The VEGF levels in H22 tumor-bearing mice were lower in each drug group,and the difference was significant(p<0.01).At high doses,the VEGF content in H22 tumor-bearing mice was not significantly different from Whitmania pigra Whitman and Hirudinaria manillensis.The results showed that the Whitmania pigra Whitman and Hirudinaria manillensis have anti-thrombin in vivo and can reduce the VEGF level.3.Determination of MVD in tumor tissue by immunohistochemistry assayTo determine the effect on the MVD of the tumor tissue,the tumor tissue was immunohistochemically stained by CD34.Results:At high dose,the MVD of the tumors in the Whitmania pigra Whitman group was significantly lower than that in the model group(p<0.01),and the MVD of the tumor in the Hirudinaria manillensis group was lower than that in the model group(p<0.05).There was no significant difference in the MVD between the tumor tissues of the Whitmania pigra Whitman and Hirudinaria manillensis.The results showed that the Whitmania pigra Whitman and Hirudinaria manillensis have the effect of reducing the MVD of tumors.Conclusion:Leech can reduce the level of VEGF,inhibit the angiogenesis of tumor and finally achieve the anti-tumor effect through its anti-thrombin t effect.