Pharmacological Mechanism Of Shugan-Liangxue Precription On Regulating Immune Metabolism Against Breast Cancer

The development of breast cancer is closely related to the immune status.The decreased immune surveillance function and enhanced immune suppression function promote breast cancer immune escape.Immune status is regulated by metabolism.Indoleamine-2,3-dioxygenase-1（IDO1）mediated tryptophan metabolic pathway plays an important role in promoting breast cancer immune escape.Shugan-Liangxue Prescription（SLP）is composed of eleven herbs,such as Bupleuri Radix,Citri Reticulatae Pericarpium,Curcumae Radix,Sophorae Flos,Moutan Cortex,Arnebiae Radix,Prunellae Spica,Salviae Miltiorrhizae Radix et Rhizoma,Curcumae Rhizoma,Astragali Radix,Bge.var.mongholicus,and Glycyrrhizae Radix et Rhizoma.It is a Chinese herbal formula for treating breast cancer in clinic.In this study,first,the rat breast cancer immune pharmacological model was established to evaluate prescription anticancer immune effects,then the metabolic mechanism promoting breast cancer immune escape was identified with omics technology,second,based on IDO1 mediated tryptophan metabolic pathway,the anticancer effects and underlying mechanism of SLP were explored,last,the quick and feasible vitro methodology of the drug immune pharmacological mechanism against breast cancer was explored.Part 1 To establish the immunopharmacological model of breast cancer1.1 To establish the immunopharmacological model of rat breast cancerObject:Complete freund’s adjuvant（CFA）was used to stimulate immunity in chemical induced breast cancer rats to establish the immunopharmacological model of rat breast cancer.Methods:230 SD rats,70 of them were in control group,breast cancer was induced by 7,112-demethylbenz[a]anthracene（DMBA）administration once in other 160 rats.At d052,rats were divided equally into model group and SLP group by tumor grade.Each group were divided into 7 subgroups（ni=10）according to the gradient interval between CFA sensitization and challenge.At d052.054,058,model group and SLP group received CFA sensitization,and each subgroup received challenge according to the gradient interval（25d-95d,k=0.8）.At d056,model group was administrated with carboxy methyl cellulose（CMC）,so was as SLP in SLP group.Control group received same volume normal saline（NS）,and was administrated with CMC.Tumor volumes were measured,tumor pathology and tumor infiltrated lymphocytes（TIL）were evaluated,and serum IL-12 was detected.The median effective interval（EI50）of tumor volume obtained from regression of tumor volume and logarithmic interval was as the optimal interval condition.EI50 of IL-12 obtained from regression of serum IL-12 and logarithmic interval was to reflect the immune surveillance status in breast cancer.Results:The tumor pathological features of this model was confirmed as invasive lobular carcinoma,the number of TIL increased significantly in SLP group.The EI50 and 95%confidence intervals of tumor volume were 33d and 26d-41d,respectively,and the equation was Y=-0.05+0.95/（1+1015.97-10.52X）,R=0.97.The tumor volume curve of SLP group was right shifted compared with that of model group.The EI50 and 95%confidence intervals of IL-12 were 40d and 25d-63d,respectively,and the equation was Y=0.25+0.73/（1+10-36.08+22.52X）,R=0.92.The IL-12 curve of SLP group was left shifted compared with that of model group.Conclusions:CFA amplified immune effect in breast cancer rats,the optimal interval of CFA sensitization and challenge was 33d.IL-12 curve showed the feed-forward mechanism of immune surveillance,and SLP group curve confirmed that this model could be used to evaluate immune response of anti-breast cancer drugs.1.2 Altered immune metabolism took part in the pathogenesis of breast cancer immune escapeObject:To observe the effects of immune metabolism in breast cancer immune escape.Methods:230 SD rats,70 of them were as control group,breast cancer was induced by DMBA administration once in other 160 rats.At d052,rats were divided equally into model group and SLP group by tumor grade.Each group were divided into 7 subgroups（ni=10）according to the gradient interval between CFA sensitization and challenge.At d052,054,058,model group and SLP group received CFA sensitization,and each subgroup received challenge according to the gradient interval（25d-95d,k=0.8）.At d056,model group was administrated with CMC,so was as SLP in SLP group.Control group received same volume NS,and was administrated with CMC.Control,model and SLP group of rats（the interval between CFA sensitization and challenge is 31d）were used for the research.The breast tumor feature and the number of TILs were calculated,and the expression of CD8,NKG2D,CCR7,foxp3 in tumor tissues were detected by immunohistochemistry staining（IHC）.Transcriptomics,proteomics,lipid omics,quantitative measurement of bile acids and amino acids were carried out in rat spleen.Transomics analysis was used to speculate the relevant targets and pathways participating in immune escape in breast cancer,the correlation analysis was carried out between differential metabolites and immunocytes molecular biomarkers.Expression of IDO1-AhR pathway,CaN-NFAT pathway and ERα,ERβ,were carried out with real-time fluorescent quantitative（RT-PCR）,Western blot（WB）,IHC and calcineurin（CaN）,IDO1 enzyme activity were detected.The correlation analysis was carried out between these critical indicators and immunocytes molecular biomarkers.Results:Several differential lipids,bile acids and amino acids were screened as immunometabolic markers by transomics.In model group,IDOl mediated tryptophan metabolism,CaN-NFAT pathway were activated,ERa upregulated while ERβdownregulated,these changes were positively correlated with immune escape biomarkers.SLP inhibited tumor growth,inhibited the immune escape associated pathway and protein expression.Conclusions:Immune metabolism related pathway（IDO1-AhR pathway）or pathogenic metabolites induced breast cancer immune escape.SLP could mediate immune metabolism to inhibit breast cancer immune escape.Part 2 To explore the effects and pharmacological mechanism of regulating immunity against breast cancer with Shugan-Liangxue prescription2.1 To study the effects of Shugan-Liangxue prescription on inhibiting immune escape against breast cancerObject:To explore the effects of SLP on inhibiting immune escape against breast cancer.Methods:At d001,DMBA was administrated to replicate rat breast cancer as olive oil in control group（ni=12）.At d052,054,058,rat suffered breast tumor received CFA sensitization,at d052 according to tumor volume,breast cancer rats were divided into 8 groups（ni=12）:model group,tamoxifen（TAM）group,glucose metabolism（alloxan）group,lipid metabolism（Met）group,cholesterol metabolism（atorvastatin）group,low（SLPL）,mediate（SLPM）and high（SLPH）dose of SLP group,at d056,rats were administrated by gavage once every day.At d085,087.091,every group received CFA challenge,as same dose of NS in control group.Tumor volume and tumor weight were calculated at d135,the expression of tumor Ki-67 was detected as cancer cell proliferation by IHC.Serum IL-12,IL-4 were detected by Elisa,and the splenic expression of CD8,CD206,NKG2D and NKG2A were detected by IHC.Results:The tumor growth and cancer cell proliferation was significantly inhibited in Met,atorvastatin,SLPM and SLPH group,with the tumor inhibition rate of 72.96%,67.27%,74.92%,69.52%,respectively.Met,atorvastatin,SLPM and SLPH group upregulated the serum IL-12 level while the downregulated IL-4 level.Met,atorvastatin and SLP group upregulated the expression of CD8 and NKG2D,while downregulated CD206 and NKG2A.Conclusions:SLP inhibited immune escape,enhanced immune surveillance against breast cancer.2.2 To explore the pharmacological mechanism of regulating immune metabolism to inhibit immune escape against breast cancer with Shugan-Liangxue prescriptionObject:To explore the pharmacological mechanism of SLP inhibiting immune escape against breast cancer.Methods:At d001,DMBA was administrated to replicate rat breast cancer as olive oil in control group（ni=12）.At d052,054,058,rat suffered breast tumor received CFA sensitization,at d052 according to tumor volume,breast cancer rats were divided into 8 groups（ni=12）:model group,tamoxifen（TAM）group,glucose metabolism group（alloxan）,lipid metabolism（Met）group,cholesterol metabolism（atorvastatin）group,low（SLPL）,mediate（SLPM）and high（SLPH）dose of SLP group,at d056,rats were administrated by gavage once every day.At d085,087,091,every group received CFA challenge,as same dose of NS in control group.Tumor volume was measured weekly,tumor volume and tumor weight were calculated at d135,rats were sacrificed,the serum kynurenine were detected by Elisa,the expression of spleen IDO1,AhR and foxp3 were detected by RT-PCR,WB and IHC,the serum glucose（GLU）,non-esterified fatty acid（NEFA）and cholesterol（CHO）were detected by biochemical analyzer,the correlation analysis was carried out between the biochemical indicators and the immunocytes molecular biomarkers.Results:SLP downregulated the expression of IDO1,AhR,foxp3 and kynurenine level.SLP downregulated GLU,NEFA level,the expression of NKG2A,NKG2D was positively,negatively correlated with GLU level,respectively,the expression of foxp3 and CD206 was positively correlated with NEFA level,the situ expression of CD8 was negatively correlated with CHO level.Conclusions:SLP inhibited IDO1 mediated tryptophan metabolism to inhibit immune escape against breast cancer,its inhibiting immune escape against breast cancer was closely related to the regulation of immune system glucose and lipid metabolism.Part 3 To explore the standard method of rapid detection of immune surveillance in breast cancer3.1 To establish the co-culture model of primary spleen cells and breast cancer cellsObject:To establish the co-culture model of primary spleen cells and breast cancer cells,confirming the appropriate culture time and density to inoculate for the research of immune metabolism mechanisms of anti-breast cancer drugs.Methods:Primary spleen cells and breast cancer cells were separated and extracted from CFA stimulated immunopharmacological model of rat breast cancer,cells were primary cultured,cancer cells group,spleen cells group and cancer-spleen co-culture group were respectively set according to the gradient culture time interval and gradient density to inoculate.The lactate dehydrogenase（LDH）activity was detected at the end of the experiment,the corresponding LDH activity in each group was used to calculate the antitumor cytolytic activity（LDH value）,the equation was LDH value=（LDHco-culture-LDHcancer cell-LDHspleencelI）/（LDHcancer cell+LDHspleen cell）,the area under curve（AUC）of LDH value for culture time and density to inoculate were calculated,median effective time（ET50）was obtained as the optimal culture time,median effective inoculation density（ED50）was obtained as the optimal density to inoculate.Cell states of different series were observed with cell slide.Results:The ET50 and 95%confidence intervals of culture time were 40h and 14h-110h,respectively,and the equation was Y=0.12+0.80/（1+1010.56-1.60X）,R=0.93.The ED50 and 95%confidence intervals of density to inoculate were 8×104/mL and 5×103/mL-1×106/mL,respectively,and the equation was Y=0.32+0.70/（1+1015.71-4.91x）,R=0.83.Conclusions:The primary spleen-breast cancer cell co-culture model was successfully established,and the optimal co-culture time and density to inoculate were 40h and 8×104/mL,respectively.3.2 To explore the immune metabolism took part in immune escape in vitro co-culture modelObject:Taking glucose metabolism as an example,to explore the vitro co-culture model of immune metabolism taking part in immune escape.Methods:Primary spleen cells and breast cancer cells were separated and extracted from CFA stimulated immunopharmacological model of rat breast cancer,cells were primary cultured,cancer cells group,spleen cells group and cancer-spleen co-culture group were respectively set according to the optimal culture time and density to inoculate.Gradient concentration of 2-deoxy-D-glucose（2-DG）were added into each group to cultivate for 20h,respectively.The LDH and IFN-y level in supernatant were detected at the end of the culture time.Median effective concentration（EC50）was obtained from regression of IFN-y level and logarithmic 2-DG concentration as optimal incubation condition.Results:The EC50 and 95%confidence intervals of 2-DG regulating glucose metabolism to promote immune escape were 50mM and l×10-3mM₂×106mM,respectively,and the equation was Y=0.29+0.57（1+10-2.94+1.75x）,R=0.59.Conclusions:The primary spleen-breast cancer cells co-culture model of immune glucose metabolism taking part in immune escape was explored.