The Mechanism Of IL-15 In Pre-exercise Training To Intervene The Pathogenesis Of Liver Cancer In Mice

Research background and purpose:Malignant tumors with high morbidity,high lethality and poor prognosis seriously threaten human life and health,and are one of the important public health problems facing human beings.Exercise training can reduce the incidence and mortality of various cancers,but the mechanism of exercise intervention for liver cancer is still unclear.The previous research of this research group confirmed that pre-exercise training can increase the content and killing effect of NK cells in vivo,and may play a role in the intervention of tumor pathogenesis by promoting tumor cell apoptosis(to be published).As an important endocrine organ of the human body,skeletal muscle can synthesize and secrete IL-15 in large quantities after exercise.IL-15 can play an anti-tumor effect by inducing the proliferation and differentiation of NK cells and enhancing the killing effect of NK cells in tumor immunotherapy,suggesting that IL-15 may play an important role in anti-tumor.In order to explore the mechanism of IL-15 in pre-exercise training to intervene in the pathogenesis of liver tumors in mice,this study first used a one-time exercise serum incubation model of liver cancer cells to verify the mechanism of NK cells in pre-exercise training to intervene in the pathogenesis of liver tumors in mice.The pre-exercise animal model explores the mechanism of IL-15-related protein signaling pathway in the tissues adjacent to cancer in the mechanism of pre-exercise training to intervene the pathogenesis of liver cancer in mice.Part one: Inhibition of the activity of the serum of mice in one-time exercise onthe activity of incubating liver cancer cells Materials and Methods:Male C57 BL / 6 mice were randomly divided into four groups: quiet + PBS injection group(group C),one-time exercise + PBS injection group(group A),quiet +IL-15 group injection(group I)and one-time exercise + Inject IL-15 group(IA group).PBS for injection is 200 ?L,IL-15 is 4.5 ?g IL-15 + 15 ?g IL-15R?-FC solutiondissolved in 200 ?L PBS,incubated at 37 ° C for 1 hour,and the one-time exercise is12 m / min × 1h treadmill exercise.TUNEL was used to determine the apoptosis of liver cells in mice,ELISA was used to determine the content of IL-15,INF-? and TNF-? in serum,and flow cytometry was used to detect the number of NK cells in serum.The serum of each group of mice was taken to incubate HePa1-6 cells.Each group of serum was subjected to two treatments,one was direct incubation,the other was the addition of epinephrine blocker ProPranolol during incubation,and the control was blank medium incubation,which was used after 48 hours.CCK-8measures cell viability.SPSS 26.0 software was used for statistical analysis,and two-way analysis of variance was used to compare the differences between groups.Research result:1.TUNEL detection of apoptosis in each groupCompared with the mice in group C,the IHS of mice in group A decreased significantly(p <0.05),the IHS of mice in group I decreased significantly(p <0.01),while the HIS of IA group combined with exercise and IL-15 injection was Lower than Group A and Group I.2.Activity changes of Hepa1-6 cells during mouse serum incubation and co-incubation with ProPranololCompared with cells cultured in normal fetal bovine serum medium,there was no significant difference in cell activity in the medium incubated with mouse serum.Incubating Hepa1-6 cells with the serum of mice in Group A and IA,the cell viability when Propranolol was not added was significantly lower than that when ProPranolol was added(p <0.05);regardless of whether Propranolol was added,Hepa1-6 cells incubated with serum of Group A The cell viability was significantly lower than the serum incubation effect of group C(p <0.01),and the viability of Hepa1-6 cells incubated with serum of group IA was significantly lower than that of group I(p<0.01).3.Serum IL-15,INF? and TNF-? levels in each groupCompared with mice in group C,the serum IL-15 of mice in groups A and I was significantly increased(p <0.05),and the contents of INF-? and TNF-? weresignificantly increased(p <0.01);compared with mice in group A In comparison,the serum IL-15,INF-?,and TNF-? levels in the IA group were significantly increased(p<0.01);compared with the group I mice,the serum IL-15 and INF-The content of ?increased significantly(p <0.01),and the content of TNF-? increased significantly(p<0.05).4.Flow cytometry was used to detect the proportion of NK cells in the serum of mice in each group.Compared with mice in group C,whether it was a one-time exercise or injection of IL-15,the proportion of NK cells in serum could be significantly increased(p<0.05),while exercise and IL-5 injection combined intervention(IA group)NK The proportion of cells was higher than that of pure exercise(group A)and IL-5 injection(group I)(p <0.01).Part two: Using the pre-exercise training model to study the mechanism of IL-15in the pathogenesis of liver cancer in mice Materials and Methods:Twenty-four 7-month-old male C57 BL / 6 mice were randomly divided into a control group(C)and an exercise group(E).The exercise group underwent 12-week moderate-intensity treadmill training at a speed of 12 m / min,and the group C was normally reared for 12 weeks.After 12 weeks,half of the mice were randomly selected from Group C and Group E,and 100 ?L of Hepa1-6 cell suspension(containing 2 × 106 cells)was injected into the abdominal cavity in situ to prepare a tumor model.Exercise tumor group(EP).All mice did not exercise during the construction of the tumor model.13 days after the injection,samples were taken for testing.Take the liver of the mouse for the following tests: observe the morphology of the liver of the mouse;detect the apoptosis of the liver of the mouse by the TUNEL method;detect the changes of various proteins in the IL-15-related protein signaling pathway by Western blot,including P-AMPK,AMPK,SIRT1,IL-15,ACP53,P53,Caspase9,Caspase3.Statistical analysis was performed using SPSS 26.0 software,and two-way analysis of variance was used to compare the differences between groups.P <0.05 was judged to be significantly different.Research result:1.Changes in liver morphology of mice in each groupCompared with normal mice,tumor mice have increased liver volume,liver lobe tissues become harder,liver lobe becomes brittle,and the arrangement is loose;liver tumors of pre-exercise mice are smaller than those of quiet mice,and the amount of hard tissue is relatively small.2.Apoptosis of tissues adjacent to cancer in each group of miceCompared with the normal quiet group of mice,the exercise group had significantly lower liver IHS(p <0.01),and the quiet tissue adjacent to cancer tissue had significantly lower IHS(p <0.01);The IHS of tissues adjacent to liver cancer was significantly reduced(p <0.01).3.Changes in IL-15-related protein signaling pathway proteins in tissues adjacent to liver cancer in various groups of miceCompared with the liver tissues of normal quiet mice,the P-AMPK / AMPK ratio and SIRT1 protein expression in the exercise group were significantly increased,and the ACP53 / P53 ratio,Caspase9 protein expression,and Caspase3 protein expression were significantly reduced;and Compared with the liver tissue of normal quiet mice,the ratio of P-AMPK / AMPK in the tissues adjacent to cancer of quiet mice was significantly reduced,the expression of SIRT1 protein was significantly increased,the ratio of ACP53 / P53,the expression of Caspase9 protein and the expression of Caspase3 protein were significant Decreased;compared with the adjacent liver cancer tissues of quiet mice,the pre-exercised mouse liver cancer tissues P-AMPK / AMPK ratio and SIRT1 protein expression were significantly increased,ACP53 / P53 ratio,Caspase9 protein expression and Caspase3 protein expression Significantly reduced.4.Changes in IL-15 mRNA expressionCompared with normal quiet mice,the expression of IL-15 mRNA in the liver of exercise mice was significantly increased(p <0.05),and the expression of IL-15 mRNA in the tissues adjacent to liver cancer in quiet mice was significantly reduced(p <0.05).The pre-exercised mouse liver cancer tissues had significantlyhigher IL-15 mRNA expression than the quiet mouse liver cancer tissues(p <0.05).Analysis conclusion:1.Both one-time exercise and injection of IL-15 / IL-15R? complex can significantly increase the level of IL-15 in the serum of mice,at the same time increase the content and killing effect of NK cells;exercise conditions serum can make liver cancer cells have significant activity decline.This indicates that pre-exercise intervention in tumorigenesis may increase the content and killing effect of NK cells by increasing IL-15 in vivo.2.Pre-exercise can significantly improve the progress of liver tumors and significantly reduce the apoptosis of tissues adjacent to liver cancer.The possible mechanism is to interfere with the pathogenesis of liver tumors in mice by interfering with IL-15-related protein signaling pathways to produce anti-apoptotic effects in adjacent tissues.