ObjectiveTo explore the host immunomodulatory effect and its molecular mechanism of gallic acid （GA） in Bacillus Calmette-Guérin （BCG） infected macrophages， and to provide experimental evidence for host-directed anti-tuberculosis research against Mycobacterium tuberculosis （Mtb）. MethodsThe minimum inhibitory concentration （MIC） of GA against BCG was determined by resazurin colorimetric assay. Bacterial growth-curve test and MTT colorimetric assay were used to evaluate the effects of GA on BCG proliferation and invasion. A BCG-infected RAW264.7 macrophage model was established， including infection control group （Ctrl）， isoniazid （INH） group， and low-， medium- and high-dose GA groups. Flow cytometry combined with colony-plate counting was performed to detect intracellular BCG survival. Cell apoptosis was measured by Annexin V-APC/7-AAD double staining， and qRT-PCR was used to detect the mRNA expression of apoptosis-related genes Caspase-3 and Bcl-2. Macrophage polarization phenotypes were analyzed by flow cytometry. Intracellular reactive oxygen species （ROS） were detected using DHE fluorescent probe. The Griess assay was applied to determine supernatant nitric oxide （NO） concentration. ELISA was used to detect the levels of TNF-α， IFN-γ， IL-6 and IL-10 in cell culture supernatant. ResultsThe MIC of GA against BCG was 1 250 μmol/L. GA inhibited BCG proliferation and invasion in a concentration-dependent manner （P＜0.05）. Safe concentrations of 20， 40 and 80 μmol/L were selected for subsequent cellular experiments. Thirty-six-hour GA pretreatment markedly reduced the intracellular BCG bacterial load （P＜0.05）. GA exerted bidirectional regulation on macrophage apoptosis： it inhibited apoptosis at the early-stage infection and promoted apoptosis at the late-stage infection. GA induced M1-type polarization in early infection and facilitated M2-type polarization in late infection （P＜0.05）. Meanwhile， GA decreased intracellular ROS and NO levels， down-regulated the secretion of pro-inflammatory cytokines and up-regulated the secretion of anti-inflammatory cytokine IL-10 （P＜0.05）. ConclusionGA inhibits BCG proliferation and invasion in vitro. It remodels host immune microenvironment via bidirectional regulation of macrophage apoptosis and polarization as well as anti-oxidative and anti-inflammatory effects， showing potential value for host-directed anti-mycobacterial therapy.

ObjectiveTo explore the host immunomodulatory effect and its molecular mechanism of gallic acid （GA） in Bacillus Calmette-Guérin （BCG） infected macrophages， and to provide experimental evidence for host-directed anti-tuberculosis research against Mycobacterium tuberculosis （Mtb）. MethodsThe minimum inhibitory concentration （MIC） of GA against BCG was determined by resazurin colorimetric assay. Bacterial growth-curve test and MTT colorimetric assay were used to evaluate the effects of GA on BCG proliferation and invasion. A BCG-infected RAW264.7 macrophage model was established， including infection control group （Ctrl）， isoniazid （INH） group， and low-， medium- and high-dose GA groups. Flow cytometry combined with colony-plate counting was performed to detect intracellular BCG survival. Cell apoptosis was measured by Annexin V-APC/7-AAD double staining， and qRT-PCR was used to detect the mRNA expression of apoptosis-related genes Caspase-3 and Bcl-2. Macrophage polarization phenotypes were analyzed by flow cytometry. Intracellular reactive oxygen species （ROS） were detected using DHE fluorescent probe. The Griess assay was applied to determine supernatant nitric oxide （NO） concentration. ELISA was used to detect the levels of TNF-α， IFN-γ， IL-6 and IL-10 in cell culture supernatant. ResultsThe MIC of GA against BCG was 1 250 μmol/L. GA inhibited BCG proliferation and invasion in a concentration-dependent manner （P＜0.05）. Safe concentrations of 20， 40 and 80 μmol/L were selected for subsequent cellular experiments. Thirty-six-hour GA pretreatment markedly reduced the intracellular BCG bacterial load （P＜0.05）. GA exerted bidirectional regulation on macrophage apoptosis： it inhibited apoptosis at the early-stage infection and promoted apoptosis at the late-stage infection. GA induced M1-type polarization in early infection and facilitated M2-type polarization in late infection （P＜0.05）. Meanwhile， GA decreased intracellular ROS and NO levels， down-regulated the secretion of pro-inflammatory cytokines and up-regulated the secretion of anti-inflammatory cytokine IL-10 （P＜0.05）. ConclusionGA inhibits BCG proliferation and invasion in vitro. It remodels host immune microenvironment via bidirectional regulation of macrophage apoptosis and polarization as well as anti-oxidative and anti-inflammatory effects， showing potential value for host-directed anti-mycobacterial therapy.

Objective : To investigate the relationship between the risk occurrence of rectal cancer and the single nucleotide polymorphisms(SNP) of zinc finger protein 6(ZBED6), and to provide the experimental basis for early diagnosis and treatment of rectal cancer. Methods: Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technology was used to genotype the ZBED6 gene in 109 randomly selected rectal cancer patients and 110 unrelated healthy controls. To evaluate the relationship between alleles, genotypes and the risk of rectal cancer, unconditional Logistic regression analysis was used toORand 95%CI. Results: The SNP rs7552670 of ZBED6 had significant correlation with the risk of rectal cancer. Compared with the population carrying the TT genotype, those carrying the TC genotype had a 2.653 fold increased risk of rectal cancer(TTvsTC:OR=2.635, 95%CI=1.501-4.690). Other SNPs had no significant correlation with the risk of rectal cancer. Conclusion There is an interaction between the polymorphisms of ZBED6(rs7552670) and rectal cancer. The population carried ZBED6 rs7552670 TC genotype had an growing risk of rectal cancer.

[Objective]To explore the origin and development relationship,core pathogenesis and medication characteristics of Wendan Decoction,and clarify its application indications.[Methods]Taking Wendan Decoction recorded in YAO Sengyuan's Set of Proven Prescriptions as the main research object,this paper adopts the method of literature search to collect and sort out medical literature related to"insomnia"during the Wei,Jin,Southern and Northern Dynasties,and makes a comparative study of the prescriptions contained therein.It traces the historical origin and the evolution of the main indications of Wendan Decoction,examines contemporary literature related to"gallbladder cold"to explore the theoretical basis of insomnia caused by gallbladder cold,and compares the recorded efficacy of the component herbs in classical materia medica to further analyze its pathological state and core pathogenesis.[Results]Wendan Decoction originated from Banxia Shumi Decoction and was related to many classical prescriptions such as Banxia Shumi Decoction,ZHANG Zhongjing's"Xiaobanxia Decoction"and Shenshi's"Dajupi Decoction".The treatment object is the combined disease of gallbladder and stomach,which is caused by the weakness of Zangfu organs after a serious disease,and the core pathogenesis is"deficiency of cold in the middle-Jiao and the inversion of Jue Qi",and the main symptom is emesis.The prescription principle of Wendan Decoction emphasizes"warming the middle and reducing inversion,regulating Qi and relieving vexation,harmonizing Ying and Wei".The medication characteristics reflect"the combination of mild reduction and warming and clearing".The application indications include nausea,dizziness,chest fullness,irritability and other manifestations of syncope and Jue Qi inversion.[Conclusion]Wendan Decoction has multiple sources,and has good clinical effect in the treatment of"insomnia"disease with the core pathogenesis of combined disease of gallbladder and stomach.The medication has the characteristics of warming and descending,which is worth further investigation for the treatment of syndromes marked by Jue Qi inversion as the core manifestation.

AIM:This study aims to investigate the regulatory effects of caffeic acid phenethyl ester(CAPE)on metabotropic glutamate receptor 5(mGluR5)and tyrosine kinase Fyn,and to explore its role in alleviating neuroinflam-mation and pathological features of Alzheimer disease(AD).METHODS:In vitro,the murine neuroblastoma N2a cell line was treated with amyloid β-protein 42 oligomers(Aβ42Os;10 nmol/L to 10 μmol/L)for 24 h.Cell viability was as-sessed by MTT assay.Western blot analyzed mGluR5 expression and Fyn phosphorylation(Tyr416).Pharmacological modulators(CHPG/MPEP)were used to evaluate mGluR5-mediated inflammatory cytokine regulation(qPCR)and Fyn ac-tivation.In vivo,wild-type(WT)and 5×FAD mice(WT,WT+CAPE,5×FAD and 5×FAD+CAPE)were analyzed for AD-related proteins,neuroinflammation(ELISA),glial activation(GFAP/Iba-1 immunofluorescence),and β-amyloid deposi-tion(thioflavin S).RESULTS:(1)Treatment with 1 μmol/L Aβ42Os increased mGluR5 expression(P<0.01)and Fyn phosphorylation(P<0.01)without affecting N2a cell viability.Intracerebral Aβ42Os injection similarly up-regulated hip-pocampal mGluR5 and Fyn(P<0.01).(2)MPEP reduced mGluR5 expression(P<0.01)and Fyn phosphorylation(P<0.01),while suppressing tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)mRNA levels(P<0.01).(3)CAPE decreased mGluR5-Fyn activation in N2a cells,neurons,and 5×FAD mice(P<0.01).(4)CAPE-treated 5×FAD mice exhibited reduced neuroinflammation markers(GFAP,Iba-1,TNF-α,IL-1β,and IL-6),Aβ plaques,and p-APP levels(P<0.01).CONCLUSION:Treatment with CAPE inhibits Aβ42Os-induced mGluR5-Fyn signaling activation,thereby attenuating neuroinflammation and the pathology associated with AD.

Objective·To Investigate the effects of salidroside(SAL)on the immune response of macrophages infected with BCG.Methods·RAW264.7 mouse macrophages were infected with BCG,and the experiments were divided into blank control(BCG),salidroside(SAL+BCG),isoniazid(INH+BCG),and salidroside+isoniazid(SAL+INH+BCG)groups.The effects of SAL and INH on the proliferation of RAW264.7 cells were detected by MTT colorimetric assay,and the experimental concentrations of SAL and INH were screened.After the establishment of the BCG-infected macrophage model,intracellular bacterial survival was detected by bacterial plate count and flow cytometry following salidroside pretreatment.The levels of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin-6(IL-6),and interleukin-10(IL-10)in the culture supernatants were measured by enzyme-linked immunosorbent assay,and apoptosis levels were assessed by flow cytometry.Results·SAL at a concentration of 800 μmol/L and INH at 10 μmol/L were selected for subsequent experiments.Compared with the BCG group,SAL pretreatment for 36 h had the best effect,and the growth of BCG in macrophages in the administered group was inhibited.The inhibitory effect of the combined application of SAL and INH was obvious and statistically significant.Compared with the BCG group,SAL alone led to decreased secretion of TNF-α,IFN-γ,IL-6,and IL-10,although the differences were not statistically significant.However,the combination of SAL and INH caused a significant decrease in the levels of TNF-α,IL-6 and IL-10 in the supernatants of the cells(all P<0.05).Compared with the BCG group,in the early stage of the infection,SAL and INH were able to significantly reduce the apoptotic levels of BCG-infected RAW264.7 cells(P=0.008,P=0.032),and the combined application of SAL and INH significantly enhanced the apoptosis levels of BCG-infected RAW264.7 cells in the late stage of infection(P=0.001).Conclusion·In BCG-infected macrophages,SAL can exhibit antimycobacterial effects on the host by enhancing macrophage immune function,suppressing intracellular bacterial survival,and concurrently mitigating the inflammatory response elicited by BCG infection.

AIM:This study aims to investigate the regulatory effects of caffeic acid phenethyl ester(CAPE)on metabotropic glutamate receptor 5(mGluR5)and tyrosine kinase Fyn,and to explore its role in alleviating neuroinflam-mation and pathological features of Alzheimer disease(AD).METHODS:In vitro,the murine neuroblastoma N2a cell line was treated with amyloid β-protein 42 oligomers(Aβ42Os;10 nmol/L to 10 μmol/L)for 24 h.Cell viability was as-sessed by MTT assay.Western blot analyzed mGluR5 expression and Fyn phosphorylation(Tyr416).Pharmacological modulators(CHPG/MPEP)were used to evaluate mGluR5-mediated inflammatory cytokine regulation(qPCR)and Fyn ac-tivation.In vivo,wild-type(WT)and 5×FAD mice(WT,WT+CAPE,5×FAD and 5×FAD+CAPE)were analyzed for AD-related proteins,neuroinflammation(ELISA),glial activation(GFAP/Iba-1 immunofluorescence),and β-amyloid deposi-tion(thioflavin S).RESULTS:(1)Treatment with 1 μmol/L Aβ42Os increased mGluR5 expression(P<0.01)and Fyn phosphorylation(P<0.01)without affecting N2a cell viability.Intracerebral Aβ42Os injection similarly up-regulated hip-pocampal mGluR5 and Fyn(P<0.01).(2)MPEP reduced mGluR5 expression(P<0.01)and Fyn phosphorylation(P<0.01),while suppressing tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)mRNA levels(P<0.01).(3)CAPE decreased mGluR5-Fyn activation in N2a cells,neurons,and 5×FAD mice(P<0.01).(4)CAPE-treated 5×FAD mice exhibited reduced neuroinflammation markers(GFAP,Iba-1,TNF-α,IL-1β,and IL-6),Aβ plaques,and p-APP levels(P<0.01).CONCLUSION:Treatment with CAPE inhibits Aβ42Os-induced mGluR5-Fyn signaling activation,thereby attenuating neuroinflammation and the pathology associated with AD.

Objective·To Investigate the effects of salidroside(SAL)on the immune response of macrophages infected with BCG.Methods·RAW264.7 mouse macrophages were infected with BCG,and the experiments were divided into blank control(BCG),salidroside(SAL+BCG),isoniazid(INH+BCG),and salidroside+isoniazid(SAL+INH+BCG)groups.The effects of SAL and INH on the proliferation of RAW264.7 cells were detected by MTT colorimetric assay,and the experimental concentrations of SAL and INH were screened.After the establishment of the BCG-infected macrophage model,intracellular bacterial survival was detected by bacterial plate count and flow cytometry following salidroside pretreatment.The levels of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin-6(IL-6),and interleukin-10(IL-10)in the culture supernatants were measured by enzyme-linked immunosorbent assay,and apoptosis levels were assessed by flow cytometry.Results·SAL at a concentration of 800 μmol/L and INH at 10 μmol/L were selected for subsequent experiments.Compared with the BCG group,SAL pretreatment for 36 h had the best effect,and the growth of BCG in macrophages in the administered group was inhibited.The inhibitory effect of the combined application of SAL and INH was obvious and statistically significant.Compared with the BCG group,SAL alone led to decreased secretion of TNF-α,IFN-γ,IL-6,and IL-10,although the differences were not statistically significant.However,the combination of SAL and INH caused a significant decrease in the levels of TNF-α,IL-6 and IL-10 in the supernatants of the cells(all P<0.05).Compared with the BCG group,in the early stage of the infection,SAL and INH were able to significantly reduce the apoptotic levels of BCG-infected RAW264.7 cells(P=0.008,P=0.032),and the combined application of SAL and INH significantly enhanced the apoptosis levels of BCG-infected RAW264.7 cells in the late stage of infection(P=0.001).Conclusion·In BCG-infected macrophages,SAL can exhibit antimycobacterial effects on the host by enhancing macrophage immune function,suppressing intracellular bacterial survival,and concurrently mitigating the inflammatory response elicited by BCG infection.

[Objective]To explore the origin and development relationship,core pathogenesis and medication characteristics of Wendan Decoction,and clarify its application indications.[Methods]Taking Wendan Decoction recorded in YAO Sengyuan's Set of Proven Prescriptions as the main research object,this paper adopts the method of literature search to collect and sort out medical literature related to"insomnia"during the Wei,Jin,Southern and Northern Dynasties,and makes a comparative study of the prescriptions contained therein.It traces the historical origin and the evolution of the main indications of Wendan Decoction,examines contemporary literature related to"gallbladder cold"to explore the theoretical basis of insomnia caused by gallbladder cold,and compares the recorded efficacy of the component herbs in classical materia medica to further analyze its pathological state and core pathogenesis.[Results]Wendan Decoction originated from Banxia Shumi Decoction and was related to many classical prescriptions such as Banxia Shumi Decoction,ZHANG Zhongjing's"Xiaobanxia Decoction"and Shenshi's"Dajupi Decoction".The treatment object is the combined disease of gallbladder and stomach,which is caused by the weakness of Zangfu organs after a serious disease,and the core pathogenesis is"deficiency of cold in the middle-Jiao and the inversion of Jue Qi",and the main symptom is emesis.The prescription principle of Wendan Decoction emphasizes"warming the middle and reducing inversion,regulating Qi and relieving vexation,harmonizing Ying and Wei".The medication characteristics reflect"the combination of mild reduction and warming and clearing".The application indications include nausea,dizziness,chest fullness,irritability and other manifestations of syncope and Jue Qi inversion.[Conclusion]Wendan Decoction has multiple sources,and has good clinical effect in the treatment of"insomnia"disease with the core pathogenesis of combined disease of gallbladder and stomach.The medication has the characteristics of warming and descending,which is worth further investigation for the treatment of syndromes marked by Jue Qi inversion as the core manifestation.

Purpose To explore the clinicopathological features,diagnosis and differential diagnosis of large B-cell lym-phoma with IRF4 rearrangement(LBCL-IRF4r).Methods Clinical data of 8 cases of LBCL-IRF4r were collect,hematoxy-lin-eosin and immunohistochemical of EnVision two-step stains,in situ hybridization and FISH was used to study the histology,immunotypes and molecular genetic characteristics.The rele-vant literatures were reviewed.Results Among 8 cases of LBCL-IRF4r,the male to female ratio was 1.67:1,with age range 10-53 years(mean 25.8 years).Five cases occurred in tonsils,2 cases in nasopharynx and 1 cases in inguinal lymph node.Microscopically,the tumors presented with a purely follic-ular,purely diffuse or a combined follicular and diffuse architec-ture.The tumor cells were typical centroblasts and less frequent-ly medium-sized blastic cells with smaller nucleoli,apoptosis and nuclear fragmentation were easily seen.Immunophenotypi-cally,the tumor cells of the eight cases of LBCL-IRF4r diffuse strongly expressed CD20(8/8),PAX5(2/2),CD79a(3/3),BCL6(8/8)and MUM-1(8/8),mostly expressed CD10(7/8),partially expressed BCL2(5/8)and CD5(4/8),and did not express Cyclin D1,CD23 and CD30.The percentage of Ki67 index ranged from 70％to 95％.EBER in situ hybridiza-tion was negative in all cases.IRF4 rearrangements were detec-ted in all cases(8/8).BCL6 rearrangements were detected in one case(1/2).MYC(0/4)and BCL2(0/3)rearrangements were not detected in all cases.Conclusion LBCL-IRF4r is more common in children and adolescents with characteristic IG::IRF4 rearrangement and a good prognosis,which needs to be differentiated from other types of large B-cell lymphoma.