Mechanism Of Emotional Stress Promotes Tumor “Susceptibility” Through Inhibiting Macrophage Function And Protective Effect Of Xiaoyaosan

Background: Chinese medicine considers that the occurrence of tumor is closely related to emotions and the root cause of tumor development is the deficiency of “Qi”.“Qi” is similar to the immunity of body in modern medicine.The relationship between emotional stress and tumorigenesis has been well defined in epidemiological studies,although the mechanism remains elusive.The pivotal role of macrophage phagocytosis in the clearance of tumor cells has been gradually discovered in recent years.It is reported that the expression and function of " eat me"-" don't eat me" signal receptors in macrophages plays a key role in the clearance of tumor cells,while the balance of “eat-me”-“don't eat-me” signal receptors is critical in determining the phagocytic capacity of macrophages.Herein,we proposed that the effect of emotional stress on tumor growth might be related with the dampened macrophage phagocytosis,as a result of the disturbed balance of “eat-me”-“don't eat-me” signal recpetors.The protective effect of Xiaoyaosan,a classic Chinese medicine compound for therapying abnormal emotional activity in clinic,was explained in this pattern.Methods and Results: Firstly,we established the correlation of psychological stress and tumor growth in 4T1-breast cancer bearing mice.In consistent with previous reports,restraint stress was found to dramatically promote tumorigenesis.In the meantime,the level of glucocorticoid(GC),an indicator of the activation of hypothalamic-pituitary-adrenal axis in response to stress,was significantly elevated in the plasma of restraint mice.To determine whether this stress hormone contributes to stress-provoked tumor growth,mice were administered with GC(corticosterone,2mg/kg,s.c.)daily for 28 d and inoculated with 4T1 cells.Results showed that GC treatment significantly elevated tumor weight and tumor volume in mice,in a similar pattern to restraint stress.Strikingly,the growth of tumor was also promoted by GC treatment in T-lymphocyte-deficient nude mice,excluding the involvement of T cells in stress-evoked tumor growth.To date,there has been several evidences to indicate that macrophage phagocytosis is a critical mediator of tumor immunosurveilance.Therefore,we monitored the detrimental effect of GC on inhibited the phagocytosis of macrophages in vivo and in vitro.Results from immunohistochemical staining demonstrated that both restraint stress and GC remarkably inhibited the phagocytosis of macrophage in tumor tissues in mice.Meanwhile,GC also decreased the phagocytosis of tumor cells by THP1-differentiated macrophages and BMDMs in a time-and dose-dependent manner.These results collectively illustrated an essential role of GC-inhibited macrophage phagocytosis in stress-caused tumor growth.Secondly,we sought to characterize the pattern how GC affects the phagocytic capacity of macrophages.Results from public database,KM plotter,showed that LRP1 mRNA level is positively correlated with the survival of breast cancer patients,while SIRP? mRNA level has no significant effect.Besides,results from Western blot and qPCR showed that stress and GC decreased the protein and mRNA levels of LRP1,while increased the protein expression of SIRP? with little mRNA change in macrophages in vivo and in vitro.These finding suggested GC disturbed that balance of LRP1-mediated “eat-me” signal receptor and SIRP?-mediated“don't eat-me” signal receptor.Next,we elucidated the molecular mechanisms of GC dependent-decrease of LRP1 in further detail.In consistent with previous study,GC can activate GR and transport it into cell nuclear in THP1-differentiated macrophages in our study.Results form THP1-differentiated macrophages treated with GC in the presence or absence of the GR antagonist(Ru486)or the protein synthesis inhibitor cycloheximide(CHX)were uncover whether LPR1 is GR-dependent trans-repression.We found that Ru486 completely abrogated LRP1 induction in response to treatment with GC,whereas CHX was not inhibitory.Besides,the decay of LRP1 mRNA in the presence or absence of GC after inhibition of transcription by actinomycin D(Act D)was analyzed to exclude GC modulate LRP1 expression on the post-transcriptional level.As we expected,GC had no significant change on the half-life of LRP1 mRNA.Furthermore,in silico promoter analyses revealed that three possibly n GRE were located at the2 kb region upstream of transcription start site of LRP1.To assess whether GR can directly transcriptionally regulate LRP1,the promoter(?2 kb)of LRP1 was cloned into a luciferase reporter(p GL3 Basic).Result from luciferase activity assay revealed that both GC and overexpression of GR can repress the transcription activity of LRP1.Briefly,these results demonstrated that GC leaded to direct,GR-dependent trans-repression of LRP1 expression.To further understand the relationship between LRP1 and SIRP? disturbed by GC,gain-and loss-of-function experiments was investigated.Similar to GC treatment,the depletion of LRP1 by siRNA in cell increased SIRP? protein level.In contrast,LRP1 overexpression in cell recovered SIRP? protein level.This indicated that LRP1 can regulate the expression of SIRP?protein.It is worthy to note that both GC and LRP1 siRNA significantly down-regulated the expression of miRNA-4695-3p through miRNA transcriptomic.This result was confirmed by q RT-PCR analyses in THP1-differentiated macrophages and Hek293 cell.The similar changes of miRNA-4695-3p were also discovered in tumor tissue of mice treated with restraint stress and GC.The results of miRNA-4695-3p inhibitor and mimic experiments verified that LRP1 regulated the expression of SIRP? protein through miRNA-4695-3p.Finally,the protective effect and mechanism of Xiaoyaosan on tumor growth in stress-mice was evaluated in the model established above.Results showed that Xiaoyaosan can enhance the phagocytosis of macrophages and inhibit the tumor growth in stress-mice by reducing the release of CORT.Further,Xiaoyaosan can increase the expression of miRNA-4695-3p in tumor tissue of stress-mice,and alleviate the disorser of “eat me”-“don't eat me” signal receptors.Conclusion: The present study clarified the role of macrophage phagocytosis in emotional stress-promoted tumor growth.Mechanistically,stress-induced GC inhibits LRP1 expression by GR-dependent transcription.The down-regulated LRP1 in turn lowers SIRP? protein level in a miRNA-4695-3p dependent manner.The disturbance of LRP1-SIRP? axis disrupts“eat-me”-“don't eat-me” signal receptors of macrophages,thereby suppressing the phagocytosis of tumor cells.Xiaoyaosan can alleviate the emotional stress-induced tumor“susceptibility” through the above mechanism.Our findings take deep insights into the mechanism for emotional stress-induced tumorigenesis and provide innovative strategies in preventing and treating cancer.