| Background:Lung cancer is the most common tumor in clinic,and its morbidity and mortality are the first in all malignant tumors.The main treatment methods of lung cancer include surgery,radiotherapy and chemotherapy,targeted treatment and immunotherapy.Dendritic cells(DCs)are the most powerful specialized antigen-presenting cells in the body,which can effectively activate T-lymphocyte immunity,and is the key to tumor immunotherapy.In tumor microenvironment,lipid peroxidation in tumor associated dendritic cells(TDCs)activated endoplasmic reticulum stress and caused unfolded protein response,UPR)can activate the downstream IRE1α-XBP1 pathway,and the resulting sheared XBP1-s can target multiple lipid synthesis genes,induce the abnormal accumulation of lipid in TDCs,show immature phenotype and dysfunction,and inhibit the anti-tumor immunity.Some studies have shown that PI3K-AKT-mTOR signaling pathway plays an important role in lipid metabolism.It can regulate lipid synthesis by regulating the expression of genes related to fat synthesis.The overexpression of XBP 1 will lead to the up regulation of PI3K/mTOR expression.After XBP1 is knocked out,the down regulation of PI3K/mTOR expression.Other studies have shown that inhibition of mTOR2 can reduce lipid production."Lipid turbidity" is not only a pathological product but also a pathogenic factor.It belongs to the pathological category of "phlegm turbidity" in traditional Chinese medicine.Feiliuping ointment is a traditional Chinese medicine compound with definite curative effect in the treatment of lung cancer,which is developed by Professor Bingkui Piao,a famous Chinese medicine doctor in China.It has the effects of benefiting qi and nourishing Yin,resolving phlegm and resolving stasis,detoxifying and promoting blood circulation.It can play an anti-tumor role through multiple targets.Previous studies have shown that FLP can significantly improve the function of DCs,play an anti-tumor effect by regulating the antigen-presenting function of DCs,and has some advantages in regulating PI3K/mTOR pathway.As the main effective component of Platycodon grandiflorum,which is the representative of expectorant in Feiliuping ointment,the study shows that Platycodon D(PD)has the functions of anti-tumor,immunoregulation,lipid-lowering and anti-oxidation,and has the functions of inhibiting ROS accumulation and regulating PI3K/mTOR signal.Therefore,we speculate that PD may be one of the effective components of FLP in regulating lipid metabolism and reversing TDCs function,which may be reversed by inhibiting the abnormal accumulation of TDCs lipid and hope to explore its mechanism.Objective(1)To construct a co culture system of TDCs and explore the effect and mechanism of Feiliuping ointment on the lipid content and function of TDCs after the ER shear XBP1s induced by Thapsigargin(TG).(2)To explore the immunomodulatory function of PD on TDCs,and to explore the effect of TG on lipid content and function of TDCs and the possible mechanism of the effect.Method(1)To establish the model of tumor associated dendritic cells co culture in vitro(2)To simulate the tumor microenvironment,the lipid content and costimulatory molecule expression of TDCs were observed by flow cytometry after intervention of TDCs model with FLP containing serum;the proliferation of mixed lymphocytes was detected by CCK8,the activation of T lymphocyte subsets was detected by flow cytometry,and the cytokine secretion of TDCs was detected by Luminex technique.(3)The effects of Dauricine on the activation of ERS,IRE1 α-XBP1 pathway,lipid content of TDCs,costimulatory molecule expression,mixed cell proliferation,subsets activation and cytokines were observed(4)FLP containing serum was used in the model of TDCs activated by ERS after TG intervention.The effects of FLP on the lipid content of TDCs,the expression of costimulatory molecules,the proliferation of mixed cells,the activation of subsets and cytokineswere observed.(5)Western blot and qPCR were used to detect the expression of BiP-IRE1α-XBP1 and PI3K-AKT-mTOR pathway mRNA and protein in FLP containing serum and explore the possible mechanism.(6)LDH and CCK8 were used to detect the toxicity of PD to DCs and the killing ability of Lewis lung cancer cells.(7)The effects of PD on lipid content,costimulatory molecule expression,mixed cell proliferation,subsets activation and cytokines of TDCs were observed.(8)The effects of PD on lipid content,costimulatory molecule expression,mixed cell proliferation,subpopulation activation and cytokines of TDCs were observed by constructing the model of TDCs activated by TG.(9)Western blot and qPCR were used to detect the expression of BiP-IRE1α-XBP1 and PI3K-AKT-mTOR pathway mRNA and protein by PD,and to explore the possible mechanism.Results(1)The positive expression rate of CD11c+on the surface of DCs was 76.5%after induction by flow cytometry,and it was co cultured with the supernatant of lung cancer cells,stimulated by freeze-thaw antigen,to construct the co culture model of TDCs.(2)In the simulated tumor microenvironment,the serum containing FLP reduced the intracellular lipid content of TDCs(P<0.01)and increased the expression of TDCs surface molecules,especially the expression of CD80 and CD86(P<0.05).FLP stimulated the proliferation of co cultured lymphocytes(P<0.05),significantly increased the proportion of Th and CTL cell subpopulation in T cells(.P<0.05)and decreased the expression of Tregs cells.The expression of IL-12p70 and IFN-γincreased(P<0.05).(3)After TG,in TDCs culture model,the mRNA and protein expression of IRE1α-XBP1(t/u/s)pathway increased(P<0.05),meanwhile,it inhibited the secretion of IL-12 and IFN-γ(P<0.05).(4)The results showed that the lipid content of TDCs decreased significantly(P<0.05)and the expression of TDCs surface molecules was restored after the treatment of TG with FLP containing serum,among which the expression level of CD80 and CD86 was increased significantly(P<0.05),the proliferation ability of mixed lymphocyte was increased(P<0.05),the proportion of Th and CTL cell subsets was increased(P<0.05),and the expression of Tregs cell subsets was decreased(P<0.05)0.05),which promoted the secretion of IL-12p70 and IFN-γ(P<0.05).(5)The results of qPCR and Western blotting showed that the mRNA and protein expression of BiP-IRE1α-XBP1 and PI3K-AKT-mTOR pathway increased after the activation of ERS by TG(P<0.05);the expression of BiP-IRE1α-XBP1 pathway mRNA and protein decreased significantly after the intervention of TDCs by FLP containing serum(P<0.05);the expression of XBP1-s,an important lipid regulatory transcription factor,decreased significantly in the FLP+TG group The mRNA and protein of PI3K-AKT-mTOR pathway activated by TG decreased to a certain extent.and the phosphorylation level of Akt protein decreased significantly(6)The results of LDH showed that the concentration of PD below 30 um had no obvious damage to DCs,or affected the release of LDH in DCs cells;CCK-8 showed that PD could effectively kill Lewis lung cancer cells,and its IC50 value was about 13 um.(7)In the co culture system of TDCs.the high,middle and low concentrations of PD can effectively reduce the lipid content of TDCs(P<0.01),increase the expression of mch-ii on the surface of TDCs(P<0.05),and increase CD80(PDH,PDM,P<0.05),CD86(PDM,PDL,P<0.05),the proliferation of PDH mixed lymphocytes(P<0.05),the expression rate of Th and CTL subsets(P<0.05),the expression of Tregs subsets(PDH,P<0.05)and the secretion of IL-12p70(PDH,PDM,P<0.05)IFN-γ(PDH,P<0.05)cytokines were increased in PD high,middle and low group(8)PDH could increase the expression of mch-ii on the surface(P<0.05),CD80 and CD86 on the surface(P<0.05),PDH could increase the proliferation of lymphoid mixed cells(P<0.05),PDH and PDM could significantly increase the expression of Th and CTL cell subsets(P<0.05)<PD could reduce the expression of Tregs cell subpopulation(P<0.05),and promote TDCs to secrete IL-12p70(PDH,P<0.05),IFN-γ(PDH,PDM,P<0.05)cytokines.(9)The results of qPCR and Western blot showed that after TG activated ERS,the expression of BiP-IRE1α-XBP1(t/u/s)pathway mRNA and protein in PD+TG group was significantly decreased(P<0.01).At the same time,the expression of PI3K-AKT-mTOR pathway mRNA and protein in PD+TG group was decreased to some extent,and the expression of p-Akt phosphorylated protein in PD+TG group was significantly decreased(P<0.05).ConclusionBased on the experimental conditions,we speculate that:(1)TG can induce ERS,activate IRE1α-XBP1 pathway,produce XBP1-s shear form,which can cause abnormal accumulation of TDCs lipids and dysfunction of TDCs.(2)FLP containing serum can reverse the abnormal accumulation of lipid and antigen presentation dysfunction of TDCs caused by the shear of XBP1 induced by TG activated ERS.(3)PD may be one of the effective components of FLP to improve the abnormal accumulation and antigen presentation of TDCs.(4)The mechanism of PD regulation of FLP containing serum and its effective component to improve the abnormal accumulation and antigen presentation function of TDCs may be realized by regulating BiP-IRE1α-XBP1 and PI3K-AKT-mTOR pathway. |
