| Lung cancer is the most common malignant tumor in the clinic,and its morbidity and mortality rank first in all malignant tumors.At present,surgical resection is still the most effective way to eradicate lung cancer,but most patients find that the tumor is already in the metastasis stage and lost the opportunity for surgery.Immunotherapy has been widely recognized as part of tumor therapy,and dendritic cells have been extensively studied in the treatment of tumor immunity due to their tumor antigen presentation function.Abnormal lipid metabolism can lead to tumor immunosuppression,and lipid accumulation induced by the IER1?-XBP1 pathway in the endoplasmic reticulum stress pathway leads to a decrease in antigen-presenting function of tumor-associated dendritic cells(TDCs),thereby promoting the immunosuppressive microenvironment.form.The lung tumor cream is a traditional Chinese medicine compound developed by Professor Piao Bingkui of Guang'anmen Hospital of China Academy of Chinese Medical Sciences.It has been found in a large number of clinical trials.The lung tumor cream can prolong the stability of the tumor,improve the quality of life of patients,and reduce the quality of life.The toxic and side effects caused by chemotherapy and radiotherapy,especially the symptoms of lung cancer patients with deficiency of Qi and Yin.A large number of basic experiments have also shown that the traditional Chinese medicine compound Feiliuping cream Luomaping cream can significantly increase the function of dendritic cells and increase the killing activity of NK cells;it can exert anti-tumor effect by regulating the antigen presentation function of dendritic cells.The mechanism of action is related to inhibiting angiogenesis,up-regulating the expression of DCs-associated membrane molecules,inducing maturation,increasing immune synapse formation,promoting migration,and enhancing T cell killing activity,and has a certain advantage in regulating PI3K/AKT signaling pathway.Based on the previous research of the research group,we conducted the following experiments:Objective:1)To study the effect of XBP1 overexpression on TDCs function and lipids in tumor tissues of Lewis lung cancer-bearing mice and the resulting mechanism of action;2)To study the effect of lung tumor cream on the function and lipid of TDCs in XBP1 overexpressing Lewis lung cancer tumor tissues and to produce an effect mechanism;3)Reveal the molecular mechanism of lipid accumulation by XBP1-mediated lung tumor cream.Method:1)XBP1 overexpressing lentivirus was prepared by cloning vector technology,and XBP1 overexpressing Lewis cell stably transfected strain was prepared by cell transfection technique;XBP1 overexpressing Lewis lung cancer mouse tumor-bearing model was established,and divided into four groups:XBP1 overexpression group(Group G),XBP1 overexpression-loaded lung tumor cream group(GF group),normal Lewis lung cancer group(N group),normal Lewis lung cancer-loaded lung tumor cream group(NF group),2)The effects of XBP1 overexpression on TDCs function,T cell subsets,Treg,inflammatory cytokines,lipid content in TDCs and transplanted tumors in Lewis lung cancer-bearing mice were observed at 14 days and 21 days.3)The effects of Feiliuping ointment on TDCs function,T cell subsets,Treg,inflammatory cytokines,lipid content in TDCs and transplanted tumor of XBP1 overexpressing Lewis lung cancer-bearing mice were observed at 14 days and 21 days.4)Using qPCR method to observe the effect of Feiliuping ointment on mRNA content of IDO1,Argl,IREla-XBP1 and PI3K-AKT-mTOR pathway in XBP1 overexpressing Lewis lung cancer/normal Lewis lung cancer-bearing mice;Western blot method to observe the effect of Feiliuping Ointment on the expression of the IRE1?-XBP1 pathway and PI3K-AKT-mTOR pathway protein in XBP1 overexpressing Lewis lung cancer-bearing mice.Result:1)XBP1 overexpressing lentiviral vector was successfully prepared by using GV358 plasmid as vector and lentiviral transfection technology;XBP1 overexpressed Lentiviral vector was successfully introduced into Lewis lung cancer cells by cell transfection technology;Mouse xenograft model;2)Compared with the N group,XBP1 overexpression inhibited the function of TDCs,with the most obvious inhibition by CD80(14day,P<0.01;21day,P<0.01);the expression of CD3+CD8+ was significantly decreased at 21 days(P<0.05);up-regulated Treg expression(14day,P<0.01;21day,P<0.05);promoted TGF-?1 secretion(P<0.01)and decreased TNF-a concentration(P<0.01)at 21 days;increased TDCs Lipid content(21 days,P<0.01)promoted the growth of transplanted tumors in mice.3)Compared with group G,GF group can significantly increase the surface marker of TDCs,with MHC-?,CD40,and CD80 being more significant(P<0.05),increasing antigen presentation function;up-regulating T cell subsets CD3+,CD3+ The expression of CD4+,CD3+CD8+(P<0.05)decreased the expression level of Treg cells(14day,P<0.01;21day,P<0.05);decreased the concentration of TGF-?1 in serum(14day,P<0.05;21day,P<0.05),the concentration of TNF-a was increased(21 days,P<0.05);the lipid accumulation in TDCs was decreased(21days,P<0.05),and tumor growth was inhibited.4)qPCR IDO1 results showed that the expression of IDO1 mRNA in group G was greater than that in group N,which was statistically significant at 14 days;the mRNA expression of IDO1 in group G was higher than that in GF group,and the difference was statistically significant(14 days,P<0.01).21 day,P<0.05);the mRNA expression level of IDO1 in NF group was lower than that in N group,and the difference was the most significant at 14 days(P<0.01).The results of qPCR Argl showed that the expression of Argl mRNA in group G was greater than that in group N,and the difference was statistically significant(14day,P<0.05;21 day,P<0.01).The expression of Argl mRNA in group G was higher than that in group GF,and the difference was Statistical significance(P<0.05);Arg1 mRNA expression in NF group was lower than N group,and the difference was most significant at 21 days(P<0.05).It indicates that the lung tumor cream can reduce the mRNA content of IDO1 and Arg1 in mouse tumor tissues.The results of the qPCR IRE1?-XBP1 pathway showed that the mRNA content of the G group was higher than that of the N group at 14 days,and the difference was statistically significant(P<0.05).The mRNA expression of the G group was higher than that of the GF group,among which Bip(The difference between P<0.01)and IRE1?(P<0.05)was the most obvious.Compared with the N group,Bip mRNA content in the NF group was decreased,and the difference was statistically significant(P<0.01).At 21 days,the mRNA content of the G group was higher than that of the N group,which was significantly different by Bip(P<0.01)and XBP1(P<0.05).The mRNA expression of the GF group was lower than that of the G group,and the difference was statistically significant.(P<0.05);the mRNA expression of the NF group was lower than that of the N group,but the difference was not statistically significant(P>0.05).The results of the qPCR PI3K-AKT-mTOR pathway showed that the mRNA content of the G group was higher than that of the N group at 14 days,which was significantly different by PI3K(P<0.05).The mRNA expression of GF group was lower than that of G group,and mTOR(P)<0.05);the mRNA expression of NF group was lower than that of N group,but the difference was not statistically significant(P>0.05).At 21 days,the mRNA content of the G group was higher than that of the N group,among which AKT(P<0.05)was significantly different.The mRNA content of G group was higher than that of N group,and the difference was statistically significant(P<0.05);NF group The expression of each mRNA was lower than that of N group,and the difference between AKT and mTOR was the most significant(P<0.05).It indicates that the lung tumor cream can reduce the mRNA content of the IRE1?-XBP1 and PI3K-AKT-mTOR pathway.The results of the Western Blot IRE1?-XBP1 pathway showed that the protein of group G in group G was greater than group N at 14 days,but the difference was not statistically significant.The protein expression of Bip and IRE1? in lung tumor filling group was smaller than that in the unloaded tumor.In the cream group,the Bip protein expression in the GF group was the most significant(P<0.01).At 21 days,the expression of each group in group G was larger than that in normal Lewis lung cancer-bearing mice,and the expression of Bip and IRE1?protein was the most significant(P<0.01).The expression of each group in group G was higher than that in the GF group.The difference was statistically significant(Bip,P<0.01;IRE1?,P<0.01;XBP1,P<0.05).Compared with the expression of each protein in group N,the expression of each protein in the NF group was lower,and Bip protein was the most obvious(P<0.05).The results of the Western Blot PI3K-AKT-mTOR pathway showed that the protein expression of pAKT in group G was higher than that in the GF group at 14 days(P<0.05).The protein expression of PI3K and mTOR in group G was higher than that in the GF group.The difference was statistically significant(P<0.05);there was no statistically significant difference between the other groups(P>0.05).At 21 days,the protein expression of PI3K,mTOR,and pmTOR in group G was higher than that in group N(P<0.01).The protein expression of PI3K,mTOR,pAKT,and pmTOR in group G was higher than that in group G,and the difference was higher.There was significant statistical significance(P<0.01).The expression of each protein in the NF cream group was lower than that in the N group,and the difference of pAKT was the most significant(P<0.01).It indicated that the lung tumor cream can reduce the expression of IRE1?-XBP1 and PI3K-AKT-mTOR pathway proteins.In Conclusion:1)XBP1 overexpression can inhibit TDCs antigen presentation function in Lewis lung cancer,resulting in increased lipid accumulation;2)The lung tumor cream can reverse the functional defects of TCDs caused by lipid accumulation and enhance the antigen-producing function of TDCs;3)The enhancement of TDCs phenotype and function by lung tumor cream may be achieved by down-regulating the PI3K-AKT-mTOR signaling pathway by reducing the activation of the IRE1?-XBP1 pathway mediated by the endoplasmic reticulum. |
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