| Part Ⅰ:Literature researchThis paper systematically analyzed the current understanding of radiation damage effect in modern medicine and the preface research on the etiology,pathogenesis and prescription of radiation damage in traditional Chinese medicine,theoretically traced the feasibility and necessity of prevention and treatment of radiation damage by traditional Chinese medicine,and sought the basis for prevention and treatment of radiation damage from the analysis of the formulation of Jianpi Bushen prescription(JBP)and the results of preliminary clinical and basic research.Part Ⅱ:Protective effect of Jianpi Bushen prescription on immune function damage and its effect on PI3K/Akt/m TOR signaling pathway and autophagyObjective:To observe the drug efficacy of JBP in anti-radiation immune function damage,and to determine whether PI3K/Akt/m TOR signaling pathway and autophagy are involved in the anti-radiation damage process of JBP.Methods:(1)Ten of the 50 SPF Kunming mice were randomly selected as the normal group,and the other 40 mice were given 6.0Gy X-ray one-time whole body irradiation to induce radiation immune damage mouse model.(2)After modeling,the mice were randomly divided into 4 groups with10 mice in each group:model group,high-dose Jianpi Bushen prescription group(H-JBP),low-dose Jianpi Bushen prescription group(L-JBP),and thymosin group.H-JBP and L-JBP were given concentrated liquid of JBP by gavage,respectively;The thymosin group was given thymosin enteric-coated tablets suspension by intragastric administration;normal group and model group were given the same amount of normal saline intragastric administration.Once a day,for 7 consecutive days.(3)After gavage,mice were weighed,peripheral blood was collected,and spleen and thymus were separated.The index of immune organs(spleen and thymus)was calculated.White blood cell count(WBC)and lymphocyte count(LYM)were detected.Interferon-γ(INF-γ)and interleukin-2(IL-2)levels were detected by ELISA.The histopathological changes of spleen were observed by HE staining.The ultrastructure and autophagosomes of spleen were observed by transmission electron microscopy.The m RNA expression levels of PI3K,Akt and m TOR were detected by real-time quantitative PCR.The expression of p-PI3K,PI3K,p-Akt,Akt,p-m TOR,m TOR and autophagy related molecules Beclin1,LC3-Ⅱ,LC3-Ⅰand p62 in spleen tissues were detected by WB.Results:(1)Effects of JBP on immune function of radiation mice:compared with normal group,mice in radiation model group did not die,but diet and water were significantly reduced,lethargy,activity was reduced,and hair was dim and sparse;The body weight,thymus index and spleen index were significantly decreased(P<0.01),the levels of IFN-γand IL-2 in spleen were significantly decreased(P<0.01),the white blood cell count(WBC)and lymphocyte count(LYM)in peripheral blood were significantly decreased(P<0.01),and HE staining of spleen showed the disappearance of spleen corpuscle structure and a large number of lymphocyte infiltration.Hyperplasia of connective tissue.After the administration of JBP,the diet and water of mice increased,the mental state,hair and activity of mice were significantly improved,the body weight of mice increased significantly,the index of thymus and spleen increased significantly,the contents of IFN-γand IL-2 in spleen tissue and the contents of WBC and LYM in peripheral blood increased significantly(P<0.05 or P<0.01).Splenic tissue damage and inflammatory cell infiltration were alleviated.The effect of JBP in low dose group was better than that in high dose group(P<0.05 or P<0.01),and was similar to that in thymosin group(P>0.05).(2)Effects of JBP on autophagy of mice spleen tissue:(1)Transmission electron microscopy(TEM):Compared with the normal group,the number of autophagosomes in spleen tissues of radiation model group was increased,and the structure of mitochondria and other organelles was seriously damaged.Compared with the model group,the autophagosomes of spleen were significantly reduced and the destruction of mitochondria and other organelles was alleviated after treatment with JBP.(2)WB detection:Compared with normal group,Beclin1 protein expression and LC3-Ⅱ/LC3-Ⅰratio in spleen tissue of mice in radiation model group were significantly increased,while p62 protein expression was significantly decreased(P<0.01).Compared with model group,Beclin1protein expression and LC3-Ⅱ/LC3-Ⅰratio were significantly decreased and p62 protein expression was increased after treatment with JBP(P<0.05 or P<0.01).The effect of JBP in low dose group was better than that in high dose group(P<0.05 or P<0.01),and was similar to that in thymosin group(P>0.05).(3)Effects of Jianpi Bushen Prescription on PI3K/Akt/m TOR axis-related molecules in mouse spleen:(1)Real-time quantitative PCR:Compared with normal group,m RNA levels of PI3K,Akt and m TOR in spleen tissues of mice in radiation model group were significantly down-regulated(P<0.01).Compared with model group,m RNA levels of PI3K,Akt and m TOR in spleen tissues of mice in JBP were significantly up-regulated(P<0.01 or P<0.05).The up-regulation effect of JBP in low-dose group on the m RNA levels of m TOR was better than that in high-dose group(P<0.05),and the effect of other indexes was similar to that in high-dose group(P>0.05).The effect of JBP in low-dose group was similar to that of thymosin group(P>0.05).(2)WB detection:Compared with normal group,the ratio of p-PI3K/PI3K,p-Akt/Akt and p-m TOR/m TOR in spleen tissue of mice in radiation model group was significantly decreased(P<0.01).Compared with model group,the ratios of p-PI3K/PI3K,p-Akt/Akt and p-m TOR/m TOR in spleen tissue of JBP were significantly increased(P<0.01 or P<0.05).The effect of L-JBP was similar to that of thymosin group(P>0.05).Conclusion:A one-time 6.0Gy total body irradiation can cause immune function impairment in mice,which is mainly manifested as the decrease of immune organ index,spleen tissue structure damage,decrease of IFN-γand IL-2 secretion levels,and decrease of WBC and LYM in peripheral blood.At the same time,radiation down-regulates m RNA and protein expression levels of related molecules in PI3K/Akt/m TOR signaling pathway,thereby inhibiting the pathway,upregulating autophagy related protein expression,and inducing autophagy over-activation.JBP can activate PI3K/Akt/m TOR signaling pathway,inhibit over-activation of autophagy,increase immune organ index of mice,reduce structural damage of spleen,promote secretion of IFN-γand IL-2,increase WBC and LYM in peripheral blood,and promote recovery of immune function after radiation.PartⅢ:Jianpi Bushen prescription regulates the molecular mechanism of T cell autophagy against radiation immune function damage through PI3K/Akt/m TOR signaling pathwayObjective:To observe the effects of JBP on T cell homeostasis and function,and to determine whether the regulation of autophagy by PI3K/Akt/m TOR signaling pathway is the internal molecular mechanism of JBP against radiation immune function damage.Methods:(1)Isolation and culture of radiation-damaged mice spleen T cells to prepare spleen-invigorating kidney prescription and thymosin drug-containing serum.The T cells cultured in vitro were divided into radiation model group,blank serum intervention group,Jianpi Bushen prescription drug-containing serum intervention group(JBP),JBP+autophagy inhibitor group(3-methyladenine),JBP+PI3K inhibitor group(LY294002),JBP+Akt inhibitor group(Bicalutamide),JBP+m TOR Inhibitor group(rapamycin),thymosin drug-containing serum group,and normal mouse T cells were isolated and cultured as normal control group.(2)Cells were pretreated with each inhibitor for 2 hours,then treated with each drug-containing serum for 48 hours,and the following tests were performed:CCK8 was used to detect cell proliferation activity;The apoptosis rate and T cell subsets(CD3~+,CD4~+,CD8~+)were determined by flow cytometry.Autophagy fluorescence intensity was observed by immunofluorescence labeling LC3 and confocal laser microscopy.INF-γ,IL-4,IL-17 and IL-10 in the supernatant of cell culture were detected by ELISA.The m RNA levels of PI3K,Akt and m TOR were detected by real-time quantitative PCR.WB was used to detect the expression of PI3K/Akt/m TOR axis-related molecules p-PI3K,PI3K,p-Akt,Akt,p-m TOR,m TOR and autophagy related molecules Beclin1,LC3-Ⅱ,LC3-Ⅰ,p62.Results:(1)Effects of JBP on T cell Homeostasis and function:(1)The effects of JBP on T cell proliferation and apoptosis:compared with normal group,the proliferative activity of T cells in radiation model group was significantly decreased,and the apoptosis rate was significantly increased(P<0.01).Compared with radiation model group,the proliferative activity of T cells was significantly increased and the apoptosis rate was significantly decreased after the intervention of serum containing JBP(P<0.01).Compared with JBP,the T cell proliferation activity of JBP+PI3K inhibitor,JBP+Akt inhibitor,JBP+m TOR inhibitor group was significantly decreased,and the cell apoptosis rate was increased(P<0.01).The T cell apoptosis rate of JBP+autophagy inhibitor group was further decreased(P<0.05),and there was no significant difference in T cell proliferation activity(P>0.05).The effect of JBP was similar to that of thymosin group(P>0.05).(2)Effects of JBP on T cell subsets:Compared with normal group,the proportions of CD3~+,CD4~+and CD8~+T cells in radiation model group were significantly decreased,while the ratio of CD4~+/CD8~+was increased(P<0.01).Compared with radiation model group,the proportions of CD3~+,CD4~+and CD8~+T cells in JBP were increased(P<0.01),while the ratio of CD4~+/CD8~+was decreased(P<0.05).Compared with JBP,the proportions of CD3~+,CD4~+and CD8~+T cells in JBP+PI3K inhibitor,JBP+Akt inhibitor,and JBP+m TOR inhibitor groups were significantly decreased(P<0.01).The ratio of CD4~+/CD8~+was increased(P<0.05),and the proportions of CD3~+,CD4~+and CD8~+T cells and the ratio of CD4~+/CD8~+in the JBP+autophagy inhibitor group were not significantly different(P>0.05).The effect of JBP was similar to that of thymosin(P>0.05).(3)Effects of JBP on immune cytokine secretion:Compared with normal group,the secretion levels of INF-γ,IL-4 and IL-10 were significantly decreased in radiation model group,while the secretion level of IL-17 was significantly increased(P<0.01).Compared with model group,the secretion levels of INF-γ,IL-4,IL-10 were significantly increased and IL-17 was significantly decreased after the intervention of JBP(P<0.01).Compared with JBP,the secretion levels of INF-γ,IL-4 and IL-10 in JBP+PI3K inhibitor,JBP+Akt inhibitor,JBP+m TOR inhibitor group were significantly decreased(P<0.01),while IL-17 secretion levels were increased in different degrees(P<0.01 or P<0.05).The INF-γsecretion level was increased(P<0.05)and the IL-17 secretion level was decreased(P<0.05),while the IL-4 and IL-10secretion levels were not significantly different(P>0.05)in JBP+autophagy inhibitor group.The effect of JBP was similar to that of thymosin(P>0.05).(2)Effects of JBP on T cell autophagy:(1)Immunofluorescence observation:Compared with the normal group,the intensity of autophagy fluorescence in the radiation model group was significantly increased(P<0.01).Compared with model group,the fluorescence intensity of autophagy in JBP was significantly decreased(P<0.01).Compared with JBP,the autophagy fluorescence intensity of JBP+PI3K inhibitor,JBP+Akt inhibitor,JBP+m TOR inhibitor group was significantly increased(P<0.01).The autophagy fluorescence intensity of JBP+autophagy inhibitor group was further decreased(P<0.05).The effect of JBP was similar to that of thymosin(P>0.05).(2)WB detection:Compared with normal group,Beclin1 protein expression and LC3-Ⅱ/LC3-Ⅰratio were significantly increased in radiation model group,while p62 protein expression was significantly decreased(P<0.01).Compared with model group,Beclin1 protein expression,LC3-Ⅱ/LC3-Ⅰratio were significantly decreased and p62 protein expression was significantly increased after intervention with JBP(P<0.01).Compared with JBP,Beclin1 protein expression and LC3-Ⅱ/LC3-Ⅰratio of JBP+PI3K inhibitor group,JBP+Akt inhibitor group,JBP+m TOR inhibitor group were significantly increased(P<0.01 or P<0.05),p62 protein expression was significantly decreased(P<0.01).Beclin1 protein expression and LC3-Ⅱ/LC3-Ⅰratio of JBP+autophagy inhibitor group were significantly decreased(P<0.05),but p62 protein expression was not significantly different(P>0.05).The protein expression level of p62 in JBP was higher than that in thymosin containing serum group(P<0.05),and the other indexes were similar to that in thymosin containing serum group(P>0.05).(2)Effects of JBP on PI3K/Akt/m TOR axis-related molecules of T cells:(1)Real-time quantitative PCR:Compared with normal group,m RNA levels of PI3K,Akt and m TOR in T cells in radiation model group were significantly down-regulated(P<0.01 or P<0.05).Compared with model group,m RNA levels of PI3K,Akt and m TOR were significantly up-regulated after intervention with JBP(P<0.01 or P<0.05).Compared with JBP,m RNA levels of PI3K,Akt and m TOR in JBP+PI3K inhibitor group,JBP+Akt inhibitor group,and JBP+m TOR inhibitor group were significantly decreased(P<0.05).There were no significant differences in the m RNA levels of PI3K,Akt and m TOR in JBP+autophagy inhibitor group(P>0.05).The effect of JBP was similar to that of thymosin(P>0.05).(2)WB detection:Compared with normal group,the ratio of p-PI3K/PI3K,p-Akt/Akt and p-m TOR/m TOR of T cells in radiation model group was significantly decreased(P<0.01).Compared with model group,the ratio of p-PI3K/PI3K,p-Akt/Akt and p-m TOR/m TOR were significantly increased after the intervention of JBP(P<0.01).Compared with the JBP,The ratios of p-PI3K/PI3K,p-Akt/Akt and p-m TOR/m TOR were significantly decreased in JBP+PI3K inhibitor group,JBP+Akt inhibitor group,and JBP+m TOR inhibitor group(P<0.01 or P<0.05).There were no significant differences in the ratios of p-PI3K/PI3K,p-Akt/Akt and p-m TOR/m TOR in JBP+autophagy inhibitor group(P<0.05).The effect of enhancing p-m TOR/m TOR ratio in JBP was better than that in thymosin drug-containing serum group(P<0.05),and the effect of other indexes was similar to that in thymosin drug-containing serum group(P>0.05).Conclusion:The radiation may weaken the inhibition of autophagy by inhibiting the PI3K/Akt/m TOR signaling pathway,and increase the ratio of the autophagy factor Beclin1 to the LC3-Ⅱ/LC3-Ⅰ,and reduce the p62induced autophagy,which destroys the T cell state and its functions,resulting in reduced immune function and immune imbalance.JBP can be reduced by activating the PI3K/Akt/m TOR signaling pathway,reducing the function of the autophagy factor Beclin1,the rate of LC3-Ⅱ/LC3-Ⅰand the p62suppression of autophagy,which promotes the recovery of the immune function and immune balance after the radiation,and provides a new target and treatment strategy for the prevention and control of radiation immune function damage. |
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