Inhibitory Effect And Mechanism Of Water Extract Of Bombax Ceiba Flowers On Human Lung Carcinoma NCI-H460 Cells In Vitro And In Vivo

Objective:This study takes human lung carcinoma NCI-H460 cells as research objects to investigate the inhibitory effect of water extract of Bombax ceiba flowers on human lung carcinoma NCI-H460 cells in vitro;and establishment of a xenograft tumor animal model of lung carcinoma NCI-H460cells in nude mice to explore the anti-tumor effect and its mechanism.Method:1.After the NCI-H460 cells were cultured routinely and treated with water extracts of Bombax ceiba flowers in different concentrations?20,40,80,160,320,640,1280,2560?g/mL?for 24,48 and 72 hours,MTT method was used to detect the inhibitory effect of water extracts of Bombax ceiba flowers on the proliferation of NCI-H460 cells,and the half inhibitory concentration(IC₅₀)was calculated.2.After human lung carcinoma NCI-H460 cells were cultured into the logarithmic growth phase,0.2mL suspension with a cell density of 1×10⁷/mL was taken when the cells were in a good state and inoculated subcutaneously on the right hind limb of nude mice,and a xenograft tumor model of NCI-H460nude mice was successfully established.3.20 tumor-bearing nude mice were selected and randomly divided into model group,high-dose water extract of Bombax ceiba flowers group,low-dose water extract of Bombax ceiba flowers group and cyclophosphamide?CTX?positive control group with 5 mice in each group.Another 5 normal nude mice were taken as normal group.The normal group and the model group were treated with intragastric administration of equal volume distilled water once a day;the water extract of Bombax ceiba flowers group was treated with intragastric administration of water extract of Bombax ceiba flowers once a day at the doses of 1.2 g/kg and 0.6 g/kg respectively;while the CTX group was given intraperitoneally of CTX at the dosage of 0.025 g/kg?ip?once every other day.The volume of xenograft tumor in nude mice was measured every other day during administration.The intervention lasted for 13 days.On the14th day,after taking blood from nude mice by eyeball extraction,the nude mice were killed by neck removal,the tumor body,lung,spleen and liver were extracted,and the tumor inhibition rate,lung index,spleen index and liver index were weighed and calculated,respectively.4.The serum levels of interleukin-6?IL-6?and tumor necrosis factor-??TNF-??were determined by enzyme-linked immunosorbent assay?ELISA?.5.The content of superoxide dismutase?SOD?in tumor tissue was determined by WST-1 method;the content of malondialdehyde?MDA?in tumor tissue was determined by thiobarbituric acid method?TBA method?.6.Hematoxylin-eosin staining?HE?was used to observe the pathological changes of the tumors.7.The effect of water extract of Bombax ceiba flowers on the expression of Caspase-9,Caspase-3,Bax,Bcl-2 and p53 protein in nude mice xenograft tumor of human lung carcinoma NCI-H460 was observed by enzyme immunohistochemistry.Result:1.MTT assay in vitro showed that the water extracts of Bombax ceiba flowers had significant inhibitory effects on the proliferation of human lung carcinoma NCI-H460 cells after 48 and 72 hours of treatment,and the inhibitory effect increased with the increase of drug concentration and action time within a certain range.After 48 hours of treatment,the inhibition rates of water extracts of Bombax ceiba flowers on human lung carcinoma NCI-H460cells were 12.2%,18.3%,19.1%,13.4%,15.8%,21.8%,34.6%and 62.6%,respectively;after 72 hours of treatment,the inhibition rates were 7.4%,14.4%,14.8%,31.3%,40.5%,48.7%,58.2%and 75.8%respectively.The half inhibitory concentration after 48h(48h-IC₅₀)was 2002.4?g/mL and the72h-IC₅₀0 was 728.4?g/mL.2.The xenograft tumor model of human lung carcinoma NCI-H460 cells in nude mice was successfully established,and the tumorigenesis rate was100%.3.The test results after killing nude mice and stripping the tumor showed that,compared with the model group,the volume and weight of nude mice xenograft tumor in the high-dose water extracts of Bombax ceiba flowers group were significantly reduced?P<0.01?,while the volume and weight of xenograft tumor in the low-dose water extracts of Bombax ceiba flowers group were not significantly different from those in the model group?P>0.05?.The tumor inhibition rates of high-dose and low-dose water extracts of Bombax ceiba flowers groups were 59.7%and 30.5%,respectively.4.The results of ELISA showed that there was no significant difference in serum TNF-?level between high-dose water extracts of Bombax ceiba flowers group and normal group?P>0.05?,but the serum TNF-?level in low-dose water extracts of Bombax ceiba flowers group was lower than that in normal group?P<0.01?;the serum TNF-?level in the high-dose water extracts of Bombax ceiba flowers group was higher compared with the model group,and the difference was statistically significant?P<0.05?;there was no significant difference in serum IL-6 level between the high-dose and low-dose water extracts of Bombax ceiba flowers group and the normal group?P>0.05?;the scrum IL-6 level in the high-dose and low-dose water extracts of Bombax ceiba flowers group was significantly higher compared with the model group,and the difference was statistically significant?P<0.01?.5.The results of antioxidant experiment showed that the MDA level in tumor tissue of high-dose and low-dose water extracts of Bombax ceiba flowers group was lower than that of model group,and the difference was statistically significant?P<0.01?;the activity of SOD in the high-dose water extracts of Bom_baxax ceiba flowers group was significantly higher than that in the model group?P<0.01?,but there was no significant difference in the activity of SOD between the low-dose group and the model group?P>0.05?.6.HE staining results showed that the cancer cells in the tumor tissue of model group were closely arranged,grew vigorously,had large nucleus and less necrosis area;while the cancer cells in the tumor tissue of water extracts of Bombax ceiba flowers group o showed necrosis in varying degrees,and the nuclear lysis and disappearance could be seen in the necrotic cells.The number of cancer cells was significantly reduced,and the size was different.The intercellular loosening,small nucleus,partial rupture and incompleteness of the envelope were observed.7.Immunohistochemical results of CTX group and high-dose and low-dose water extracts of Bombax ceiba flowers group showed that the integral optical density?IOD?of Caspase-3 in tumor tissue of high-dose and low-dose water extracts of Bombax ceiba flowers group was higher than that of model group,and the difference was statistically significant?P<0.01?;the integral optical density?IOD?of Caspase-9,Bax and p53 in the high-dose water extracts of Bombax ceiba flowers group was higher than that in the model group,while the integral optical density?IOD?of Bcl-2 was lower than that in the model group,the difference was statistically significant?P<0.05 or P<0.01?;and there was no significant difference in the integrated optical density?IOD?of Caspase-9,Bax,p53 and Bcl-2 between the low-dose water extracts of Bombax ceiba flowers group and the model group?P>0.05?.Conclusion:The water extracts of Bombax ceiba flowers has anti-proliferation effect on human lung cancer NCI-H460 cells,which increases with the increase of drug concentration and action time.The water extracts of Bombax ceiba flowers can also inhibit the xenograft tumor of human lung carcinoma NCI-H460 cells in nude mice and induce apoptosis.Its mechanism may be related to antioxidant stress,scavenging free radicals,increasing the level of IL-6 and TNF-?,inducing up-regulation of Caspase-9,Caspase-3,Bax,p53 and down-regulation of Bcl-2 protein expression.