Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype

Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE^(-/-)mice.However,little is known about the role of lnc_000048 in classically activated macrophage(M1)polarization.In this study,we established THP-1-derived testing state macrophages(M0),M1 macrophages,and alternately activated macrophages(M2).Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages.Flow cytometry was used to detect phenotypic proteins(CD11b,CD38,CD80).We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048.Flow cytometry,western blot,and real-time fluorescence quantitative PCR results showed that down-regulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response,while over-expression of lnc_000048 led to the opposite effect.Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization.Moreover,catRAPID prediction,RNA-pull down,and mass spectrometry were used to identify and screen the protein kinase RNA-activated(PKR),then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR.Immunofluorescence(IF)-RNA fluorescence in situ hybridization(FISH)double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage.We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation,leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression.Taken together,these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke.

Neutrophil peptide 1 accelerates the clearance of degenerative axons during Wallerian degeneration by activating macrophages after peripheral nerve crush injury

Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.

血清HbA1c、VCAM-1、VEGF表达水平与后循环脑梗死的相关性

目的 探讨血清糖化血红蛋白(HbA1c)、血管细胞粘附分子1(VCAM-1)、血管内皮生长因子(VEGF)表达水平与后循环脑梗死的相关性。方法 选取80例后循环脑梗死患者作为观察组,依据椎基底动脉狭窄程度分为轻度组42例、中度组22例、重度组16例,同时选取同期50名健康体检者作为对照组。比较观察组和对照组血清HbA1c、VCAM-1、VEGF表达水平;不同程度后循环脑梗死患者血清HbA1c、VCAM-1、VEGF表达水平;血清HbA1c、VCAM-1、VEGF表达水平与椎基底动脉狭窄程度的相关性分析;血清HbA1c、VCAM-1、VEGF表达水平对脑梗死的诊断价值。结果 观察组血清HbA1c、VCAM-1、VEGF表达水平显著高于对照组,差异有统计学意义(P<0.05)。轻度组、中度组和重度组后循环脑梗死患者血清HbA1c、VCAM-1、VEGF表达水平差异均有统计学意义(P<0.05)。重度组血清HbA1c、VCAM-1、VEGF表达水平显著高于中度组和轻度组,中度组血清HbA1c、VCAM-1、VEGF表达水平显著高于轻度组,差异均有统计学意义(P<0.05)。血清HbA1c、VCAM-1、VEGF表达水平与椎基底动脉狭窄程度均呈正相关(P<0.05)。血清HbA1c、VCAM-1、VEGF水平联合检测椎基底动脉狭窄程度AUC及敏感度均高于单独检测,差异有统计学意义(P<0.05)。结论 血清HbA1c、VCAM-1、VEGF表达水平与后循环脑梗死病情严重程度呈正相关,三者对后循环脑梗死患者具有较好的诊断价值。

子痫前期孕妇血清中激活素A、抑制素A及VCAM-1的表达及与病情严重程度关系

探讨子痫前期(PE)孕妇血清中激活素A、抑制素A及血管细胞黏附因子-1(VCAM-1)的表达及与病情严重程度关系。方法 选取我院收治的PE孕妇为研究对象,分为轻度PE组(30人),严重PE组(15人),对照组为同期正常孕妇(30人)。收集血清后应用酶联吸附免疫法检测血清中激活素A、抑制素A及VCAM-1表达水平,并分析激活素A、抑制素A与VCAM-1表达相关性。结果 3 组孕妇血清中激活素A、抑制素A及VCAM-1的表达水平有显著差异( P<0. 05) 。重度PE组各血清指标水平显著高于轻度PE组和对照组,轻度PE组血清学各指标显著高于对照组(P<0. 05)。重度PE组和轻度PE组孕妇血清激活素 A和抑制素A与VCAM-1表达水平呈正相关 (P<0. 05 ) ,对照组血清各指标表达无相关性( P>0. 05)。结论 PE孕妇血清中激活素A、抑制素A和VCAM-1表达水平显著上调,且与PE严重程度密切相关,PE发生可能与激活素A和抑制素A诱导VCAM-1表达,进而影响内皮功能有关。

Knockout of C6orf120 in Rats Alleviates Concanavalin A-induced Autoimmune Hepatitis by Regulating Macrophage Polarization

Objective The effect of the functionally unknown gene C6orf120 on autoimmune hepatitis was investigated on C6orf120 knockout rats(C6orf120^(-/-))and THP-1 cells.Method Six–eight-week-old C6orf120^(-/-)and wild-type(WT)SD rats were injected with Con A(16 mg/kg),and euthanized after 24 h.The sera,livers,and spleens were collected.THP-1 cells and the recombinant protein(rC6ORF120)were used to explore the mechanism in vitro.The frequency of M1 and M2 macrophages was analyzed using flow cytometry.Western blotting and PCR were used to detect macrophage polarization-associated factors.Results C6orf120 knockout attenuated Con A-induced autoimmune hepatitis.Flow cytometry indicated that the proportion of CD68^(+)CD86^(+)M1 macrophages from the liver and spleen in the C6orf120^(-/-)rats decreased.C6orf120 knockout induced downregulation of CD86 protein and the mRNA levels of related inflammatory factors TNF-α,IL-1β,and IL-6 in the liver.C6orf120 knockout did not affect the polarization of THP-1 cells.However,rC6ORF120 promoted the THP-1 cells toward CD68^(+)CD80^(+)M1 macrophages and inhibited the CD68^(+)CD206^(+)M2 phenotype.Conclusion C6orf120 knockout alleviates Con A-induced autoimmune hepatitis by inhibiting macrophage polarization toward M1 macrophages and reducing the expression of related inflammatory factors in C6orf120^(-/-)rats.

Th17/Treg balance and macrophage polarization ratio in lower extremity arteriosclerosis obliterans

Objective:To explore the balance of peripheral blood T helper 17 cells/regulatory T cell(Th17/Treg)ratio and the polarization ratio of M1 and M2 macrophages in lower extremity arteriosclerosis obliterans(ASO).Methods:A rat model of lower extremity ASO was established,and blood samples from patients with lower extremity ASO before and after surgery were obtained.ELISA was used to detect interleukin 6(IL-6),IL-10,and IL-17.Real-time RCR and Western blot analyses were used to detect Foxp3,IL-6,IL-10,and IL-17 expression.Moreover,flow cytometry was applied to detect the Th17/Treg ratio and M1/M2 ratio.Results:Compared with the control group,the iliac artery wall of ASO rats showed significant hyperplasia,and the concentrations of cholesterol and triglyceride were significantly increased(P<0.01),indicating the successful establishment of ASO.Moreover,the levels of IL-6 and IL-17 in ASO rats were pronouncedly increased(P<0.05),while the IL-10 level was significantly decreased(P<0.05).In addition to increased IL-6 and IL-17 levels,the mRNA and protein levels of Foxp3 and IL-10 in ASO rats were significantly decreased compared with the control group.The Th17/Treg and M1/M2 ratios in the ASO group were markedly increased(P<0.05).These alternations were also observed in ASO patients.After endovascular surgery(such as percutaneous transluminal angioplasty and arterial stenting),all these changes were significantly improved(P<0.05).Conclusions:The Th17/Treg and M1/M2 ratios were significantly increased in ASO,and surgery can effectively improve the balance of Th17/Treg,and reduce the ratio of M1/M2,and the expression of inflammatory factors.

ATAT1 deficiency enhances microglia/macrophage-mediated erythrophagocytosis and hematoma absorption following intracerebral hemorrhage

MIcroglia/macrophage-mediated erythrophagocytosis plays a crucial role in hematoma clearance after intracerebral hemorrhage.Dynamic cytoskeletal changes accompany phagocytosis.However,whether and how these changes are associated with microglia/macrophage-mediated erythrophagocytosis remain unclear.In this study,we investigated the function of acetylatedα-tubulin,a stabilized microtubule form,in microglia/macrophage erythrophagocytosis after intracerebral hemorrhage both in vitro and in vivo.We first assessed the function of acetylatedα-tubulin in erythrophagocytosis using primary DiO GFP-labeled red blood cells co-cultured with the BV2 microglia or RAW264.7 macrophage cell lines.Acetylatedα-tubulin expression was significantly decreased in BV2 and RAW264.7 cells during erythrophagocytosis.Moreover,silencingα-tubulin acetyltransferase 1(ATAT1),a newly discoveredα-tubulin acetyltransferase,decreased Ac-α-tub levels and enhanced the erythrophagocytosis by BV2 and RAW264.7 cells.Consistent with these findings,in ATAT1-/-mice,we observed increased ionized calcium binding adapter molecule 1(Iba1)and Perls-positive microglia/macrophage phagocytes of red blood cells in peri-hematoma and reduced hematoma volume in mice with intracerebral hemorrhage.Additionally,knocking out ATAT1 alleviated neuronal apoptosis and pro-inflammatory cytokines and increased anti-inflammatory cytokines around the hematoma,ultimately improving neurological recovery of mice after intracerebral hemorrhage.These findings suggest that ATAT1 deficiency accelerates erythrophagocytosis by microglia/macrophages and hematoma absorption after intracerebral hemorrhage.These results provide novel insights into the mechanisms of hematoma clearance and suggest ATAT1 as a potential target for the treatment of intracerebral hemorrhage.

Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage

Preclinical and clinical studies have shown that microglia and macrophages participate in a multiphasic brain damage repair process following intracerebral hemorrhage.The E26 transformation-specific sequence-related transcription factor Spi1 regulates microglial/macrophage commitment and maturation.However,the effect of Spi1 on intracerebral hemorrhage remains unclear.In this study,we found that Spi1 may regulate recovery from the neuroinflammation and neurofunctional damage caused by intracerebral hemorrhage by modulating the microglial/macrophage transcriptome.We showed that high Spi1expression in microglia/macrophages after intracerebral hemorrhage is associated with the activation of many pathways that promote phagocytosis,glycolysis,and autophagy,as well as debris clearance and sustained remyelination.Notably,microglia with higher levels of Soil expression were chara cterized by activation of pathways associated with a variety of hemorrhage-related cellular processes,such as complement activation,angiogenesis,and coagulation.In conclusion,our results suggest that Spi1 plays a vital role in the microglial/macrophage inflammatory response following intracerebral hemorrhage.This new insight into the regulation of Spi1 and its target genes may advance our understanding of neuroinflammation in intracerebral hemorrhage and provide therapeutic targets for patients with intracerebral hemorrhage.

Effect of Mnk1 on lipopolysaccharide-induced inflammatory responses in macrophages

Objective:To investigate the effect of mitogen-activated protein kinase interaction serine kinase 1(Mnk1)gene deletion on lipopolysaccharide(LPS)-induced inflammatory response in mouse macrophages(Mφ)and the possible mechanism.Methods:Healthy male wildtype C57BL/6J(WT)and Mnk1 knockout(KO)mice were selected at 8-10 weeks of age and divided into WT+PBS,KO+PBS,WT+LPS and KO+LPS groups,and the serum levels of IL-1βwere measured by ELISA after 24 h intraperitoneal injection of PBS or LPS.The mRNA expression levels of IL-1βand Sprouty2(Spry2)in the spleen Mφwere measured by qRTPCR.Mφwas also extracted from the peritoneal cavity of two strains of mice for in vitro experiments to detect macrophage adhesion function and stimulated with equal volumes of LPS or PBS solution for 24 h,divided into WT+PBS group,KO+PBS group,WT+LPS group and KO+LPS group,and transfected with adenovirus expressing Spry2.qRT-PCR was used to detect the mRNA expression levels of LFA-1α,IL-1β,iNOS,CD206,Arg1 and Spry2 in Mφ.Mnk1,ERK1/2,P-ERK1/2,P-p38,P-JNK and Spry2 protein levels in Mφwere detected by western blot.Results:In the in vivo experiments,the concentration of IL-1βin the serum of the KO+LPS group was more significantly elevated than that of the WT+LPS group in mice injected intraperitoneally with LPS.The expression level of splenic MφIL-1βwas higher and the mRNA expression level of Spry2 was decreased in the KO+LPS group compared to the WT+LPS group.In the in vitro experiments,the mRNA expression levels of IL-1βand iNOS were elevated and those of CD206,Arg1 and Spry2 were decreased in the KO+LPS group compared with the WT+LPS group;the expression of LFA-1αwas not significantly different in the WT+PBS and WT+LPS groups,while the expression level of LFA-1αwas significantly increased in the KO+LPS group compared with the WT+LPS group.The results of the macrophage adhesion function assay showed that the adhesion rate of Mφin the KO group was increased at several time points compared to the WT group.After LPS stimulation,the expression of MφSpry2 decreased in Mnk1 KO group compared to WT group,while the expression of P-ERK1/2 increased compared to WT group.After Mφwas transfected with adenovirus overexpressing Spry2 and stimulated with LPS,MφSpry2 expression increased in the KO+AdSpry2 group and P-ERK1/2 expression decreased significantly compared to KO+AdGFP.Conclusion:Mnk1 knockdown enhances LPS-induced inflammatory responses in macrophages,and the mechanism may be related to the involvement of Spry2,a substrate of Mnk1,in regulating macrophage function.

Role of Cyclin D1b in Inducing Macrophages Toward a Tumor-associated Macrophage-like Phenotype in Murine Breast Cancer

Objective:Tumor-associated macrophages(TAMs)of the M2 phenotype are frequently associated with cancer progression.Invasive cancer cells undergoing epithelial-mesenchymal transition(EMT)have a selective advantage as TAM activators.Cyclin D1b is a highly oncogenic splice variant of cyclin D1.We previously reported that cyclin D1b enhances the invasiveness of breast cancer cells by inducing EMT.However,the role of cyclin D1b in inducing macrophage differentiation toward tumor-associated macrophage-like cells remains unknown.This study aimed to explore the relationship between breast cancer cells overexpressing cyclin Dlb and TAMs.Methods:Mouse breast cancer 4T1 cells were transfected with cyclin D1b variant and co-cultured with macrophage cells in a Transwell coculture system.The expression of characteristic cytokines in differentiated macrophages was detected using qRT-PCR,ELISA and zymography assay.Tumor-associated macrophage distribution in a transplanted tumor was detected by immunofluorescence staining.The proliferation and migration ability of breast cancer cells was detected using the cell counting kit-8(CCK-8)assay,wound healing assay,Transwell invasion assay,and lung metastasis assay.Expression levels of mRNAs were detected by qRT-PCR.Protein expression levels were detected by Western blotting.The integrated analyses of The Cancer Genome Atlas(TCGA)datasets and bioinformatics methods were adopted to discover gene expression,gene coexpression,and overall survival in patients with breast cancer.Results:After co-culture with breast cancer cells overexpressing cyclin D1b,RAW264.7 macrophages were differentiated into an M2 phenotype.Moreover,differentiated M2-like macrophages promoted the proliferation and migration of breast cancer cells in turn.Notably,these macrophages facilitated the migration of breast cancer cells in vivo.Further investigations indicated that differentiated M2-like macrophages induced EMT of breast cancer cells accompanied with upregulation of TGF-β1 and integrinβ3 expression.Conclusion:Breast cancer cells transfected with cyclin D1b can induce the differentiation of macrophages into a tumor-associated macrophage-like phenotype,which promotes tumor metastasis in vitro and in vivo.

Scavenger receptor A-mediated nanoparticles target M1 macrophages for acute liver injury

Acute liver injury(ALI)has an elevated fatality rate due to untimely and ineffective treatment.Although,schisandrin B(SchB)has been extensively used to treat diverse liver diseases,its therapeutic efficacy on ALI was limited due to its high hydrophobicity.Palmitic acid-modified serum albumin(PSA)is not only an effective carrier for hydrophobic drugs,but also has a superb targeting effect via scavenger receptor-A(SR-A)on the M1 macrophages,which are potential therapeutic targets for ALI.Compared with the common macrophage-targeted delivery systems,PSA enables site-specific drug delivery to reduce off-target toxicity.Herein,we prepared SchB-PSA nanoparticles and further assessed their therapeutic effect on ALI.In vitro,compared with human serum albumin encapsulated SchB nanoparticles(SchB-HSA NPs),the SchB-PSA NPs exhibited more potent cytotoxicity on lipopolysaccharide(LPS)stimulated Raw264.7(LAR)cells,and LAR cells took up PSA NPs 8.79 times more than HSA NPs.As expected,the PSA NPs also accumulated more in the liver.Moreover,SchB-PSA NPs dramatically reduced the activation of NF-κB signaling,and significantly relieved inflammatory response and hepatic necrosis.Notably,the high dose of SchB-PSA NPs improved the survival rate in 72 h of ALI mice to 75%.Hence,SchB-PSA NPs are promising to treat ALI.

Exosome-derived miR-146a-5p from decidual macrophages in preeclampsia inhibits the viability and invasive ability of trophoblast cells by targeting HIF1α

Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1αprotein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1αby double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1αin trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1αprotein expression of trophoblast cells.

THE ROLE OF VCAM-1/VLA-4 IN THE ACTIVATION OF ALLOGENIC T CELLS BY MURINE MACROPHAGES

This work was supported by grants from the National Natural Science Foundation of China.No. (39730420). ** To whom requests for reprints should be addressed.This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Vascular cell adhesion molecule 1 (VCAM 1) is a member of immunoglobulin superfamily. The principal ligand for VCAM 1 is integrin α4β1/VLA 4 (very late antigen 4). It was reported that VCAM 1 was expressed on macrophages and dendritic cells, but little is known about its function on these professional antigen pre senting cells (APC). The present study was performed to investigate the expression of VCAM 1 on macrophages and the role of VCAM 1/VLA 4 in the activation of allogenic T cells by murine macrophages. We analyzed VCAM 1 expression on peritoneal macrophages and macrophage cell line J774A.1 by fluorescence activated cell sorting (FACS). Using neutralizing antibodies, we further analyzed the role of VCAM 1/VLA 4 interaction in macrophage and allogenic T cell mixed lymphocyte reaction (MLR). We found that VCAM 1 was consti tutively expressed on macrophages and its expression level was upregulated by soluble tumor associated antigen (freeze thaw lysates of FBL 3 leukemia cells) and TNF α. In MLR assays, we observed that blocking VCAM 1/VLA 4 interaction with anti VCAM 1 or anti VLA 4 mAbs caused significant inhibition of the proliferative response and IL 2 production. These results suggest that VCAM 1on macrophages not only facilitates the cell to cell contact through adhesive interaction but also plays a role in the costimulation of T cells via its interaction with VLA 4 on the T cells.

血红素加氧酶1(HO-1)基因敲除影响小鼠肺脏免疫细胞组成平衡并加重脂多糖诱导的急性肺损伤

目的 评价血红素加氧酶1(HO-1)基因缺失对脂多糖(LPS)诱导急性肺损伤(ALI)小鼠肺脏免疫细胞组成及炎症性损伤的影响。方法 选取C57BL/6野生型(WT)小鼠和同背景HO-1条件敲除(HO-1^(-/-))小鼠,按照随机数字法分为WT对照组、 LPS处理的WT组、 HO-1^(-/-)对照组和LPS处理的HO-1^(-/-)组。LPS处理的WT组和LPS处理的HO-1^(-/-)组分别经尾静脉注射LPS(15 mg/kg)建立ALI模型,WT对照组和HO-1^(-/-)对照组经尾静脉注射同等体积生理盐水。造模12 h后,处死小鼠并收集各组肺组织。HE染色观察肺组织病理变化。PCR检测肺组织肿瘤坏死因子α (TNF-α)、白细胞介素1β (IL-1β)和IL-6 mRNA表达。流式细胞术检测肺组织中性粒细胞(CD45^(+)CD11b^(+)Ly6G^(+)Ly6C^(-))、总单核细胞(CD45^(+)CD11b^(+)Ly6C^(hi))、促炎性单核细胞亚群(CD45^(+)CD11b^(+)Ly6C^(hi)CCR2^(hi))、总巨噬细胞(CD45^(+)CD11b^(+)F4/80^(+))、 M1巨噬细胞亚群(CD45^(+)CD11b^(+)F4/80^(+)CD86^(+))、 M2巨噬细胞亚群(CD45^(+)CD11b^(+)F4/80^(+)CD206^(+))、总T细胞(CD45^(+)CD3^(+))、 CD3^(+)CD4^(+)T细胞亚群、 CD3^(+)CD8^(+) T细胞亚群和髓源性抑制细胞(MDSC, CD45^(+)CD11b^(+)Gr1^(+))百分比。结果 与相应对照组相比,LPS处理的WT和HO-1^(-/-)小鼠,肺组织炎症损伤加重;TNF-α、 IL-1β和IL-6 mRNA水平增加;中性粒细胞、总单核细胞、促炎性单核细胞亚群、 MDSC和总巨噬细胞比例显著增加;CD3^(+)、 CD3^(+)CD4^(+)和CD3^(+)CD8^(+) T细胞比例显著降低。静息状态下,与WT对照组小鼠相比,HO-1^(-/-)对照组小鼠肺脏中性粒细胞、单核细胞、促炎性单核细胞比例增加;CD3^(+)和CD3^(+)CD8^(+) T细胞比例降低。与LPS处理的WT小鼠相比,LPS处理的HO-1^(-/-)小鼠肺组织TNF-α和IL-1β mRNA表达水平更高,总单核细胞、促炎性单核细胞亚群、 M1巨噬细胞和M1/M2比值显著增加;CD3^(+)CD8^(+) T细胞百分比显著降低。结论 HO-1的缺失影响ALI小鼠肺脏免疫系统功能,加重LPS刺激后的炎症性损伤。

sVCAM-1、NT-pro BNP、miR-126-5p在不稳定性心绞痛中的表达及相关性研究

目的探讨可溶性VCAM-1分子(sVCAM-1)、N末端脑钠肽前体(NT-pro BNP)、微小核糖核苷酸126-5p(miR-126-5p)在不稳定性心绞痛中的表达及相关性研究。方法选取2021年3月至2022年3月我院收治的胸痛患者160例,其中不稳定性心绞痛患者90例作为研究组,另外70例为对照组。对比两组临床特征病例特征的相关性;分析对比研究组和对照组sVCAM-1、NT-pro BNP、miR-126-5p的水平;比较sVCAM-1、NT-pro BNP、miR-126-5p在不同冠状病变血管支数与不同Gensini评分的相关性,并采用ROC曲线分析sVCAM-1、NT-pro BNP、miR-126-5p的诊断价值。结果研究组总胆固醇(CHOL)、低密度脂蛋白胆固醇(LDL-C)水平明显高于对照组(P<0.05);研究组sVCAM-1、NT-pro BNP水平均高于对照组,而miR-126-5p的表达低于对照组(P<0.05);三支病变组患者sVCAM-1、NT-pro BNP水平明显高于双支病变组、单支病变组,且双支病变组高于单支病变组,而miR-126-5p表达低于双支病变组、单支病变组,且双支病变组低于单支病变组(P<0.05);高分组sVCAM-1、NT-pro BNP水平明显高于低分组、中分组,且中分组高于低分组,而miR-126-5p表达低于低分组、中分组,且中分组低于低分变组(P<0.05);ROC曲线分析结果显示,sVCAM-1、NT-pro BNP、miR-126-5p联合检测在不稳定型心绞痛具有较高的诊断价值。结论sVCAM-1、NT-pro BNP表达水平与不稳定心绞痛患者病变情况及严重程度呈正相关,而与miR-126-5p的表达水平呈负相关,联合检测具有较高的诊断价值。

抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用

目的探讨敲低长链非编码RNA(lncRNA)牛磺酸上调基因1(TUG1)抑制核苷酸结合寡聚结构域样受体蛋白1(NLRP1)炎症小体在缓解阿尔茨海默病进展中的作用。方法选取9~10周龄遗传背景为C57/BL6的野生型小鼠(WT组,10只)或淀粉样前体蛋白(APP)/早老素1(PS1)转基因小鼠(30只)。APP/PS1转基因小鼠随机分为模型(model)组,模型+敲低lncRNA TUG1组[model+lncRNA TUG1短发夹RNA(shRNA)组]和model+shRNA非靶标(NT)组,每组10只。分别采集12周龄第1天(3月龄)和32周龄第1天(8月龄)小鼠外周血和脑皮质组织,并分离皮质中的原代小胶质细胞和原代星形胶质细胞,每个时间点每组5只小鼠。Real-time PCR分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和巨噬细胞移动抑制因子(MIF)mRNA的水平,以及原代星形胶质细胞中补体蛋白C1r和C1s mRNA的水平。ELISA法测定其外周血浆中MIF含量。对3月龄和8月龄小鼠脑皮质原代小胶质细胞和原代星形胶质细胞共培养。CCK-8法测定上述2种细胞的增殖能力。Western blotting分别测定3月龄和8月龄上述4个分组小鼠脑皮质组织中MIF、白细胞介素1β前体(pro-IL-1β)、凋亡相关斑点样蛋白(ASC)、Caspase-1(p20)、Caspase-1(full)、NLRP1及NLRP3蛋白的表达水平。采用免疫荧光染色法测定8月龄各分组小鼠脑皮质组织中β淀粉样蛋白(Aβ)表达。结果3月龄和8月龄时,与WT组小鼠相比,model组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF相对表达水平显著上调,原代小胶质细胞和原代星形胶质细胞增殖能力增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF的相对表达水平显著降低,原代小胶质细胞和原代星形胶质细胞增殖能力降低(P<0.05)。与WT组相比,model组小鼠外周血浆中MIF含量显著升高;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)、NLRP1以及NLRP3的蛋白表达水平显著升高;Aβ免疫荧光强度明显增强(P<0.05)。与model组相比,model+lncRNA TUG1 shRNA组小鼠外周血浆中MIF含量显著降低;小鼠脑皮质组织中pro-IL-1β、ASC、Caspase-1(p20)、Caspase-1(full)和NLRP1的蛋白表达水平显著降低,Aβ免疫荧光强度明显降低(P<0.05),而NLRP3蛋白质的表达水平无明显变化(P>0.05)。与model组相比,model+shRNA NT组小鼠上述所有检测指标差异均无显著性(P>0.05)。结论APP/PS1转基因小鼠脑皮质组织和原代小胶质细胞中lncRNA TUG1和MIF因子表达上调与脑皮质内NLRP1炎症小体激活成正相关,敲低lncRNA TUG1可缓解阿尔茨海默病的进展。

M1型肿瘤相关巨噬细胞在肝细胞癌组织中浸润的意义

背景与目的:肿瘤相关巨噬细胞(tumor-associated macrophages,TAM)是肿瘤微环境中的主要基质细胞,在肿瘤进展过程中发挥重要作用,本研究旨在探究肝细胞癌(hepatocellular carcinoma,HCC)中M1型TAM浸润的临床意义。方法:收集2012年1月—2020年12月在南通大学附属南通第三医院接受手术的HCC患者石蜡包埋组织样本320例,采用免疫组织化学法检测CD86标记的M1型TAM在HCC组织中分布情况,计算阳性细胞密度,根据细胞密度分组:大于平均密度(29个/mm^(2))判定为高密度组,小于或等于平均密度为低密度组;统计分析M1型TAM密度与HCC临床病理学特征、肿瘤浸润CD8^(+)T淋巴细胞之间的相关性及预后意义;采用免疫组织化学法检测程序性死亡配体-1(programmed death ligand-1,PD-L1)的表达情况,根据CD86、PD-L1细胞密度将病例分4组:CD86^(+)高密度组中PD-L1高密度(CD86^(high)PD-L1^(high))和PD-L1低密度(CD86^(high)PD-L1^(low))组;CD86^(+)低密度组中PD-L1高密度(CD86^(low)PD-L1^(high))和PD-L1低密度(CD86^(low)PDL1^(low))组,分析CD86^(+)M1型TAM密度联合PD-L1表达的预后意义。本研究通过南通大学附属南通第三医院伦理委员会批准(伦理编号:EK2022005)。结果:CD86^(+)M1型TAM主要分布于肿瘤间质中;其高密度率为44.7%(143/320)。CD86^(+)M1型TAM密度与CD8^(+)肿瘤浸润细胞毒性T淋巴细胞密度呈正相关(P<0.001)、与乙型肝炎病毒表面抗原(hepatitis B virus surface antigen,HBsAg)阳性呈负相关(P=0.003),与患者性别、年龄、肝硬化、肿瘤大小、组织学分级、微血管侵犯等临床病理学指标均无明显相关性;CD86^(+)M1型TAM高密度组患者总生存期(overall survival,OS)、无病生存期(disease-free survival,DFS)优于低密度组,差异均有统计学意义(P均<0.001)。多因素Cox比例风险回归模型分析显示,低密度CD86^(+)M1型TAM是评估OS和DFS的独立风险因子(OS:HR=1.468,P=0.022;DFS:HR=2.233,P<0.001)。CD86^(high)PD-L1^(high)组HCC患者OS、DFS差于CD86^(high)PD-L1^(low)组,两者差异有统计学意义(P均<0.05)。CD86^(low)PD-L1^(high)组OS、DFS差于CD86^(low)PD-L1^(low)组,两者OS差异有统计学意义(P<0.05),DFS差异无统计学意义。结论:HCC组织中存在高密度CD86^(+)M1型TAM提示患者预后良好,并且是独立的预后因子。HCC组织表达PD-L1提示肿瘤侵袭性增强,患者预后差。

右美托咪定通过下调Dectin-1表达抑制免疫细胞浸润保护缺血/再灌注损伤的心肌

目的:探索右美托咪定(Dex)保护缺血/再灌注(I/R)诱导的心肌损伤的分子机制。方法:小鼠在体实验分组如下:野生型小鼠对照(Control)组、假手术(Sham)组、野生型小鼠缺血/再灌注模型(WT I/R)组和Dex预处理(WT Dex)组,Dectin-1基因敲除小鼠缺血/再灌注模型(KO I/R)组和Dex预处理(KO Dex)组,每组6只。TTC染色并测定小鼠心脏梗死区域(%),HE染色和病理学分析小鼠心肌损伤情况,ELISA测定小鼠血清TNF-α、IL-6和IL-10的水平,流式细胞术(FCM)计数和分选小鼠心肌浸润M2巨噬细胞和中性粒细胞,qPCR测定上述分选细胞中Dectin-1 mRNA表达情况。结果:TTC结果显示Control组和Sham组小鼠心肌未发生梗死;与WT I/R组相比,WT Dex组、KO I/R组和KO Dex组小鼠心肌梗死体积显著减少(P<0.05)。与KO I/R组相比,KO Dex组小鼠心肌梗死体积也明显减小(P<0.05)。HE染色结果显示WT I/R组小鼠心肌排列杂乱,出现大量断裂心肌纤维,WT Dex组、KO I/R组和KO Dex组小鼠心肌纤维存在少许断裂的情况,结构破坏不显著,心肌排列较为整齐;KO Dex组小鼠心肌损伤程度小于KO I/R组。ELISA结果显示,与Sham组相比,WT I/R组小鼠血清TNF-α和IL-6水平显著升高,IL-10水平显著降低;与WT I/R组相比,WT Dex组和KO I/R组小鼠血清TNF-α和IL-6水平显著降低,IL-10水平显著升高;与KO I/R组相比,KO Dex组小鼠血清TNF-α和IL-6水平显著降低,IL-10水平显著升高(P<0.05)。FCM结果显示,与Sham组相比,WT I/R组小鼠心肌浸润大量M2巨噬细胞和中性粒细胞(P<0.05);与WT I/R组相比,WT Dex组、KO I/R组和KO Dex组小鼠心肌浸润的M2巨噬细胞和中性粒细胞显著降低(P<0.05);KO I/R组和KO Dex组之间差异无统计学意义(P>0.05)。qPCR结果显示,与Sham组相比,WT I/R组小鼠心肌浸润的M2巨噬细胞和中性粒细胞中Dectin-1 mRNA表达量显著上调(P<0.05);与WT I/R组相比,Dex组则显著降低(P<0.05);KO I/R组和KO Dex组小鼠不表达Dectin-1。结论:Dex预处理对I/R损伤心肌的保护机制涉及降低I/R后小鼠心肌浸润的M2巨噬细胞和中性粒细胞数量,这一过程可能是通过抑制Dectin-1的表达来实现的。

补体因子H相关蛋白1促进巨噬细胞分泌肿瘤坏死因子-α调控足细胞增殖和迁移实验研究

目的:探讨补体因子H相关蛋白1(CFHR1)通过巨噬细胞分泌肿瘤坏死因子-α(TNF-α)调控足细胞增殖和迁移的作用。方法:体外培养人单核巨噬细胞和人肾足细胞。巨噬细胞分为对照组和CFHR1干预组,分别进行牛血清白蛋白或CFHR1重组蛋白干预24 h,ELISA法测定上清液TNF-α水平。足细胞分为空白组、TNF-α干预组、对照上清液干预组、CFHR1上清液干预组、CFHR1上清液+TNF-α中和抗体干预组。CCK8法检测各组细胞增殖。Transwell法检测各组细胞迁移。Wb法检测各组细胞中相关蛋白变化。结果:巨噬细胞的CFHR1干预组上清液中TNF-α含量显著增加(P<0.05)。与空白组比较,TNF-α干预组、CFHR1上清液干预组的细胞增殖比率和迁移数量均显著降低(均P<0.05)。与CFHR1上清液干预组比较,CFHR1上清液+TNF-α中和抗体干预组的细胞增殖比率和迁移数量均显著提高(均P<0.05)。与空白组比较,TNF-α干预组、CFHR1上清液干预组的足细胞裂孔膜蛋白(Nephrin)、足突蛋白(Podocin)、纤维状肌动蛋白(F-Actin)、整合素α3β1蛋白(α3β1)表达均显著降低(均P<0.05)。与CFHR1上清液干预组比较,CFHR1上清液+TNF-α中和抗体干预组的Nephrin、Podocin、F-actin、α3β1蛋白表达均显著增多(均P<0.05)。结论:CFHR1促进巨噬细胞分泌的TNF-α可显著抑制足细胞增殖水平和迁移能力,这可能是高浓度CFHR1促进肾病综合征发展的途径。