Inhibitory Effect Of Dihydromyricetin On Choriocarcinoma Growth Through TGF?-Smads Signaling Pathway In Vivo

Choriocarcinoma(CC)is a highly malignant and invasive trophoblastic neoplasm,most of which are secondary to normal or abnormal pregnancy.China and Southeast Asian countries have relatively high incidence,mainly in women of childbearing age,but also in men.The pathogenesis of choriocarcinoma is still unclear.It is easy to psss through the blood metastasisd and the condition deteriorates.But it is sensitive to chemotherapeutic drugs.Therefore,chemotherapy is the main treatment.Surgery and radiotherapy were used as supplementary means.Chemotherapy is the first choice for choriocarcinoma treatment,but the dosage must reach the maximum tolerance of patients and patients need continuous treatment for multiple courses to achieve the desired effect.With the increase of side effects received at the same time,some patients were forced to terminate treatment due to intolerance of the side effects,and some patients developed drug resistance during treatment,which ultimately affected the cure rate.Therefore,it is an urgent problem to study the mechanism of choriocarcinoma and search for effective and low toxic chemotherapeutic drugs for the treatment of choriocarcinom.Dihydromyricetin(DMY),also known as ampelopsin,is a dihydroflavonol compound isolated from the vine tea of Ampelopsis,which contains the highest content in the young stems and leaves of ampelopsis.Ampelopsis is a rich and available plant resource in China.It has been found that dihydromyricetin has many physiological activities,such as anti-oxidation,anti-inflammatory,bacteriostasis,hypoglycemia,hypolipidemia,liver protection and immunity enhancement.In recent years,with the in-depth study of dihydromyricetin,it has been found that it has other physiological activities in anti-cancer,protection of cardiovascular system,protection of skin against aging and improvement of metabolic diseases.Especially its role in anti-cancer has been paid more and more attention.Transforming growth factor?(TGF?)is a multifunctional cytokine,which plays a role in tumors.It can affect the proliferation,differentiation,metastasis,apoptosis and tumorigenesis of tumor cells.The TGF?-Smads signal transduction pathway is composed of ligands,receptors,Smads proteins and regulated target genes of this pathway.Abnormalities in any part of the pathway may lead to disturbances in the transduction signal,which in turn contribute to the development and progression of the tumor.Previous in vitro cell experiments of research group showed that TGF?-Smads signaling pathway is involved in the proliferation of choriocarcinoma cells;dihydromyricetin inhibited the growth of choriocarcinoma cells;dihydromyricetin may inhibit the proliferation of choriocarcinoma cells through TGF?-Smads signaling pathway.In order to further study the anti-tumor effect and mechanism of dihydromyricetin,the following studies were carried out in vivo.Part ? Establishment of xenograft model of human choriocarcinoma JAR cells in nude miceObjective:A nude mouse xenograft model of human choriocarcinoma JAR cells was established to study its biological characteristics.Method:1 Choriocarcinoma JAR cells were cultured and cell suspensions were prepared.2 12 Balb/c female nude mice were randomly divided into control group and experimental group.The suspension containing 1 x 10~7 choriocarcinoma JAR cells was injected subcutaneously into the dorsal femur of nude mice in experimental group,and the same volume of saline was injected into the same part of the control group.The tumorigenesis rate was recorded.The diameter of tumors was measured.The survival status of nude mice was observed.3 HE staining method was used to observe the histopathological changes of JAR cells,transplanted tumors and mainly metastasis organs.4 Immunohistochemistry was used to detect the expression level of?-HCG protein in transplanted tumor tissues.5 The serum level of?-HCG was detected by ELISAResult:1 All the 8 nude mice in the experimental group were tumorigenic,and the tumorigenic rate of transplanted tumors was 100%.The survival time of nude mice with tumors was(24.75+3.41)days.2 HE staining showed that the tissue structure of choriocarcinoma cells in transplanted tumor tissues was similar to that of JAR cells cultured in vitro.the histological structure of choriocarcinoma cells in transplanted tumors was similar to that of JAR cells.No metastasis was found in the main organs of the body.3 Immunohistochemistry showed that?-HCG was mainly expressed in the cytoplasm of choriocarcinoma cells in the experimental group.4 The ELISA method showed that the serum levels of?-HCG in experimental nude mice(7.11±2.47)ng/ml were significantly higher than those in the control group(4.57±1.31).ng/m.(P<0.05).Conclusion:A xenograft model of choriocarcinoma JAR cells in nude mice was established by subcutaneous injection of choriocarcinoma cell suspension in nude mice.This method has the characteristics of easy operation,high tumorigenesis rate and short cycle.Part ?:Inhibitory effect of dihydromyricetin on choriocarcinoma growth through TGF?-Smads signaling pathway in vivoObjective:To investigate the effects of dihydromyricetin and combined methotrexate on the growth of human choriocarcinoma xenografts in nude mice and observe the toxicity and side effects of dihydromyricetin.In order to explore its mechanism,the effects of dihydromyricetin on the expression of?-HCG,TGF?R II,Smad2,P-smad2 and C-myc in choriocarcinoma xenografts were detected.Method:1 Human choriocarcinoma JAR cells were cultured and cell suspension was made.2 Nude mice were randomly divided into six groups:control group,low concentration group of dihydromyricetin(50mg/kg),medium concentration group of dihydromyricetin(100mg/kg),high concentration group of dihydromyricetin(150mg/kg),methotrexate group(0.5mg/kg),dihydromyricetin(100mg/kg)combined with methotrexate group(0.5mg/kg).Dihydromyricetin and methotrexate were administered by intragastric and intraperitoneal injection respectively.The condition of the nude mice and the general characteristics of the transplanted tumor were observed.3 After 14 days of treatment,the mice were killed.Body weight,spleen weight,tumor weight,tumor length and diameter were measured.The spleen index,tumor volume and mass inhibition rate were calculated.4 Serum biochemistry was detected the alanine aminotransferase(ALT),aspartate aminotransferase(AST),lactate dehydrogenase(LDH)and creatine kinase(CK).5 The serum level of?-HCG was detected by ELISA.6 The pathological changes of transplanted tumor tissues in each group were observed by HE staining.7 The expression levels of?-HCG,TGF?RII,Smad2,P-smad2 and C-myc in the transplanted tumor tissues of each group were detected by immunohistochemistry.8 The protein expression levels of TGF?RII,Smad2,P-smad2 and C-myc in the transplanted tumor tissues of each group were detected by Western blot method.Result:1 Dihydromyricetin can significantly inhibit the growth of subcutaneous tumors of choriocarcinoma in nude mice in a dose-dependent manner.The tumor inhibition rates of low concentration,medium concentration and high concentration of dihydromyricetin in nude mice were 18.28%,23.62%and24.31%,respectively.In addition,histopathological examination results showed that the cell necrosis in the transplanted tumor tissues of the methotrexate group and the combination group was more serious than that of the other groups,and the number of tumor cells was significantly lower than that of the other groups.2 Dihydromyricetin significantly reduced alanine aminotransferase(ALT),aspartate aminotransferase(AST),lactate dehydrogenase(LDH)and creatine kinase(CK)levels(P<0.05).Dihydromyricetin has a protective effect on hepatocytes and cardiomyocytes.It can also reduce the damage of hepatocytes and cardiomyocytes caused by the side effects of methotrexate.3 Immunohistochemistry results showed that the expression levels of?-HCG,TGF?RII,Smad2,P-smad2 and C-myc in the dihydromyricetin group were lower than those in the control group(P<0.05).4 The results of Western blot showed that dihydromyricetin combined with methotrexate could decrease the expression of TGF?RII,Smad2,P-smad2 and C-myc protein(P<0.05).Conclusion:1 Dihydromyricetin can significantly inhibit the growth of choriocarcinoma transplanted tumors.Combination of methotrexate and dihydromyricetin has synergistic anti-tumor effect.2 Dihydromyricetin can alleviate the damage of hepatocytes and cardiac myocytes,and alleviate the toxic and side effects caused by methotrexate.3 The mechanism of dihydromyricetin inhibiting the growth of choriocarcinoma may be related to the regulation of TGF?-Smads signaling pathway and down-regulation of the expression of TGF?RII,Smad2,P-smad2 and C-myc.