The Mechanism Of Berberine Regulating The Neutrophil Phenotype To Improve Doxorubicin Resistance

Cancer is a leading cause of human disease-related death,and chemotherapy is currently the standard of care,but chemotherapy resistance is the main reason for clinical patients to be forced to terminate treatment and causes chemotherapy to fail.Previous studies of cancer chemotherapeutic resistance have been focused on cancer cells themselves,including changes in drug targets,ABC transporter expression,DNA damage repair,and apoptotic pathways,that leads to poor treatment.Tumor immune microenvironment is closely related to tumor development.However,the relationship between chemotherapeutic resistance and immune response had not yet received enough attention.Neutrophils,the most important effectors of innate immunity,have been reported to have a crucial role in tumor initiation and progression.The accumulation of tumor-associated neutrophil was positively correlated with poor prognosis.Moreover,neutrophil is heterogeneous,with an anti-tumor N1 phenotype and a tumor-promoting N2 phenotype.Berberine is an isoquinoline alkaloid from Coptidis rhizomes,and has regulation properties of neutrophil function.Doxorubicin is the representative drug of chemotherapy resistance.But the effect of berberine on neutrophil phenotype and doxorubicin resistance has not been reported.The aim of this study is to explore the relationship between neutrophil phenotype and chemotherapeutic resistance and the mechanism of berberine regulating the neutrophil phenotype to improve doxorubicin resistance.This study is divided into in vitro,in vivo and mechanism.In vitro,to obtain neutrophils,ATRA were induced HL-60 to differentiate into HL-60N1,and TGF-? were induced HL60-N1 to differentiate into HL60-N2.ELISA kits(IFN-?,IL-2,IL-6,PD-L1,IL-10,TGF-?.)were used to detect the levels of cytokines.MTT assay was used to detect the effect of HL60-N1 and HL60-N2 cells on tumor cell viability.HL60,HL60-N1 or HL60-N2 cells were treated with berberine or doxorubicin at different concentrations,and the effect of berberine and doxorubicin on cell viability was detected by MTT.Laser holography was used to detect the effect of doxorubicin and berberine alone or in combination on the morphology of neutrophils of different types;Wright-Giemsa staining was used to detect its effect on nuclear morphology;Annexin V-FITC / PI was used to detect the effect on apoptosis.The phagocytic ability was detected by intracellular phagocytosis of E.coli experiment;CD66b expression was detected by immunofluorescence.In vivo,we first established urethane induced lung cancer model in mice.To evaluate the effects of doxorubicin and berberine alone or in combination on tumor growth and lung tumor nodules;the pathological changes of lung in mice were examined by HE staining;immunofluorescence and Evans blue staining was used to detect changes in vascular permeability of lung tissue in mice;immunofluorescence was used to detect changes in neutrophil typing in lung alveolar lavage fluid of mice;immunochemical detection of PD-1 expression in spleen tissue;flow cytometry was used to detect the ratio of cytotoxic T cells;the effect of NLR levels in each group were analyzed by blood routine examination.And neutrophils depletion was used to analyze the role of neutrophils in the development of lung cancer.After that,we evaluate the tumor growth of H22 xenograft tumor mouse model;immunofluorescence was used to detect bone marrow neutrophil CD66 b and CD309 expression;the tumor vascular integrity was assayed with the Evans blue dye;blood routine examination detection of mouse NLR levels in each group;the ratio of cytotoxic T cells was detected by flow cytometry.LY6G-m Ab depleted neutrophils and adoptive infusion of HL60-N1 and HL-60-N2 cells were used to evaluate the role of neutrophils in doxorubicin resistance in tumor progression.Finally,immune challenge model was established to record the tumor growth of tumor,the expression of CD66 b and CD309 in bone marrow neutrophils,the permeability of tumor tissue,the level of NLR levels and the ratio of memory T cells.Evaluate the immunosuppressive effect of neutrophils and the regulatory effect of berberine on neutrophils.In the mechanism research,the expression of autophagy related LC3-B and P62 protein was observed by immunofluorescence and AO staining,DCFH-DA,Mito-Tracker Green and Lyso-Tracker Red were used to detect intracellular ROS,mitochondria,and lysosomal function,respectively;NBT was used to detect doxorubicin or berberine alone or in combination on the differentiation of HL60 cells;CFSE-7AAD staining was used to detect the effect of doxorubicin and berberine alone or in combination on cell lysing ability;immune clearance was assessed using a calcein-release assay;the effect of different antibodies on CD8+T cell surface markers by co-culture system.After that,molecular mechanism of berberine and doxorubicin regulating neutrophil phenotype predicted by network pharmacology;immunofluorescence and western bolt were used to confirm network FOXO signaling pathway and JAK2-STAT3 signaling pathway;molecule inhibitor WP1066 were used to verify molecular mechanism of berberine and doxorubicin regulating neutrophils.And meta-analysis was used to analyze the prognostic relationship between NLR levels and cancer chemotherapy.In vitro results showed that different type of neutrophils had significant differences in the nucleus,cell volume,related cytokines and tumor cell viability after inducing by ATRA and TGF-?;continuous doxorubicin treatment led to HL-60 cell resistance to doxorubicin accompanied by the shift of HL-60 cells to the N2 phenotype.Unexpectedly,the combination treatment with doxorubicin and berberine resulted in the polarization of HL-60 cells towards the N1 phenotype.Berberine had little effect on cell viability in HL-60 and HL-60-N1 cells,improved apoptosis of HL60-N2 cells,accompanied by an increment in obvious cell nucleus changed and phagocytic capacity,a reduction in expression of CD66 b and cell volume.In vivo result indicated that both berberine treatment and LY6G-m Ab prevented lung carcinogenesis.Unexpectedly,doxorubicin alone promoted neutrophil polarization to N2 and lung carcinogenesis.Berberine alone or in combination can make N1 neutrophils cell recovery,preventing tumor development,at the same time reduced NLR levels,serum PD-L1 levels,PD-1 expression on the surface of splenic T lymphocytes,reduced the expression of CD31,tie2 and CD309 in lung tissues and the vascular permeability shown by Evans blue,increases the rate of spleen cytotoxic CD8 + T cells.In the H22 xenograft model,the HL60-N1 cell injection prevented tumor growth,whereas the HL60-N2 cell injection promoted tumor growth.Berberine or LY6G-m Ab had a negligible effect on suppression of tumor growth,but maintain the neutrophil N1 phenotype and promote the tumor suppressive effect of doxorubicin.The results of bone marrow neutrophil immunotyping,serum cytokines,and spleen T lymphocytes were similar to those in the urethane-induced lung cancer model.In the H22 cell rechallenge immune study where tumor immune rejection was suppressed by doxorubicin but was promoted by berberine.Importantly,the combination of doxorubicin and berberine synergistically stimulated H22 cell immune rejection accompanied by a reduction in bone marrow N2 neutrophils,intratumor permeability to Evans blue dye,NLR levels,serum levels of PDL1,Th2 cytokines,and spleen T lymphocyte surface PD-1 expression.Conversely,this combination increased spleen memory CD8+ cell rates.The mechanism research results show that berberine reduces HL60-N2 cell autophagy,enhances mitochondrial and lysosome functions,reduces ROS levels,and promotes HL60 cells differentiation.Berberine alone or in combination with doxorubicin can promote cell lysis of HL60 cells,accompanied by immune clearance of CD8 +T cells.Consistent with function results,HL-60-N2 cells produce more PD-L1,and promoted CD8+ T cell expression of PD-1 surface receptor.CD309 antibodies but not CD66 b and CD133 antibodies prevented PD-L1 production in HL-60-N2 cells.Network pharmacology analysis revealed that berberine and doxorubicin regulating neutrophil phenotype was related to the FOXO signaling pathway and the JAK2-STAT3 signaling pathway.Immunofluorescence and Western bolt results show that the molecular mechanism is related to JAK2-STAT3;the JAK2-STAT3 pathway was verified as an important regulatory mechanism by using WP1066.Meta-analysis results show that NLR levels are positively correlated with poor prognosis for chemotherapy in cancer patients.In summary,neutrophils play a key role in doxorubicin resistance.Berberine selectively regulates the phenotype of neutrophils to maintain immune monitoring and the sensitivity of cancer cells to doxorubicin.The mechanism is related to JAK2 / STAT3-CD309-PD-L1 signaling cascades.