Study On The Anti-tumor Effect And The Mechanism Of The Ethanol Extracts From Ageratum Conyzoides L. From Guangxi

Objective:In this experiment,human lung cancer NCI-H460 cells, human cervical cancer He La cells and human liver cancer SMMC-7721 cells were cultured in vitro,and NCI-H460 nude mice transplanted tumor model of human lung cancer was established in vivo,in order to study the antitumor effect and mechanism of ethanol extract from ageratum conyzoides L.from Guangxi in vitro and in vivo.Methods:?1?With different concentrations of ethanol extract of from ageratum conyzoides L.from Guangxi in vitro cultured human lung cancer NCI-H460 cells,human cervical cancer He La cells and human liver cancer SMMC-7721 cells,the cell morphology,growth were observed under microscope,and CCK-8 assay was used to detect the inhibitory effect of drugs on tumor cell proliferation.The apoptosis morphology of human lung cancer NCI-H460 cells was observed by using acridine orange/ethidium bromide?AO/EB?double fluorescence staining;cell apoptosis was detection by flow cytometry Annexin V-FITC/PI double staining;Real-time PCR method was used to detect the effects of ethanol extract from ageratum conyzoides L.from Guangxi on the expression of Bcl-2,Bax and caspase-3m RNA in NCI-H460 cells;in cell culture medium,the activity of superoxide dismutase?SOD?was measured by WST-1 method,and the malondialdehyde?MDA?content was measured by TBA method;The content of inter Leukin-6?IL-6?and tumor necrosis factor?TNF-??in NCI-H460 lysate was detected by enzyme linked immunosorbent assay?ELISA?.?2?Twenty Kunming mice were randomly divided into two groups:medication administration team?n=10?:the maximum concentration?containing crude drug 1.80 g/m L by gavage for blockage limits?gavage;The control group?n=10?:gavage of equal volume of physiological saline,use the maximum tolerance dose?MTD?method.In 24 hours,the mice were given 2times by gavage,weighed every 7 days,mice were observed continuously for14 days,and the acute toxicity of the mice after the action of ethanol extract from ageratum conyzoides L.from Guangxi was observed.Establishing human lung cancer NCI-H460 tumor-burdened nude mice model,they were randomly divided into 6 groups,blank control?healthy mice?,model group,positive control group,low dose group,middle dose group,high dose group.After the model was successfully established,the blank control group?healthy nude mice?was given the same volume of physiological saline,the positive control group was given cisplatin intraperitoneally,and the ageratum conyzoides L.group?low,medium and high dose?Dosage gavage respectively,in addition to the positive group administered on the next day,the other groups administered once daily.During the 14 consecutive days,the weight of the nude mice was weighed and the tumor growth was observed.After 14days,blood samples were taken from naked eyes to detect the related indexes.The tumor growth was observed and the weight of nude mice in nude mice was measured and the tumor suppressor rate was calculated.The contents of IL-6 and TNF-?in serum of tumor bearing mice were detected by ELISA.The activity of SOD and glutathione peroxidase?GSH-PX?and MDA content were measured by kit,and hematoxylin and eosin stain?HE?was used to observe the pathomorphological changes of tumor tissues in each group under light microscope.Results:?1?Experiments results showed that different concentrations of ethanol extract from ageratum conyzoides L.from Guangxi had a certain proliferation inhibition effect on human lung cancer NCI-H460 cells,human cervical cancer He La cells and human liver cancer SMMC-7721 cells,showing the obvious dose and time dependent effect.The most sensitive cell line was human lung cancer H460 cells,and the inhibitory effect was significant?P<0.05?.The IC₅₀ value of 24h?36h and 48h was 118.26?g/m L,99.15?g/m L and 92.24?g/m L respectively.After the drug treatment,AO/EB observed treatment group tumor cell morphology changed significantly under the fluorescence microscope,apoptotic bodies and cytoplasmic fragments,Annexin V-FITC/PI double staining method significantly increased the apoptosis rate?P<0.05?,treated by different concentration?The concentration is 50?g/m L,100?g/m L,200?g/m L?of 24h,cell apoptosis rates were:?2.33±0.07?%,?6.51±0.25?%and?11.87±0.56?%,There is an obvious dose-dependence.The results of RT-PCR showed that ethanol extract from ageratum conyzoides L.from Guangxi could down-regulate the expression of Bcl-2 m RNA in the cells,increase the expression of Bax,caspase-3 m RNA,and the ratio of Bcl-2/Bax decreased.After dealing with the drug of NCI-H460 cell lysis solution,ethanol extract from ageratum conyzoides L.from Guangxi,TNF-?levels of high dose group and positive group significantly higher than the model group?P<0.05?,drug low,medium and high dose group and positive group content of IL-6 is lower than the model group?P<0.01?;The supernatant of NCI-H460 cells after drug treatment,ethanol extract from ageratum conyzoides L.from Guangxi,the content of MDA in low and high dose group and positive group was significantly lower than that model group?P<0.05?,high dose group and positive group SOD activity was significantly higher than that in the model group?P<0.05??The results of acute toxicity test showed that:compared with the control group,the body weight of the administration group was not significantly reduced?P>0.05?,and no obvious toxic symptoms were found.All the mice survived.Establishment of human lung cancer NCI-H460 in nude mice,tumor formation rate is 100%,the low,medium and high dose group the inhibition rates were 16.84%,17.87%,48.26%,high dose group and positive group of nude mice transplanted tumor weight less than the model group significantly?P<0.05?,low dose of transplanted tumor the tumor weight of mice compared with the model group,the difference was not statistically significant?P>0.05?;The content of TNF-?in medium,high dose group and positive group was higher than the model group?P<0.05?.The contents of IL-6 in medium and high dose was lower than model group?P<0.05?;middle,high dose group and positive group of MDA content significantly lower than the model group?P<0.05?,high dose group,the positive group of SOD?GSH-PX vitality was obviously higher than the model group?P<0.05?;HE staining showed that the tumor cells in the model group were heteromorphic.Compared with the model group,the cancer cells in the low,middle and high dose groups of ethanol extract from ageratum conyzoides L.from Guangxi showed different degrees of necrosis and apoptosis.Conclusions:?1?In vitro experiments showed that ethanol extracts from ageratum conyzoides L.from Guangxi could inhibit the proliferation of human lung cancer NCI-H460 cells,human cervical cancer He La cells and human liver SMMC-7721 cells in a time and concentration dependent manner,The inhibitory effect of lung cancer NCI-H460 cells is the most obvious.In vivo experiments also showed that the ethanol extracts from ageratum conyzoides L.from Guangxi has a significant inhibitory effect on human lung cancer NCI-H460 cells.?2?The inhibitory effect of ethanol extract of ageratum conyzoides from Guangxi on human lung cancer NCI-H460 cells may be related to inducing apoptosis of tumor cells,scavenging free radicals and regulating or enhancing immune function.By increasing the expression of Bax and Caspase-3 m RNA and down regulating the expression of Bcl-2 m RNA,the decrease of Bcl-2/Bax ratio is a possible pathway to induce apoptosis of tumor cells.