| Lung cancer ranks first in terms of incidence and mortality among malignant tumors in China,yet the direct cause of death for most lung cancer patients is lung cancer co-morbidities,not the cancer itself.Cancer co-comorbidities are usually defined as acute or chronic diseases other than cancer,including cardiovascular disease,liver disease,kidney disease,diabetes,connective tissue disease,neurological disease,and autoimmune disease.More than 90%of lung cancer patients suffer from one or more lung cancer co-morbidities.Cancer co-morbidities not only seriously affect the quality of life but also largely limit the development of cancer treatment strategies.It is believed that the development of cancer co-morbidities may be related to cancerinduced metabolic abnormalities and inflammation,but the molecular mechanisms are unknown and effective therapeutic agents are not available.Immune checkpoint inhibitor(ICI)attenuates suppressive immune checkpoint signaling,leading to the activation of anti-tumor immune responses,and thus offers potent efficacy against a wide range of cancers.Cytotoxic T-lymphocyte-associated protein 4(CTLA4)and/or programmed cell death receptor 1(PD-1)/programmed cell death 1-ligand 1(PD-L1)monoclonal antibodies have been approved for the treatment of a variety of cancers;however,while ICIs have changed the treatment strategy for solid and hematologic cancers,they can also lead to a range of immune-mediated toxicities that are referred to as immune-related adverse events(irAEs).irAEs can lead to treatment discontinuation,permanent tissue and organ damage,and even fatal consequences.irAEs can lead to treatment discontinuation,permanent tissue and organ damage,and even fatal consequences.Given the increasingly widespread use of ICIs,the incidence of irAEs is like further increase,therefore,it is particularly important to investigate the pathogenesis of irAEs in detail.Ginsenoside Rd(G-Rd)is a protopanaxadiol saponin with biological activities on the nervous system,digestive system,endocrine system,cardiovascular system and cancer.Our research group found that G-Rd could inhibit the release of inflammatory mediators from Raw 264.7 cells induced by lipopolysaccharide,and its antiinflammatory activity was comparable to the glucocorticoid dexamethasone.Therefore,it is speculated that G-Rd may be effective in inflammation and immune-related diseases.The liver facilitates the exchange of macromolecules required for blood metabolism,promotes the aggregation of immune cells and cytokine storm in pathological situations,and serves as a target organ for most solid tumor metastases,predicting that the liver may be more susceptible to distal tumors and immune cells.In the current study,we explored the mechanism of Lewis lung cancer(LLC)and/or PD1 monoclonal antibody exacerbated IH in mice based on the immune hepatitis(IH)model induced by concanavalin A(ConA)and explored the interventional effect of GRd,with the aim to provide a theoretical basis for the search of therapeutic targets of lung cancer with immune hepatitis co-morbidity and PD-1 monoclonal antibody hepatotoxicity,and also provide a scientific basis for the development and utilization of innovative G-Rd drugs.Ⅰ.The role and mechanism of Lewis lung cancer aggravated ConA-induced immune hepatitis in miceMethods:(1)10 male C57BL/6 mice were randomly divided into blank control(Ctrl)group and LLC group according to body weight,5 mice in each group.Subcutaneous injection(s.c.)of LLC cells in the LLC group established a mouse LLC transplantation tumor model,while the Ctrl group was left untreated.14 d later,ELISA was performed to detect IL-1β and IFN-β in heart,lung,liver,kidney,gastrocnemius and colon tissues of each group.(2)32 male C57BL/6 mice,of which 16 were s.c.inoculated with LLC cells and the other 16 were left untreated.14 d later the tumor-bearing mice were randomly divided by body weight into LLC group and ConA-induced IH with LLC co-morbidity(LLC+ConA)group,and the uninoculated mice were divided into Ctrl group and ConA group,8 mice in each group.mice in ConA group and LLC+ConA group were tail intravenous injection(i.v.)of 0.075%ConA solution,20 mL/kg,to establish the ConAinduced IH model in mice.Mice in the Ctrl and LLC groups were i.v.an equal volume of saline,after 24 h,serum,plasma and liver were collected from mice in each group.Photographs were taken to observe the gross morphology of liver;liver index was calculated;H&E staining was performed to detect liver histopathology;serum ALT,AST and LDH activity were detected spectrophotometrically;liver tissue IL-1β,IL-18,IFN-β,cGAMP content and serum MPO-DNA level were detected by ELISA;liver tissue CCL2,Cxcll and IL-8 mRNA levels were detected by QPCR;plasma cfDNA levels were detected by fluorescence spectrophotometry;multiplex fluorescence immunohistochemistry for NETs levels;flow cytometry for F4/80+macrophage percentage;transcriptome sequencing for whole gene transcript levels and differential gene kegg signaling pathway enrichment;the expression levels of cGAS,STING,NLRP3,ASC,Casp1 and cleaved Casp1 were detected by Western Blot.(3)24 male C57BL/6 mice,12 of which were s.c.inoculated with LLC cells and the other 12 were left untreated.14 d later the tumor-bearing mice were randomly divided into LLC+ConA and DNase Ⅰ(LLC+ConA+DNase Ⅰ)groups according to body weight,and the uninoculated mice were divided into Ctrl and ConA groups,6 mice in each group.LLC+ConA+DNase Ⅰ group mice were injected intraperitoneally(i.p.)with 1 U/μL DNase I,100μL/each,30 min later ConA,LLC+ConA and LLC+ConA+DNaseⅠ group mice i.v.0.075%ConA solution,20 mL/kg,to establish a ConA-induced IH model in mice,and Ctrl group mice i.v.an equal volume of saline,24 h later serum,plasma and liver were collected from each group of mice.liver index was calculated;H&E staining was performed to detect liver histopathology;serum ALT,AST and LDH activity were detected spectrophotometrically;the percentage of F4/80+macrophages in liver tissue was detected by flow cytometry;plasma cfDNA level was detected by fluorescence spectrophotometry;liver tissue IL-1β,IL-18,IFN-β and serum MPODNA levels by ELISA.(4)24 male C57BL/6 mice were inoculated with LLC cells,and 14 d later were randomly divided by body weight into LLC+ConA group,ConA-induced IH with LLC co-morbidity+control clodronate liposome(LLC+ConA+PBS-lip)group,and ConAinduced IH with LLC co-morbidity+clodronate liposome(LLC+ConA+Clo-lip)group,8 mice in each group.Mice in the LLC+ConA+PBS-lip group i.v.control clodronate liposome suspension,0.2 mL/each,mice in the LLC+ConA+Clo-lip group i.v.clodronate liposome suspension,0.2 mL/each,mice in each group i.v.0.075%ConA solution after 24 h to establish the ConA-induced IH model in mice.and serum,plasma and liver were collected after 24 h.The percentage of F4/80+macrophages in liver tissue was detected by flow cytometry;liver index was calculated;H&E staining was used to detect liver histopathology;serum ALT,AST and LDH activities were detected spectrophotometrically;plasma cfDNA levels were detected by fluorescence spectrophotometry;liver tissue IL-1β,IL-18,IFN-β contents,MPO-DNA levels and serum MPO-DNA levels were detected by ELISA.(5)32 male C57BL/6 mice,16 of which were i.v.adeno-associated virus(AAV)knockdown intrahepatic cGAS and the other 16 were i.v.AAV control vector.3 w later,8 control vector mice and 8 cGAS knockdown mice s.c.were randomly inoculated with LLC according to body weight and divided into ConA-induced IH and LLC comorbidity+control vector(LLC+ConA+Vec.)group and ConA-induced IH-LLC comorbidity+cGAS knockdown(LLC+ConA+cGAS-/-)group,the remaining 8 control vector mice and 8 cGAS knockdown mice were left untreated and divided into ConAinduced IH+control vector(ConA+Vec.)group,ConA-induced IH+cGAS knockdown(ConA+cGAS-/-).After 14 d,mice in each group were treated with i.v.0.075%ConA solution to establish the ConA-induced IH model,and serum,plasma and liver were collected 24 h later.cGAS mRNA levels in liver tissues were detected by QPCR;liver index was calculated;liver histopathology was detected by H&E staining;serum ALT,AST and LDH were detected spectrophotometrically.The levels of IL-1β,IL-18,IFNβ,cGAMP and serum MPO-DNA in liver tissues were measured by ELISA;cGAS,STING,NLRP3,ASC,Casp1 and cleaved Casp1 protein expression levels in liver tissues were measured by Western Blot.Results:(1)Compared with the Ctrl group,the levels of IL-1β and IFN-β in liver tissue and IL-1β in colon tissue were significantly higher in the LLC group(P<0.01),while the levels of inflammatory cytokines in the rest of the tissues did not show significant changes(P>0.05)(2)Compared with the Ctrl group,the liver index of mice in the LLC group were significantly enlarged(P<0.05),liver tissues were not significantly abnormal,liver tissue IL-1β,IFN-β and cGAMP levels were significantly increased(P<0.05 or P<0.001),the percentage of F4/80+macrophages was increased,cGAS,STING,ASC,Casp1 and cleaved Casp1 protein expression were significantly upregulated(P<0.05),serum ALT,AST and LDH activities,MPO-DNA levels,plasma cfDNA fluorescence intensity,liver tissue IL-18 content,CCL2,Cxcll,IL-8 mRNA levels and NLRP3 protein expression were not significantly changed(P>0.05),the liver index of ConA group mice were significantly increased(P<0.001),liver tissue had coagulative necrosis,serum ALT,AST and LDH activities were significantly increased,MPODNA levels were significantly increased(P<0.001),plasma cfDNA fluorescence intensity was significantly enhanced(P<0.001),liver tissue IL-1β,IL-18,IFN-β and cGAMP contents were significantly elevated,CCL2 and IL-8 mRNA levels were significantly elevated(P<0.05,P<0.01 or P<0.001),MPO and H3cit positive expression was increased,F4/80+macrophage percentage was elevated,cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression was significantly upregulated(P<0.05 or P<0.001).In comparison with the ConA group,the liver tissues of mice in the LLC+ConA group had massive inflammatory cell infiltration,extensive hemorrhage and coagulative necrosis,serum ALT,AST and LDH activities,and MPO-DNA levels were significantly increased(P<0.05 or P<0.001),and plasma cfDNA fluorescence intensity was significantly enhanced(P<0.001),liver tissue IL-1β,IL-18,IFN-β and cGAMP levels,CCL2,Cxcll and IL-8 mRNA levels were significantly increased(P<0.05,P<0.01 or P<0.001),MPO and H3cit positive expression was further increased,and the percentage of F4/80+ macrophages was increased.Transcriptome sequencing showed that LLC aggravated ConA induced IH in mice associated with NOD-like receptor signaling pathway and cytoplasmic DNA sensing pathway,and cGAS/STING/NLRP3 inflammasome signaling pathway related protein expression were significantly upregulated(P<0.05 or P<0.001),with no significant change in liver index(P>0.05).(3)Compared with LLC+ConA group,DNase I significantly reduced liver coefficient(P<0.001),improved liver histopathological damage,significantly reduced serum ALT,AST,LDH activity and MPO-DNA levels(P<0.001),significantly attenuated plasma cfDNA fluorescence intensity(P<0.001),significantly reduced liver tissue IL-1β,IL-18 and IFN-β contents in liver tissue(P<0.001).(4)Compared with the LLC+ConA group,the liver index,serum ALT,AST,LDH activity and MPO-DNA levels,plasma cfDNA fluorescence intensity,liver tissue IL1β,IL-18,IFN-β contents and MPO-DNA levels in the LLC+ConA+PBS-lip group mice were not significantly changed(P>0.05),and liver tissue F4/80+macrophage percentage was comparable to LLC+ConA group;compared with LLC+ConA+PBSlip group,Clo-lip reduced the percentage of F4/80+ macrophages in mouse liver tissue,significantly reduced liver index(P<0.05),improved liver histopathological damage,significantly reduced serum ALT,AST and LDH activity(P<0.001),significantly reduced liver tissue IL-1β,IL-18,IFN-β content and MPO-DNA levels(P<0.001),but had no significant effect on plasma cfDNA fluorescence intensity and serum MPODNA levels(P>0.05).(5)Compared with the ConA+Vec.group,the liver index of mice in the ConA+cGAS-/-group were significantly reduced(P<0.01),a small amount of punctate and focal necrosis was seen in liver histopathology,serum ALT and AST activities were significantly reduced(P<0.001),liver tissue cGAS mRNA levels,IL-18 and cGAMP levels were significantly reduced(P<0.05),cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression were significantly downregulated(P<0.01 or P<0.001),but plasma cfDNA fluorescence intensity,serum LDH activity and MPO-DNA level,liver tissue IL-1β and IFN-β content were not significantly changed(P>0.05),and LLC+ConA+Vec.group mice showed significant increase in liver index(P<0.01),large focal necrosis in liver tissue,serum ALT,AST and LDH activity were significantly increased,MPO-DNA level was significantly increased(P<0.001),plasma cfDNA fluorescence intensity was significantly enhanced(P<0.01),liver tissue cGAS mRNA level,IL-1β,IL-18 IFN-β and cGAMP levels were significantly increased(P<0.05,P<0.01 or P<0.001),and cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression were significantly upregulated(P<0.05 or P<0.001);compared with the LLC+ConA+Vec.group,the LLC+ConA+cGAS-/-group mice had significantly reduced liver index(P<0.001),most of the liver tissues had punctate necrosis,serum ALT,AST and LDH activities were significantly reduced(P<0.001),liver tissue cGAS mRNA levels,IL-1β,IL-18,IFN-βand cGAMP contents were significantly reduced(P<0.001),cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression were significantly downregulated(P<0.001),but plasma cfDNA fluorescence intensity and serum MPODNA levels were not significantly changed(P>0.05).Ⅱ.The role and mechanism of anti-PD-1 in aggravating ConA-induced immune hepatitis in miceMethods:(1)32 male C57BL/6 mice were randomly divided into Ctrl group,ConA group,IgG+ConA induced IH(IgG+ConA)group and PD-1 monoclonal antibody+ConA induced IH(anti-PD-1+ConA)group according to body weight,8 mice in each group.anti-PD-1+ConA group i.p.0.05%PD-1 monoclonal antibody,0.2 mL/each,IgG+ConA group i.p.equal volume of IgG control,30 min later,mice in each group except Ctrl group i.v.0.075%ConA solution,20 mL/kg,to establish ConA-induced IH model in mice,Ctrl group i.v.equal volume of saline.24 h later,serum,plasma and liver were collected.Photographs were taken to observe the gross morphology of liver;liver index was calculated;H&E staining was performed to detect liver histopathology;serum ALT,AST and LDH activity were detected spectrophotometrically;liver tissue IL-1β,IL-18,IFN-β,cGAMP content and serum MPO-DNA level were detected by ELISA;liver tissue CCL2,Cxcll and IL-8 mRNA levels were detected by QPCR;fluorescence spectrophotometry for plasma cfDNA fluorescence intensity;multiplex fluorescence immunohistochemistry for liver NETs levels;flow cytometry for liver F4/80+macrophages and PD-1+/F4/80+macrophages percentage;The protein expression levels of PD-1,cGAS,STING,NLRP3,ASC,Casp1 and cleaved Casp1 were detected by Western Blot.(2)24 male C57BL/6 mice were randomly divided into anti-PD-1+ConA group,PD-1 monoclonal antibody+ConA-induced IH+PBS-lip(anti-PD-1+ConA+PBS-lip)group and PD-1 monoclonal antibody+ConA-induced IH+Clo-lip(anti-PD1+ConA+Clo-lip)group according to body weight,8 mice in each group.anti-PD1+ConA+PBS-lip group mice i.v.control clodronate liposome suspension,0.2 mL/each,and LLC+ConA+Clo-lip group mice i.v.clodronate liposome suspension,0.2 mL/each,24 h later mice in each group i.p.0.05%PD-1 monoclonal antibody.After 30 min,each group of mice i.v.0.075%ConA solution was used to establish the ConA-induced IH model in mice,and serum and liver were collected after 24 h.The percentage of F4/80+macrophages in liver tissues was detected by flow cytometry;the liver index was calculated;the liver histopathology was detected by H&E staining;the serum ALT,AST and LDH activities were detected by spectrophotometry;the contents of IL-1β,IL-18,IFN-β and MPO-DNA level in liver tissues were detected by ELISA.(3)32 male C57BL/6 mice,16 of which were i.v.AAV knockdown intrahepatic cGAS and the other 16 were i.v.AAV control vector.3 w later,they were randomly divided by body weight into ConA+Vec.group,ConA+cGAS-/-group,PD-1 monoclonal antibody+ConA-induced IH co-morbidity+control vector(anti-PD1+ConA+Vec.)group and PD-1 monoclonal antibody+ConA induced IH co-morbidity+cGAS knockdown(anti-PD-1+ConA+cGAS-/-)group,8 mice in each group.anti-PD1+ConA+Vec.group and anti-PD-1+ConA+cGAS-/-group mice i.p.0.05%PD-1 monoclonal antibody,30 min later,the mice i.v.0.075%ConA solution in each group was used to establish the ConA-induced IH model in mice,and serum and liver were collected after 24 h.QPCR was performed to detect liver cGAS mRNA levels;liver index was calculated;H&E staining was performed to detect liver histopathology;serum ALT,AST and LDH activities were measured spectrophotometrically;liver tissue IL-1β,IL-18,IFN-β and cGAS were measured by ELISA;cGAS,STING,NLRP3,ASC,Caspl and cleaved Caspl protein expression levels in liver tissue were detected by Western Blot.Results:(1)Compared with the ConA group,liver tissue pitting and focal necrosis,liver index,serum ALT,AST,LDH activity and MPO-DNA levels,plasma cfDNA fluorescence intensity,liver tissue IL-1β,IL-18,IFN-β,cGAMP content,PD-1 protein expression,PD-1+/F4/80+macrophage percentage and cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression were not significantly changed(P>0.05);compared with the IgG+ConA group,PD-1 monoclonal antibody aggravated liver histopathological damage in mice,significantly increased serum ALT,AST and LDH activities(P<0.001),significantly increased liver tissue IL-1β,IL-18,IFN-β and cGAMP levels,CCL2 and Cxcll mRNA levels(P<0.01 or P<0.001),decreased MPO and H3cit positive expression,and significantly increased STING,NLRP3,Casp1 and cleaved Casp1 protein expression(P<0.01 or P<0.001),but had no effect on liver coefficient,plasma cfDNA fluorescence intensity and serum MPO-DNA level,liver tissue IL-8 mRNA level,PD-1 protein expression,PD-1+/F4/80+macrophage percentage and cGAS and ASC protein expression were not significantly increased(P>0.05).(2)Compared with the anti-PD-1+ConA group,there were no significant changes in liver index,serum ALT,AST and LDH activities,liver tissue F4/80+macrophage percentage,MPO-DNA levels,IL-1β,IL-18 and IFN-β contents in the anti-PD1+ConA+PBS-lip group mice(P>0.05);compared with anti-PD-1+ConA+PBS-lip group,Clo-lip significantly reduced liver index(P<0.05),improved liver histopathological damage,significantly reduced serum ALT,AST and LDH activities(P<0.001),and significantly reduced liver tissue IL-1β,IL-18,IFN-β levels and MPODNA levels(P<0.05 or P<0.001)and reduced the percentage of F4/80+macrophages in liver tissues.(3)Compared with the IgG+ConA+Vec.group,a small amount of punctate and focal necrosis was seen in liver tissues of the IgG+ConA+cGAS-/-group mice,and serum ALT,AST and LDH activities were significantly reduced(P<0.001).cGAS mRNA levels,IL-1β,IL-18,IFN-β and cGAMP contents in liver tissues were significantly reduced.cGAS,STING and NLRP3 protein expression were significantly downregulated(P<0.05,P<0.01 or P<0.001),liver index,liver tissue ASC,Caspl and cleaved Caspl protein expression tended to be reduced but not significantly different(P>0.05),and the anti-PD-1+ConA+Vec.group serum ALT,AST and LDH activities were significantly increased(P<0.001),liver tissue IL-1β,IL-18,IFN-β and cGAMP levels were significantly increased,NLRP3,Casp1 and cleaved Casp1 protein expression were significantly upregulated(P<0.05 or P<0.001),but liver coefficients,liver tissue cGAS mRNA levels,the cGAS,STING and ASC protein expression were not significantly changed(P>0.05);compared with the anti-PD-1+ConA+Vec.group,the liver index of mice in the anti-PD-1+ConA+cGAS-/-group was significantly reduced(P<0.05),and serum ALT,AST and LDH activities were significantly reduced(P<0.001),and liver tissue cGAS mRNA levels,IL-1β,IL-18,IFN-β and cGAMP levels were significantly reduced,and cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression was significantly downregulated(P<0.05,P<0.01 or P<0.001).Ⅲ.Study of PD-1 monoclonal antibody aggravated ConA induced immune hepatitis in Lewis lung cancer miceMethods:32 male C57BL/6 mice,16 of which were s.c.inoculated with LLC cells and the other 16 were left untreated.14 d later,the tumor-bearing mice were randomly divided into LLC+ConA group and PD-1 monoclonal antibody+ConA induced IH co-morbidity with LLC(anti-PD-1+LLC+ConA)group according to body weight,and the uninoculated mice were divided into Ctrl and ConA groups,8 mice in each group.Mice in the anti-PD-1+LLC+ConA group were i.p.0.05%PD-1 monoclonal antibody,0.2 mL/each,and after 30 min,mice in each group except the Ctrl group were i.v.0.075%ConA solution,20 mL/kg,to establish a ConA-induced IH model in mice,and the Ctrl group was i.v.an equal volume of saline.serum and liver were collected 24 h later.H&E staining for liver histopathology;spectrophotometry for serum ALT,AST and LDH activity;ELISA for liver tissue IL-1β,IL-18,IFN-β and cGAMP content;Western Blot for liver tissue cGAS,STING,NLRP3,ASC,Casp1 and cleaved Casp1 protein expression levels were detected by Western Blot.Results:Compared with LLC+ConA group,PD-1 monoclonal antibody aggravated liver histopathological damage in mice,significantly increased serum ALT,AST and LDH activities(P<0.05,P<0.01 or P<0.001),significantly increased liver tissue IL-1β,IL18,IFN-β and cGAMP contents,and significantly upregulated STING,NLRP3,Casp1 and cleaved Caspl protein expression(P<0.05,P<0.01 or P<0.001),but had no significant effect on cGAS and ASC protein expression(P>0.05).IV.Exploring the intervention effect of ginsenoside Rd on immune hepatitis mice based on cGAS/STING/NLRP3 inflammasome signaling pathwayMethods.32 male C57BL/6 mice were randomly divided into Ctrl group,ConA group and G-Rd 10 and 20 mg/kg group according to body weight,8 mice in each group.The mice in each group were i.p.G-Rd or equal volume of propylene glycol-saline,20 mL/kg,once/d for 2 d.1 h after the first administration,mice in each group except Ctrl group were i.v.0.075%ConA solution,20 mL/kg,to establish the ConA-induced mouse IH model,and Ctrl group i.v.equal volume of saline.24 h later,serum and liver were collected.H&E staining was performed to detect liver histopathology;serum ALT,AST and LDH activity were detected spectrophotometrically;IL-1β,IL-18,IFN-β and cGAMP were detected by ELISA;cGAS,STING,NLRP3,ASC,Casp1 and cleaved Caspl were detected by Western Blot.Results:Compared with the ConA group,G-Rd 10 and 20 mg/kg could reduce liver histopathological damage,significantly reduced serum ALT,AST and LDH activities in mice(P<0.001),significantly reduced liver tissue IL-1β,IL-18,IFN-β and cGAMP contents(P<0.05 or P<0.001),reduced liver tissue F4/80+macrophage percentage,GRd 20 mg/kg could significantly downregulated liver tissue cGAS,STING,NLRP3,ASC,Casp1 and cleaved Casp1 protein expression(P<0.05,P<0.01 or P<0.001),GRd 10 mg/kg significantly downregulated liver tissue STING protein expression(P<0.01),cGAS,NLRP3,ASC,Casp1 and cleaved Casp1 protein expression tended to decrease,but there was no significant difference(P>0.05).V.Exploring the intervention of ginsenoside Rd on Lewis lung cancer aggravated ConA-induced immune hepatitis in mice based on cGAS/STING/NLRP3 inflammasome signaling pathwayMethods:(1)32 male C57BL/6 mice,of which 24 were s.c.inoculated with LLC cells and the other 8 were left untreated.14 d later the tumor-bearing mice were randomly divided into LLC+ConA group and G-Rd 10 and 20 mg/kg group according to body weight,and uninoculated mice were ConA group,8 mice in each group.Mice in each group i.p.corresponding dose of G-Rd or equal volume of propylene glycol-saline,20 mL/kg,1 time/d,were administered continuously for 2 d.1 h after the first administration mice in each group i.v.0.075%ConA solution,20 mL/kg,to establish the ConA-induced IH model in mice,and serum and liver were collected 24 h later.H&E staining was performed to detect liver histopathology;serum ALT,AST and LDH activity were detected by spectrophotometry;ELISA was performed to detect IL-1β,IL-18,IFN-βand cGAMP contents in liver tissues;Western Blot was performed to detect cGAS,STING,NLRP3,ASC,Caspl and cleaved Caspl protein expression levels in liver tissues.(2)32 male C57BL/6 mice,of which 16 i.v.AAV overexpressed cGAS in liver and the other 16 i.v.AAV control vector.3 w later,the 16 cGAS overexpressing mice were randomly divided into ConA-induced IH with LLC co-morbidity+cGAS overexpression(LLC+ConA+cGASOE)group and ConA-induced IH with LLC comorbidity+G-Rd 20 mg/kg+cGAS overexpression(LLC+ConA+G-Rd+cGASOE)group according to body weight,and 16 control vector mice were randomly divided into ConA-induced IH and LLC co-morbidity+control vector(LLC+ConA+Vec.)group and ConA-induced IH and LLC co-morbidity+G-Rd 20 mg/kg+control vector(LLC+ConA+G-Rd+Vec.)groups according to body weight,8 mice in each group.Mice in each group i.p.20 mg/kg G-Rd or equal volume of propylene glycol-saline,20 mL/kg,1 time/d,were administered continuously for 2 d.1 h after the first administration of each group i.v.0.075%ConA solution was used to establish the ConA-induced IH model in mice,and serum and liver were collected 24 h later.cGAS mRNA levels in liver tissue were detected by QPCR;H&E staining was performed to detect liver histopathology;serum ALT,AST and LDH activity were detected spectrophotometrically;IL-1β,IL-18,IFN-β and cGAMP levels were detected by ELISA;cGAS,STING,NLRP3,ASC,Casp1 and cleaved Casp1 protein expression levels in liver tissues were detected by Western Blot.Results:(1)Compared with LLC+ConA group,G-Rd 10 and 20 mg/kg both improved liver histopathological damage in mice,significantly reduced serum ALT,AST and LDH activities(P<0.001),significantly reduced liver tissue IL-1β,IL-18,IFN-β and cGAMP contents(P<0.001),and significantly downregulated cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression(P<0.05,P<0.01 or P<0.001).(2)Compared with LLC+ConA+Vec.group,mice in LLC+ConA+cGASOE group had large coagulative necrosis in liver tissues,and serum ALT,AST and LDH activities were significantly increased(P<0.001).cGAS mRNA levels,IL-1β,IL-18,IFN-β and cGAMP contents in liver tissues were significantly increased.cGAS,STING,Caspl and cleaved Caspl protein expression were significantly upregulated(P<0.05,P<0.01 or P<0.001),but NLRP3 and ASC protein expression did not change significantly(P>0.05),and liver tissue morphology of mice in LLC+ConA+G-Rd+Vec.group tended to be normal,and serum ALT,AST and LDH activities were significantly reduced(P<0.05 or P<0.001),liver tissue cGAS mRNA levels,IL-1β,IL-18,IFN-βand cGAMP contents were significantly reduced,cGAS,NLRP3,ASC,Casp1 and cleaved Caspl protein expression were significantly downregulated(P<0.05,P<0.01 or P<0.001),and no significant changes in STING protein expression(P>0.05);compared with LLC+ConA+G-Rd+Vec.group,mice in LLC+ConA+G-Rd+cGASOE group had coagulative necrosis,massive inflammatory cell infiltration,significantly increased serum ALT,AST and LDH activities(P<0.001),liver tissue cGAS mRNA levels,IL-1β,IL-18,IFN-β and cGAMP levels were significantly increased(P<0.05,P<0.01 or P<0.001),and cGAS/STING/NLRP3 inflammasome signaling pathwayrelated protein expression were significantly upregulated(P<0.01 or P<0.001).Ⅵ.Exploring the intervention of ginsenoside Rd on PD-1 monoclonal antibody aggravated ConA-induced immune hepatitis in Lewis lung cancer mice based on cGAS/STING/NLRP3 inflammasome signaling pathwayMethods:(1)40 male C57BL/6 mice,of which 32 were s.c.inoculated with LLC cells and the other 8 were left untreated.14 d later the tumor-bearing mice were randomly divided into LLC+ConA group,anti-PD-1+LLC+ConA group and G-Rd 10 and 20 mg/kg group according to body weight,and uninoculated mice were ConA group,8 mice in each group.Mice in each group i.p.corresponding dose of G-Rd or equal volume of saline,20 mL/kg,1 time/d,were administered continuously for 2 d.30 min after the first administration,mice in anti-PD-1+LLC+ConA group and G-Rd 10,20 mg/kg group i.p.0.05%PD-1 monoclonal antibody,0.2 mL/each,30 min after each group i.v.0.075%ConA solution,20 mL/kg,to establish ConA-induced IH model in mice,and serum and liver were collected after 24 h.H&E staining was performed to detect liver histopathology;serum ALT,AST and LDH activities were detected spectrophotometrically;liver tissue IL-1β,IL-18,IFN-β and cGAMP contents were detected by ELISA;Western Blot was performed to detect the expression levels of cGAS,STING,NLRP3,ASC,Caspl and cleaved Caspl in liver tissues.(2)32 male C57BL/6 mice,16 of which i.v.AAV overexpressed cGAS in the liver and the other 16 i.v.AAV control vector.3 w later all mice were inoculated with LLC,and 14 d later the 16 cGAS overexpressing mice were randomly divided by body weight into PD-1 monoclonal antibody+LLC and ConA induced IH co-morbidities+cGAS overexpression(anti-PD-1+LLC+C on A+cGA SOE)and PD-1 monoclonal antibody+ConA induced IH with LLC co-morbidity+cGAS overexpression+G-Rd 20 mg/kg(anti-PD-1+LLC+ConA+G-Rd+cGASOE)groups,and 16 control vector mice were randomly divided by body weight into PD-1 monoclonal antibody+ConA induced IH with LLC co-morbidity+control vector(anti-PD-1+LLC+ConA+Vec.)group and PD-1 monoclonal antibody+ConA induced IH and LLC co-morbidity+control vector+G-Rd 20 mg/kg(anti-PD-1+LLC+ConA+G-Rd+Vec.)group,8 mice in each group.Mice in each group i.p.G-Rd 20 mg/kg or an equal volume of propylene glycolsaline,20 mL/kg,once/d,were administered for 2 d.30 min after the first administration mice in each group i.p.0.05%PD-1 monoclonal antibody,0.2 mL/only,and 30 min later mice in each group i.v.0.075%ConA solution,20 mL/kg,to establish ConA induced IH model in mice,and serum and liver were collected 24 h later.cGAS mRNA levels in liver tissues were detected by QPCR;liver histopathology was detected by H&E staining;serum ALT,AST and LDH activities were detected by spectrophotometry;IL-1β,IL-18,IFN-β and cGAMP contents in liver tissues were detected by ELISA;The expression levels of cGAS,STING,NLRP3,ASC,Casp1 and cleaved Caspl were detected by Western Blot.Results:(1)Compared with the anti-PD-1+LLC+ConA group,G-Rd 10 and 20 mg/kg improved liver histopathological damage in mice,significantly reduced serum ALT,AST and LDH activities(P<0.01 or P<0.001),significantly reduced liver tissue IL-1β,IL-18,IFN-β and cGAMP contents(P<0.001),G-Rd 20 mg/kg significantly downregulated cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression(P<0.05 or P<0.001),G-Rd 10 mg/kg significantly downregulated STING,NLRP3,ASC and cleaved Caspl protein expression(P<0.05,P<0.01 or P<0.001),and could down-regulate cGAS and Casp1 protein expression,but there was no significant difference(P>0.05).(2)Compared with the anti-PD-1+LLC+ConA+Vec.group,mice in the anti-PD1+LLC+ConA+cGASOE group had a larger area of coagulative necrosis in liver tissue,significantly higher serum ALT,AST and LDH activities(P<0.01 or P<0.001),and liver tissue cGAS mRNA levels,IL-1β,IL-18,IFN-β and cGAMP levels were significantly increased(P<0.05,P<0.01 or P<0.001),cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression were significantly upregulated(P<0.05,P<0.01 or P<0.001),anti-PD-1+LLC+ConA+G-Rd+Vec.group mice showed reduced liver histopathological damage,serum ALT,AST and LDH activities were significantly reduced(P<0.01 or P<0.001),liver tissue IL-1β,IL-18,IFN-β and cGAMP contents were significantly reduced(P<0.05 or P<0.001),cGAS,NLRP3 and cleaved Caspl protein expression were significantly down-regulated(P<0.05 or P<0.001),cGAS mRNA levels,STING,ASC and Caspl protein expression had a tendency to be down-regulated,but there was no significant difference(P>0.05);compared with the anti-PD-1+LLC+ConA+G-Rd+Vec.group,the anti-PD1+LLC+ConA+G Rd+cGASOE group mice had a large infiltration of inflammatory cells in liver tissue,and serum ALT,AST and LDH activities were significantly increased(P<0.05,P<0.01 or P<0.001),liver tissue cGAS mRNA levels,IL-1β,IL-18,IFN-β and cGAMP contents were significantly increased,and cGAS/STING/NLRP3 inflammasome signaling pathway-related protein expression were significantly upregulated(P<0.01 or P<0.001).Conclusions:1.LLC promotes increased liver macrophage... |
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