| BackgroundHeat shcok proteins(HSPs)compose a super-falimy,which includes several subfamiliers of HSPA(50-90kDa),HSPB(10-30kDa),HSPH(90-110kDa)and DNAJ(20-50kDa).Since the initial discocvery in early 1960s,the heat shock respones have been investigated substantially and been demonstrated complex roles in many aspects.Intracellular proteins may misfold,aggragate and even cause cell death when are exposed to stimuli such as heat shock,havey mental,ischemia/hypoxia,infection,odidants,and harmfil chemicals.How cells response to?Haet shok proteins serve as a first-line defence inresponse to these stimuli.Mounting evidence demonstrates that some HSPs such as HPB1(HSP25/27),HSAlA(HSP70/72)protect against ischemic disease,septic/sepsis shock,cardiac hypertrophy and heart failure.However,the extracellular HSPB1 and HSPAlA that released from crells are harmful and serve as independent risk factors of cardiovascular diseases.Moreover,HSP90 has been shown to facilitate tumor growth and macrophage activation.Therefore,HSPs exert heterogeneous function dependent on the the HSP itself,location and pathologic conditions.Ischemic stroke is a leading cause of disability and main cause of mortality,due to neunal death and hyperpermeability at acute phase.Thus,promoting neurogenesis and angiogenesis is the main approaches for the management.Non-alcoholic fatty liver disease(NAFLD)is a high morbidity disease.The"two-hits" therory is recognized as main mechanism for the development of NAFLD.The first hit is metabolic stress that leading to fat accumulation in hepatocytes.The second hit inflammation that not only increase heaptocyte fat accumulation but also cause hepatosyte injury.Evidence reveals that macrophage-mediated inflammatory responses plays critical roles in the NAFLD development,among which,M1 macrophages are pro-inflammatory and M2 macrophages are anti-infammatory.Heat shock protein A12A is a novel member of HSPs family.It was first cloned from Atherosclerotic plaque of aorta by Han et al in 2003.The subsequent studies demonstrated that Hspa12a mRNA was decreased in the brains of schizophrenia humans.A recent study reported that the HSPA12A expression shows a negative correllation with survival time in liver cancer patients.The data suggest a role of HSAP12A in the homeostasis regulation.However,the causal relationship of HSPA12A with any disorder is unknown.ObjectiveIn the present study,we investigated HSPA12A expression profile and the role of HSPA12A in the pathogenesis of ischemic stroke and NAFLD using HSPA12A knockout mice in vivo and cell cultures in vitro.Materials and Methods1?Protein expression profile of HSPA12A1.1 Expressin of HSPA12A protein in mouse tissues The tissues of heart,liver,splees,lung,kidney,brain,pancrea,white fat,skeletal muscles and bone were collected from 8-week old C57BL/6J mice.The expression of HSPA12A was analyzed using Western blot.1.2 Expression of HSPA12A in brain neurons Brain tissues were collected from8-week old,C57BL/6J mice.Cryosections were parepared for co-immunostaining against HSPA12A and neuronal marker ?-Tubulin? or NueN.1.3 Expression of HSPA12A in hepatosytes and hepatic macrophages Hepatocytes.Human normal hepatic tissues(without fibrosis,tumor,infection and fat accumulation)were collected from resection of hepatic hemangioma.After fixed and embedded in OTC,Cryosections were parepared for co-immunostaining against HSPA12A and hepatocyte marker HEP1.Macrophages.All the processes were the same as the above mentioned human hepatocyte staining except for using macrophgae marker CD68 to replace HEP1 as primary antibody.1.4 Relative expression levels between hepatocytes and hepatic macrophaes Primary hepatocytes and macrophages were separated from mouse then Western blot was used to analyse the HSPA12A protein expression in hepatocytes and macrophages.2?Alterations of HSPA12A expression in ischemic brains and NAFLD livers2.1 Alterations of HSPA12A expression in ischemic brains Ischemic troke was induced by middle cerebral artery occlusion(MCAO)for 1 h in 8-10 week old,male C57BL/6J mice.Sham mice served as controls.The successful ischemic stroke was verified using TTC staining 24 h after MCAO.Hspa12a mRNA was analysed using real-time PCR 24 h after MCAO.HSPA12A protein expression was analysed in cytosolic and pellet fractions using Western blot at 0.5,3,9 and 24 h after MCAO.2.2 Alterations of HSPA12A expression in NAFLD livers Five-week old male C57BL/6J mice were fed with high fat diet(HFD)that containing 60%fat for 14 weeks.Chow diet-fed mice served as controls.After then,the following experiments were subsequently performed.(1)Oil Red O staining was performed on hepatic cryosections to indicate fat accomulation.(2)HSPA12A protein expression was analysed in cytosolic and pellet fractions of hepatic tissues using Western blot.3?Effects of HSPA12A deletion on ischemic stroke injury3.1 Deletion of HSPA12A expression in HSPA12A knockout mcie HSPA12A conditional knockout mice were generated using Cre-loxP recombinant system.To delete HSPA12A expression(Hspa12a-/-),the HSPA12A conditional knockout mice were cross bred with EIIa-Cre transgenic mice.The wild type littermates served as controls.3.2 Effects of HSPA12A deletion on the infarct size of stroke mice Twenty-four hours after stroke,barins were collected from WT and Hspa12a-/-mice for TTC staining.The infacrt volume was quantified using a soft-ware(AlphaEaseFC,San Leandro,CA)and expressed as a percentage of total brain volume.3.3 Effects of HSPA12A deletion on the functional recovery after stroke Twenty days after stroke,motor-function was analyzed using open-field tests.Total distance and averaged moving speed was recorded and analyzed within 20 min test using an ANY-maze analysis soft ware.3.4 Effects of HSPA12A deletion on neurogenesis after stroke The first dosage of BrdU(100 ?g/gm)was adminstrated by intraperitoneal injection 1 h after stroke.One injection each day was given to mice for the next 4 days.Twelve hours after the last injection of BrdU,brain tissues for paraffin-embedded sectioning.After dewaxed,rehydrated,and DNA denatured,the sections were incubated with the primary antibodies against BrdU and NeuN followed by incubation with Cy3-and FITC-conjugated secondary antibodies,respectively.The staining was observed.The BrdU-positive cells in the ischaemic hemisphere and hippocampus were counted using computerising software(Cellsens Dimension,Olympus).4?Effects of HSPA12A deletion on NAFLD development and progress4.1 Expression and distribution of HSPA12A in human NAFLD livers Human NAFLD hepatic tissues(obvious fat accumulation,but no fibrosis,tumor,and infection)were collected from resection of hepatic hemangioma in the patients without alcohol addiction histry.The controls were the same as NAFLD sampling except for no obvious fat deposition.Hspa12a mRNA was analysed using real-time PCR.HSPA12A protein expression was analysed in cytosolic and pellet fractions using Western blot.4.2 Correlation beteen serum HSPA12A levels and hepatic function injury in NAFLD patients Serum from NAFLD patients were subjected to ELISA analysis for measuring HSPA 12A protein levels(pg/ml).The correlations beteen serum HSPA12A levels and activities of ALT or AST were calculated.4.3 HSPA12A deletion in livers of HSPA12A knockout mice Liver tissues were collected from adult WT and Hspa12a-/-mice for HSPA12A protein expression analysis using Western blot.4.4 Induction of NAFLD with HFD Four-week old male WT and Hspa12a-/-mice were fed HFD for 14 weeks.Chow diet-fed mice served as controls.4.5 Effects of HSPA12A deletion on liver weight and hepatocyte areas in mice after HFD feeding After HFD experiments,livers were weighed and liver weighte were expressed as the ratio to tibia length.Also,hepatic tissues were prepared for paraffin-sectionning and H&E staining was perfomed on sections for measuring hepatocyte area.4.6 Effects of HSPA12A deletion on HFD-induced liver fat accumulation After HFD experiments,liver fat accumulation was analysed using Oil Red O staining and triglyceride(TAG,mmol/mg tissue)content measuring.4.7 Effects of HSPA12A deletion on HFD-induced liver functional injury After HFD experiments,serum was collected from mice for measurng activities of ALT and AST(IU/L).4.8 Effects of HSPA12A deletion on expression of lipid metabolic genes in HFD-fed mcie After HFD experiments,hepatic tissues were collected from mice for measurng mRNA expression of lipid metabolic genes,including nuclear transcriptional factors(Chrebp,Srebp1c,Ppara,Ppary),lipid synthesis(Gck,L-pk,Acc,Fas,Scd1,Gpat,Dgat2 and Elovl6),lipid storage and transportaiton(Cidea,Plin1,Apob,Hsl and Cgi58).Protein expression of PPAR?,SCD1 was also analysed using Western blot.4.9 Effects of HSPA12A deletion on expression of chemokines and pro-inflammatory cytokines in HFD-fed mice Hepatic tissues were collected from mice after HFD experiments.The mRNA expression of pro-inflammatory cytokines(Il-1?,Il-6 and Tnf?),chemokines(Mcp1)and inflammatory sinaling mediators(Tlr4 and Nfkb)was analysed using real-time PCR.4.10 Effects of HSPA12A deletion on macrophage amount in livers of mice after HFD feeding Hepatic tissues were collected from mice after HFD experiments.Cryosections were parepared for immunostaining agaist macrophage marker F4/80.4.11 Effects of HSPA12A deletion on macrophage polarization in livers of mice after HFD feeding After HFD experiments,hepatic tissues were collected from mice for measurng mRNA expression of M1 macrophage markers(Inos,Tnf?,Mcp1,Ifn?,Il-12,Cd86 and Cd68)and M2 macrophage markers(Cd163,Arg1 and Cd206).Protein expression of CD206,iNOS,TNF?,TLR4 and p-NF?B was also analysed using Western blot.4.12 Effects of hepatocyte HSPA12A on lipid accumulation in hepatocytes(1)Primary hepatocytes were isolated from Hspa12a-/-and WT.Lipid accumulation was induced by OA(200 ?M)incubation for 24 h and indicated by increase of TAG contents.(2)HSPA12A was overexpressed in three types of hepatocytes:primary mouse hepatocyte from WT mice,mise AML-12 cells and human hepatocellular carcinoma cells.After stimulated with OA for 24 h,lipid accumulation in hepatocytes was evaluated by Oil Red O staining and quantification.4.13 Paracrine effects of macrophage HSPA12A on lipid accumulation in hepatocytes(1)Conditioned meduium(CM)were collected from HSPA12A overexpressed or normal expressed mice RAW264.7 macrophages that stimulated with LPS(500ng/ml)for 16 h.(2)CM were applied to AML-12 hepatocytes.After incubation with OA for 24 h,lipid accumulation was analysed wih Oil Red O staining and quantification.4.14 Regution of HSPA12A in macrophage polarization4.14.1 Distribution of HSPA12A in liver macrophages of NAFLD patients Liver cryosections of NAFLD patients were co-immunostained with HSPA12A and macrophage marker CD68.4.14.2 Effects of macrophage polarization on HSPA12A expression and distribution M1 macrophage polarization was induced by LPS stimulation for 16 h in RAW264.7 macrophages.HSPA12A protein expression was analysed in cytosolic and pellet fractions using Western blot.The blots against GAPDH and Histone H3 were used as loading respective controls.Immunostaining for HSPA12A were also performed in cells.4.14.3 Effects of HSPA12A on macrophage polarization HSPA12A-overexpressed or normal-expressed RAW264.7 macrophages were incubated with LPS(500ng/ml)for 16 h.The mRNA expression of M1 macrophage markers(Inos,Tnf?,Mcp1,Il-1?,Ccl3 and Ccl4)was analysed by real-time PCR.Protein expression of iNOS,TNF?,TLR4 and p-NF?B was also analysed using Western blot.4.15 Regulation of nuclear PKM2 in HSPA12A-mediated macrophage activation 4.15.1 PKM2 nuclear translocation in liver macrophages of human and mice NAFLD(1)Cryosections of human NAFLD livers were co-immunostained with PKM2 and macrophage markers CD68.Hoechst 33342 was counterstained nuclei.(2)Cryosections of HFD-induced NAFLD of mice were co-immunostained with PKM2 and macrophage markers F4/80.4.15.2 Effects of HSPA12A knockout on HFD-induced PKM2 nuclear translocation in liver macrophages of mice Cryosections of liver tissues of HFD-fed WT and Hspa12a-/-mice were subjected to co-immunostained with PKM2 and macrophage markers F4/80.Hoechst 33342 was counterstained nuclei.Protein expression was also analysed in cytosolic and pellet fractions using Western blot.4.15.3 Regulation of nuclear PKM2 in HSPA12A-promoted macrophage polarization HSPA12A-overexpressed or normal-expressed RAW264.7 macrophages were incubated with LPS(500ng/ml)for 16 h.PKM2 inhibitor DASA-58 was adminstrated prior to LPS stimulation.The mRNA expression of M1 macrophage markers(Tnf? and Mcp1)was analysed by real-time PCR.4.16 Mediation of nuclear PKM2 in paracrine effects of macrophage HSPA12A on hepatocyte lipid deposition Conditioned medium(CM)were collected from DASA-58 and LPS-treated RAW264.7 macrophages with HSPA12A overexpression.The CM collected from LPS-treated and HSPA12A overexpressed RAW264.7 macrophages served as controls CM.After incubated with CM and LPS for 16 h,AML-12 hepatocytes were collected for analysing lipid accumulation by Oil Red O staining and mRNA expression of Ppar?,Ppar?,Acc,Elovl6,Dgat2 and Hsl.5?Statistic analysis Results are expressed as the meanąstandard deviation.Groups were compared using Student's two-tailed unpaired t-test,one-way analyses of variance(ANOVA)or two-way analysis of variance analysis followed by a Tukey test as a post-hoc test.P<0.05 was considered statistically significant.SPSS 17.0 was used.Results1?Protein expression profile of HSPA12A1.1 Expressin of HSPA12A protein in mouse tissues Immunobloting analysis demonstrated that in adult C57BL/6J mice,HSPA12A expression was highest in brains,followed by kidney,white fat,heart,lung,skeletal muscles and slpeen.The liver,pancrea and bone showed the lowest HSPA12A expreslon.1.2 Expression of HSPA12A in brain neurons Immunostaining demonstrated that in adult C57BL/6J mice brains,HSPA12A protein was presented in the cells positive with neuronal marker ?-Tubulin ? or NueN,suggesting that HSPA12A was expressed abountly in brain neurones.1.3 Expression of HSPA12A in hepatosytes and hepatic macrophages Hepatocytes.Immuno staining demosntrated that in human normal hepatic tissues,HSPA12A signals were hardly detected in the cells positive with hepatocyte marker HEP1,suggeting that HSPA12A was expressied at a very low level in human hepatocytes.Macrophages.Immunostaining demosntrated that in human normal hepatic tissues,HSPA12A signals were strong in the cells positive with macrophage marker CD68,suggeting that HSPA12A was expressed abountly in human liver macrophages.1.4 Relative expression levels between hepatocytes and hepatic macrophaes Immunostaining demonstrated that in mice livers,HSPA12A expresison was increased by about 16 fold in macrophaes compared to hepatocytes(P<0.01).2?Alterations of HSPA12A expression in ischemic brains and NAFLD livers2.1 Alterations of HSPA12A expression in ischemic brains TTC staining indicated that ischemic stroke was induced succefully by MCAO.Comapred to sham mice,ischemic stroke decraesed Hspa12a mRNA expression and HSPA12A protein contents in both cytosolic and pellet fractions(P<0.01 or 0.05).2.2 Alterations of HSPA12A expression in NAFLD livers Oil Red O staining demonstrated that NAFLD was succesfully by HFD feeding.Compared to chow diet controls,HSPA12A protein contents was dcreased in cytosolic but increased in pellet fractions in livers of HFD-fed mice(P<0.01 or 0.05).3?Effects of HSPA12A deletion on ischemic stroke injury3.1 Deletion of HSPA12A expression in HSPA12A knockout mcie HSPA12A protein was absent in brains of Hspa12a-/-mice analysed by Western blot comapred to WT controls,suggesting conditional knockout mice can be used in the following experiments.3.2 HSPA12A deletion enlarged infarct volumemic stroke TTC staining demonstrated a significant enlarged infarct volume in Hspa12a-/-mice comapred WT mice 24 h after stroke(P<0.01).3.3 HSPA12A deletion impaired functional recovery after stroke Open filed tests demonstrated that 20 days after stroke,moving didtance and averaged moving speed was recovered to normal levels in WT mice compared to its sham controls.Importantly,a significant reduced moving didtance and averaged moving speed was detected in in Hspa12a-/-mice comapred WT mice 20 days after stroke(P<0.05).3.4 HSPA12A deletion impaired neurogenesis after stroke Immunostaining against BrdU demonstrated that 5 days after stroke,a significant reduced number of the total cells positive with BrdU and the cells cells double-positive with BrdU and neuronal marker NeuN(P<0.01),suggesting that HSPA 12A deletion impaired neurogenesis after stroke.4?Effects of HSPA12A deletion on NAFLD development and progress4.1 Expression and distribution of HSPA12A in human NAFLD livers Compared to human normal livers,Hspa12a mRNA was incaresed in NAFLD livers.Also,HSPA12A protein expression was decraesed in cytosolic but increased in pellet fractions in NAFLD livres compared to normal livers in humans(P<0.01 or 0.05).4.2 Correlation beteen serum HSPA12A levels and hepatic function injury in NAFLD patients NAFLD patients with abnormal elevated activities of ALT and AST showed higher serum HSPA12A levels(P<0.0.05).4.3 HSPA12A deletion in livers of HSPA12A knockout mice Liver tissues showed absence of HSPA12A protein expression in Hspa12a-/-mice.4.4 HSPA12A deletion attenuated increases of liver weight and hepatocyte areas in mice after HFD feeding HFD for 14 weeks increased liver weight and hepatocyte sreas in WT mice.However,the HFD-induced these increases were attenuated in Hspa12a-/-mice compared to WT mice(P<0.01).4.5 Effects of HSPA12A deletion on HFD-induced liver fat accumulation The HFD-induced liver fat accumulation was attenuated in Hspa1 2a-/-mice compared to WT mice(P<0.01),as indicated by analysis of Oil Red O staining and triglyceride content measuring.4.6 Effects of HSPA12A deletion on HFD-induced liver functional injury HFD significantly increased serum activities of AST and ALT in WT mice while did not change the activitiee in Hspa12a-/-mice(P<0.01).Compared to HFD-fed WT mice,HFD-fed Hspa12a-/-mice also showed significantly lower activities of AST and ALT in serum(P<0.01).4.7 Effects of HSPA12A deletion on expression of lipid metabolic genes in HFD-fed mcie After HFD feeding for 14 weeks,compared to WT mice,Hspa12a-/-mice demonstrated significantly reduced mRNA levels of Chrebp,Srebp1c,Ppar?,Ppar?,Gck,L-pk,Acc,Fas,Scd1,Gpat,Dgat2,Elovl6,Cidea,Plin1,Apob,Hsl and Cgi58.By contrast,Hspa12a-/-mice demonstrated increased Atgl mRNA levels compared to WT mice after HFD feeding.Also,reduced protein expression of PPARy and SCD1 was detected in Hspa12a-/-mice compared to WT mice after HFD feeding(P<0.01 or 0.05).4.8 Effects of HSPA12A deletion on expression of chemokines and pro-inflammatory cytokines in HFD-fed mice After HFD feeding for 14 weeks,compared to WT mice,Hspal2a-/-mice demonstrated significantly reduced mRNA levels of pro-inflammatory cytokines(Il-1?,Il-6 and Tnf?),chemokines(Mcpl)and inflammatory sinaling mediators(Tlr4 and Nfkb)(P<0.01 or 0.05).4.9 Effects of HSPA12A deletion on macrophage amount in livers of mice after HFD feeding After HFD feeding for 14 weeks,compared to WT mice,Hspa12a-/-mice demonstrated significantly reduced macrophage aboundance in livers(P<0.01).4.10 Effects of HSPA12A deletion on macrophage polarization in livers of mice after HFD feeding After HFD feeding for 14 weeks,compared to WT mice,Hspa12a-/-mice demonstrated significant reduced mRNA expression of M1 macrophage markers(Inos,Tnf?,Mcp1,Ifn?,Il-12,Cd86 and Cd68)and increased M2 macrophage markers(Cd163,Arg1 and Cd206).The increased protein expression of CD206 and decreased protein iNOS,TNF?,TLR4 and p-NF?B was also observed in Hspa12a-/mice compared to WT mice after HFD feeding.4.11 Effects of hepatocyte HSPA12A on lipid accumulation in hepatocytes The fat deposition induced by OA in primary hepatocytes was not significantly changed by either HSPA12A knockout or HPSA12A overexpression.The same results were observed in both mice AML-12 cells and human hepatocellular carcinoma cells.4.12 Paracrine effects of macrophage HSPA12A on lipid accumulation in hepatocytes The Conditioned meduium from HSPA12A-overexpressed RAW264.7 macrophages signifcantly promoted the OA-induced fat accumulation in AML-12 hepatocytes(P<0.01).4.13 Regution of HSPA12A in macrophage polarization4.13.1 Distribution of HSPA12A in liver macrophages of NAFLD patients HSPA12A showed significant nuclear translocation in liver macrophages compared to normal controls.4.13.2 Effects of macrophage polarization on HSPA12A expression and distribution Western blot analysis demonstrated a significant upregulated HSPA12A expressionin both cytosolic and pellet fractions of LPS-treated RAW264.7 macrophages,compared to vehicle cells(P<0.01).This result was confirmed by immuno s taining.4.13.3 Effects of HSPA12A on macrophage polarization Compared to LPS-treated RAW264.7 macrophages with normal HSPA12A expression,significant increases of M1 macrophage marker mRNA(Inos,Tnf?,Mcp1,Il-1?,Ccl3 and Ccl4)and proteins(iNOS,TNF?,TLR4 and p-NF?B)were detected in HSPA12A overexpressed,LPS-treated RAW264.7 macrophages(P<0.01 or 0.05).4.14 Regulation of nuclear PKM2 in HSPA12A-mediated macrophage activation4.14.1 PKM2 nuclear translocation in liver macrophages of human and mice NAFLD PKM2 showed significant increases of nuclear translocation in liver macrophgaes of both human and mouse HFD livers(P<0.01).4.14.2 Effects of HSPA12A knockout on HFD-induced PKM2 nuclear translocation in liver macrophages of mice Results of immunostaining showed that after HFD feeding,macrophage nuclear contents of PKM2 were significantly reduced in Hspa1 2a-/-mice livers compared to WT conreols(P<0.01).The immunoblotting analysis demonstrated a lower level of PKM2 in pallet farctions of Hspa12a-/-mice livers compared to WT controls after fed with HFD(P<0.05).4.14.3 Regulation of nuclear PKM2 in HSPA12A-promoted macrophage polarization The results of immunobloting analysis demonstrated that in HSPA12A-overexpressed RAW264.7 macrophages,DASA-58 inhibited the LPS-evoked PKM2 nuclear translocation,when compared to the LPS-treated and HSPA12A-overexpressed RAW264.7 macrophage without DASA-58 treatment.Meanwhile,DASA-58 also abolished the HSPA12A overexpression-induced exaggaration of Tnfa and Mcp1 mRNA expression in LPS-trated macrophages.4.15 Mediation of nuclear PKM2 in paracrine effects of macrophage HSPA12A on hepatocyte lipid deposition Oil Red O staining demonstrated that the conditioned medium collected from DASA-58 and LPS-treated RAW264.7 macrophages with HSPA12A overexpression showed reduced lipid accumulation in OA-treated AML-12 hepatocytes,when compared to OA-treated AML-12 hepatocytes that incubated with condition medium from LPS-treated RAW264.7 macrophages with HSPA12A overexpression that without DASA-58 treatment.The macrophage HSPA12A-induced enhancement of mRNA expression of Ppar?,Ppar?,Acc,Elovl6,Dgat2 and Hsl in OA-treated AML-12 cells was also abolished by DASA-58(P<0.01).Conclusion1?HSPA12A protein was expressed at a high level in brains and a low level in livers.We also observed that HSPA12A showed abount expression in brain neurons and liver macrophages.2?Ischemic stroke redudced HSPA12A expression in brains.HSPA12A knockout exaggarated the ischemic barin injury as indicated by enlarged infarcrt size at acute phase,and also impired functional recovery at chronic phase.The data suggest that HSPA12A is a novel pro-survival gene against ischemic stroke.3?HSPA12A expression was upregulated in NAFLD liver macrophages.HSPA12A knockout attenutaed the HFD-induced NAFLD progression.However,the lipid deposition was not affected by its own HSPA12A,but was regulated by paracrine effects of macrophage HSPA12A.This effects of macrophage HSPA12A was mediated by nuclear PKM2-dependent macrophage M1 polarization. |
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