Mechanism Of HIF-1 In The Pathogenesis Of Liver Tumors In Mice During Early Exercise Training

Objective: Tumors have become one of the important public health issues facing humanity.Malignant tumors,with their serious fatality rate,seriously threaten human life and health.The incidence and mortality of malignant tumors in China are increasing year by year.In recent years,some researchers believe that the focus of research at this stage is not how to kill tumor cells,but how to effectively control the occurrence and development of tumor cells,so that they do not spread in the body and evolve into cancer.The hypoxic microenvironment is an important feature of solid tumors.As an important way to improve the ability of cells to aerobic oxidation,hypoxia-inducible factors may play an important role in tumor cell metastasis.Exercise has been reported in many literatures for the improvement of hypoxia-inducible factors.In this experiment,we established early liver training to intervene mouse liver tumor model and mouse liver tumor cell model cultured in different oxygen content environment to explore the effect of pre-exercise on HIF-1?-BNIP3 pathway in mouse liver tumor,and different oxygen content microenvironment.The changes of HIF-1?-BNIP3 pathway in mouse liver tumor cells provide new ideas for the control of mouse liver tumors.Method:1.24 male C57BL/6J mice were randomly divided into 2 groups: control group(C)and exercise group(E).The exercise group was trained in 12-week medium-intensity treadmill(speed: 12m/min).After group C was kept for 12 weeks,the two groups were separately divided into four groups: control group.(C),control tumor group(CT),exercise group(E),and exercise tumor group(ET).A tumor model was constructed using Hepa 1-6(mouse liver cancer cell line),and Hepa 1-6 cells were cultured in DMEM medium containing 10% fetal bovine serum in a 5% CO 2 incubator.Hepa1-6 cells in logarithmic growth phase were taken andthe mice were anesthetized with chloral hydrate.The mice were placed in the supine position on the mouse plate of the operating table.The rats and the surgical instruments used were routinely disinfected.The anesthetized mice were cut from left to right along the lower edge of the left rib to an incision of about 2 to 3 cm.The leftmost hepatic lobules of the liver were exposed to the incision field of view,and100 ?L of Hepa 1-6 cell suspension(containing 2 x 106 cells)was pipetted with a 1ml sterile syringe.Puncture the liver parenchyma about 0.5cm at an angle of 20,,slowly inject the cell suspension,stay for a while until no liquid and blood oozes,pull out the needle,and immediately press the needle hole with hemostatic gauze until the surface of the liver no longer oozes.The layer is closed.Continue to raise after the operation,the mouse breeding environment is clean,free to eat,water.After successful tumor formation,the material was taken for subsequent experiments.2.Construction of mouse liver tumor cell culture model: Hepa1-6 liver cancer cells were used,and mice were inoculated intraperitoneally,each 0.2 mL.The inoculation method was the same as that of the above mouse tumor model.After 14 days of free diet,the neck was sacrificed.The liver tissue of the liver was aseptically extracted,washed with PBS,and appropriately cut and placed on a 200-mesh sieve.The tissue was gently pressed with a 10 ml syringe needle.After extrusion,10% fetal bovine serum(FBS)was used.The cell screen was washed with DMEM medium containing1% penicillin(100 U/ml)and streptomycin(100 ag/ml)(1:1)to obtain a tumor cell suspension.Dilute into 1×107/mL tumor cell suspension with this medium,culture in medium,place in a constant temperature incubator with 370 C and 5% CO2 saturated humidity,and take tumor cells in logarithmic growth phase to 7 per well.×106inoculated on 24-well plates in normoxia(21% O2,5% CO2,74% N2)(O group),hypoxia(14% O2,5% CO2,81% N2)(Group F)Hypoxia(7% O2,5% CO2,88% N2)(Group D)incubator for 24 h.3.MTT colorimetry,Western Blotting Western blotting,cell immunofluorescence detection of mouse liver tumor tissue and mouse tumor cells autophagy-related proteins,apoptosis-related proteins,hypoxia-inducible factor-related proteins and tumor cell growth,etc.Such changes.4.Using the Quantity One software for protein gray value analysis,using GraphPad Prism 5.0 software for chart production,SPSS 19.0 software for statistical analysis,mean ± standard deviation for measurement data,one-way analysis of variance(One-way analysis)ANOVA)The difference between the experimental group and the control group was compared,and P < 0.05 was judged to be significantly different.Result:1.Compared with group C,HIF-1? was significantly increased in CT group(P<0.01).Compared with group E,HIF-1? was significantly increased in ET group(P<0.05).Compared with the CT group,HIF-1? was significantly decreased in the ET group(P <0.01).Compared with group C,BNIP3 in group E liver tumors was significantly lower(P <0.01).The BNIP3 of liver tumors in the CT group was significantly lower(P <0.01).Compared with the CT group,the BNIP3 of the liver tumor in the ET group was significantly lower(P <0.01).2.Compared with group C,the expression level of BCL-2 in liver tumors of group E and CT was significantly increased.(P < 0.05,P < 0.01).Compared with the CT group,the expression level of BCL-2 in liver tumors of ET group was significantly increased(P <0.05).Compared with group E,the expression level of BCL-2 in liver tumors of ET group was significantly increased(P <0.01).Compared with group C,the expression levels of BAX in liver tumors of group E and CT were significantly increased(P <0.01).Compared with the CT group,the Bax of the liver tumor in the ET group was significantly increased(P <0.05).Compared with group E,the expression level of Bax in liver tumor of ET group was significantly increased(P<0.01).Compared with group C,the ratio of BCL-2 to BAX in group E was significantly higher(P <0.05),and the ratio of CT group was significantly lower(P<0.01).Compared with the CT group,the ratio of the ET group was significantly lower(P <0.01);compared with the E group,the ratio of the ET group was significantly decreased(P <0.01).3.Compared with group C,the ratio of LC3II/LC3 I in liver tumors of group E and CT was significantly increased(P <0.01).Compared with the CT group,the LC3II/LC3 I ratio in the ET group was significantly higher(P < 0.05);compared withthe E group,the LC3II/LC3 I ratio in the ET group was significantly lower(P < 0.05).4.Compared with group D,the content of HIF-1? in tumor cells of group F was significantly lower(P <0.01).Compared with group D,the content of HIF-1? in tumor cells of group O was significantly lower(P <0.05).Compared with the F group,the HIF-1? content in the O group tumor cells was significantly decreased(P <0.01).Compared with group D,the content of BNIP3 in tumor cells of group F was significantly lower(P <0.01).Compared with group D,the content of BNIP3 in group O tumor cells was significantly lower(P <0.01).Compared with the F group,the content of BNIP3 in the O group tumor cells was significantly lower(P <0.01).5.Compared with group D,the content of BCL-2 in tumor cells of group F was significantly lower(P <0.01).Compared with group D,the content of BCL-2 in tumor cells of group O was significantly lower(P <0.01).Compared with group F,the content of BCL-2 in tumor cells of group O was significantly lower(P <0.01).Compared with group D,BAX content in tumor cells of group F was significantly lower(P <0.01).Compared with group D,the BAX content in group O tumor cells was significantly lower(P <0.01).Compared with the F group,the BAX content in the O group tumor cells was significantly decreased(P <0.01).Compared with group D,the ratio of BCL-2 to BAX in group F was significantly lower(P <0.05),and the ratio of group O was significantly lower(P <0.01).Compared with the F group,the O group ratio was significantly lower(P < 0.01).6.Compared with group D,the ratio of LC3II/LC3 I in tumor cells of group F and group O decreased significantly(P <0.01).Compared with group F,the ratio of LC3II/LC3 I in group O tumor cells was significantly decreased(P <0.01).7.Compared with group D,the viability of tumor cells in group F decreased significantly(P<0.01),and the activity of tumor cells in group O decreased significantly(P<0.01).Compared with group F,the viability of tumor cells in group O decreased significantly(P< 0.01).Conclusion:This experiment to establish early exercise training intervention mouse liver tumor model and different oxygen environment cultivating model of mice with livertumor cells,and explore the HIF-1?/BNIP3 pathway in the movement of mouse liver tumor and the effect of different oxygen content and environment:HIF-1?/BNIP3 pathway,autophagy and apoptosis in mouse liver tumor cells changes,through the experiment we get the following conclusion:1,After the establishment of the mouse liver tumor model,we found that compared with the liver of normal mice,the liver of the tumor group showed obvious pathological changes,and the mouse liver tumor model was successfully constructed.2,after building the liver tumor model in mice,we through to the hypoxia related protein and the change of autophagic apoptosis related proteins analysis,we found that compared with normal liver tissue in mice,the tumor tissue relative in hypoxia condition and the autophagy and apoptosis are at a higher level,pre exercise intervention can improve the hypoxia state of tumor tissue,improve the expression of tumor autophagy and apoptosis related proteins,hinders the development of liver tumors in mice.3,in mice after tumor cell culture model is constructed,we through to the cell vitality,related protein expression and the change of cell immunofluorescence staining results analysis,we found that the low oxygen environment to cultivate the mice liver tumor cell vitality is higher,in a relatively lower oxygen content training environment,the survival state of the tumor cells,the better.The growth of mouse liver tumor cells is affected by the oxygen content in the environment.The relatively low oxygen environment can improve the expression of hypoxia inducible factor in mouse liver tumor cells and promote the survival of tumor cells.