Experimental Study On The Anti-tumor And Immunoregulatory Activity Of Ethanol Extract Of Forsythia Suspensa Root

Objective: To study the effects of different parts (roots, leaves, fruit) ofForsythia suspensa extract on the proliferation of esophageal carcinoma, andfilter out the most effective parts, the induction of apoptosis effects of FSEERon TE-13cells and the underlying mechanisms were analized. Forthermore,the immunoregulatory activity of FSEER in vivo was studied, to providing thereference theory for Forsythia suspensa as a new anti-tumor drug.Methods:1Abstraction water extract and ethanol extract of root, leaves and fruit ofForsythia suspensa, the growth-inhibitory effect of the six extract of Forsythiasuspensa (FSWER,FSWEL,FSWEF,FSEER,FSEEL,FSEEF) on esophagealcancer cell lines(TE-13,TE-1,Yes-2,Eca-109) were examined by MTT assay,morphological changes of tumor cells were analyzed by light microscope.2After treated by FSEER (0.3,0.4,0.5,0.6,0.7,0.8mg/ml) for6,12,24h,the inhibitory effect was detected by MTT assay.3The morphological changes of TE-13cells were observed by Wright-Giemsa staining after treated by FSEER (0.4,0.5,0.6mg/ml) for24h.4Changes of mitochondrial membrane potential and apoptosis rate in TE-13cell were examined by Flow cytometry(FCM) after treated with differentconcentration of FSEER (0.4,0.5,0.6mg/ml) for defferent time (6,12,24h).5The effects of apoptosis associated protein (PARP, caspase-3, caspase-8,caspase-9and Cyt-c) and p-JAK, p-STAT3, p-ERK1/2in FSEER-inducedapoptosis of esophageal cancer cells were investigated by Western blot.6The transcriptional levels of Bax, Bad, Noxa, Bcl-2, Bcl-xl, Mcl-1inTE-13cells treated with different dose of FSEER (0.4,0.5,0.6mg/ml) for24hwere detected by RT-PCR.7The sixteen BALB/c mice of8-10-week-old were divided into four groups randomly, and constructed immunosuppression models on them besidescontrol group, and then the experimental group and model group were injectedwith cyclophosphamide(Cy) in abdominal cavity one time (200mg/kg). In thesecond day, the mice of experimental group were given FSEER (120,60mg/kg·day) by intragastric administration, the model group were givennormal saline(NS). All the mice were treated for10days. The mice of controlgroup were raised normally. To observe the survival condition of the miceeveryday, after the lasted given drugs for12hours, all the mice were killedafter weighing, and the spleen, bone marrow and peripheral blood wereremoved for the succedent experiments.8After sterile separated and the residual blood were sucked up, each micespleen were weighted to calculate the spleen index(SI), Formula: SI=spleenweight (mg)/body weight (g).9The changes of the ratio for lymphocytes of CD3+, CD4+, CD8+T cellsfrom peripheral blood of the mice were detected by Flow cytometry(FCM).10The level of TNF-α and IL-2that were produced by peripheral bloodlymphocyte of mice were detected by ELISA.11Morphological images of bone marrow of the mice were detected byWright-Giemsa's staining.Results:1The Forsythia suspense extract(FSWER, FSWEL, FSWEF. FSEER, FSEEL,FSEEF) could inhibit the proliferation of TE-13cell to some extent. Effect ofFSEER for proliferation of tumor cells is the most obvious compared withother group (P<0.05).2Compared to the controll group, FSEER(0.3,0.4,0.5,0.6,0.7,0.8mg/ml)can significantly inhibit the proliferation of TE-13cells in vitro, the differencewas significant (P <0.05).3Giemsa staining showed that cells growth sparseness,loss of volumol,cytoplasm concentration, karyokinesis and deformation into round in TE-13cells treated with FSEER for24h. While the cells in control group wereobserved appearance regularity, growth intensively and maintain polygon shapes.4FCM analyses showed that the apoptosis rate of TE-13cell was increasedafter treated with different dose of FSEER (0.4,0.5,0.6mg/ml) for differenttimes(6h,12h,24h), compared to the controll group, the difference wassignificant (P <0.05).5FSEER(0.4,0.5,0.6mg/ml) can significantly reduced the mitochondrialmembrane potential of TE-13cells. With the concentration increasing, thenumber of cells whose mitochondrial membrane potential were damaged grewup, compared with controll group, the difference was significant (P <0.01).6Western blot analyses showed the expression of cleaved caspase-3, cleavedPARP,cleaved caspase-9and Cyt-c was increased compared to the controlgroup, the difference was significant (P<0.05). While the level of caspase-8did not change significantly, compared to the control group, the difference wasnot significant (P>0.05). This results suggested that the apoptosis induced byFSEER associated with mitochondrial pathway. In addition, FSEER can alsodown-regulate the expression of p-JAK, p-STAT3and p-ERK.7RT-PCR analyses showed that FSEER could up-rdgulate the expression ofBax, Bad, Noxa and down-regulate the expression of Bcl-2, Bcl-xl, Mcl-1,which confirmed that apoptosis induced by FSEER is via the mitochondrialpathway.8The immunosuppression models of the mice that were induced bycyclophosphamide were successfully established. The locomotor activity forthe mice of the model group were feebly,the foodintake and hydroposia werenot so good, one of them was died in the7thday, however, the control groupand experimental group were normal, none of them was died.9The spleen index(SI) was increased in both of the two experimental group,compared to the model group, the difference was significant (P <0.05).10compared to the model group, the ratio of CD3+CD4+T lymphocyte andthe ratio of CD4+/CD8+was increased significantly in the low doseexperimental group(P <0.05), while the high dose experimental group have nochanges (P>0.05). 11ELISA analyses showed that the level of TNF-α and IL-2in peripheralblood lymphocyte of mice that were produced in experimental group and itwas increased markedly compared with control group, the difference wassignificant (P <0.05). In addition, the level of TNF-α and IL-2in high dose ofFSEER group is higher than low dose of FSEER group, and the difference wassignificant (P <0.05).12In the bone marrow smear, the karyocyte hyperplasia in the control group,but hematopoietic depression in the model group. After treated with FSEER,the hematopoietic depression was improved.Conclusion:1Root is the most effective part of Forsythia suspense in anti-tumor aspect, aswell as its ethanol extract(FSEER) could significantly inhibit TE-13cellgrowth.2FSEER could induced esophageal cancer cells apoptosis in a dose andtime-related manner, the underlying mechanism may be related with themitochondria-dependent pathway which is trigerd by the JAK/STAT and ERKsignaling pathways.3FSEER could significantly improve the immune function of mice, and canimproved the immunosupression induced by cyclophosphamide.