Investigation on the mechanism of Qiangxinhuoli prescription in the treatment of chronic heart failure based on p38-MAPK signaling pathway

Background:The aim of this study is to investigate the mechanism of action underlying the therapeutic effects of the national patent Chinese medicine compound“Qiangxinhuoli prescription(QXHLF)”on chronic heart failure(CHF).Methods:In vitro,the H_(9)C_(2) cell model was induced by ANGII,and cell proliferation and related protein expression were detected by Cell Counting Kit-8 and Western blot.In vivo,A rat model of CHF was prepared by ligation of the left anterior descending coronary artery.The effects of QXHLF on cardiac function in CHF rats were evaluated by cardiac index,hemodynamic changes,enzyme-linked immunosorbent assay,hematoxylin-eosin staining,immunohistochemistry,Western blot and RT-PCR.The expression of pro-apoptotic factors and anti-apoptotic factors,as well as TGFβ1,p-p38,TAK 1 mRNA,and protein,were detected.Results:In vitro,QXHLF has a significant inhibitory effect on the proliferation of H_(9)C_(2) cells.QXHLF can reduce the expression levels of TAK 1,TGFβ1,p-p38,Caspase3 and BAX proteins in H_(9)C_(2) cells,and increase the expression level of BCL_(2) protein.In vivo,QXHLF has the potential to increase left ventricular systolic pressure,m aximum rate of change in left ventricular pressure while decreasing left ventricular end diastolic pressure,and inhibiting the serum levels of brain natriuretic peptide.Moreover,QXHLF exhibits significant improvements in the pathological alterations of myocardial cells and fibers in CHF rats,leading to enhanced myocardial tissue morphology and notable advantages in combating myocardial fibrosis.QXHLF can reduce the levels of BAX and Caspase3 and up-regulate the expression of BCL_(2),thereby inhibiting cardiomyocyte apoptosis.Furthermore,QXHLF demonstrates inhibitory effects on the mRNA and protein expression levels of TGFβ_(1),TAK_(1),and p-p38 in the heart tissue of the CHF rat model.Conclusion:These findings indicate that QXHLF has a therapeutic effect on CHF by inhibiting the p38-MAPK signaling pathway,reducing myocardial fibrosis,preventing apoptosis,inhibiting cell proliferation,and restoring myocardial injury.

黑逍遥散调控p38MAPK/ATF2/COX-2信号通路对阿尔茨海默病大鼠神经炎症的影响

目的 观察黑逍遥散对Aβ_(1-42)所致阿尔茨海默病(AD)大鼠学习记忆能力的影响,并从p38MAPK/ATF2/COX-2信号通路介导的炎症级联反应探讨其作用机制。方法 双侧海马注射1μL Aβ_(1-42)溶液复刻AD大鼠模型。筛选造模成功的大鼠50只,随机分为模型组、盐酸多奈哌齐组(0.5 mg/kg)和黑逍遥散低、中、高剂量组(4.25、8.5、17 g/kg),连续给药42 d。Morris水迷宫实验检测定位航行与空间探索能力,HE染色观察海马神经元病理结构改变,ELISA法检测海马组织Aβ_(1-42)、iNOS、PGE_(2)表达,RT-qPCR法检测海马组织p38、ATF2、COX-2 mRNA表达,Western blot法检测海马组织p-p38、p-ATF2、COX-2蛋白表达。结果 与模型组比较,黑逍遥散中、高剂量组和盐酸多奈哌齐组逃避潜伏期缩短(P<0.01),有效区域运动距离、目标象限滞留时间百分比增加(P<0.01),海马CA1区神经元排列有序,胞体清晰,凋亡细胞减少,海马组织Aβ_(1-42)、iNOS、PGE_(2)水平降低(P<0.05,P<0.01),p38、COX-2 mRNA表达降低(P<0.05,P<0.01),p38、p-p38、p-ATF2、COX-2蛋白表达降低(P<0.01)。结论 黑逍遥散可改善Aβ_(1-42)所致AD大鼠的认知能力,其机制可能与阻断p38MAPK/ATF2/COX-2信号通路传导,进而减轻炎症反应有关。

Betulinic acid protects against ovarian impairment by decreasing F-2 toxin-induced oxidative stress and inflammation associated with the downregulation of p38 expression in mice

F-2 toxin is an estrogenic mycotoxin that causes reproductive disorders in animals.Betulinic acid(BA)is a natural pentacyclic lupane-structure triterpenoid that has diverse pharmacological activities.In this study,the antioxidative and anti-inflammatory effects of BA and its underlying mechanism are explored in F-2 toxin-triggered mouse ovarian damage.We found that BA alleviated the F-2 toxin-induced ovarian impairment by stimulating follicle growth,reducing inflammatory cell infiltration,repairing damaged mitochondria and endoplasmic reticulum.Simultaneously,BA not only reversed F-2 toxin-induced reduction of follicle stimulating hormone(FSH)and luteinizing hormone(LH)levels in the serum,but also restrained the protein expression of the estrogen receptors a(ERa)and ERβ.Moreover,BA restored the balance of F-2 toxin-induced ovarian redox system disorders.Subsequently,we found that 0.25 mg/kg BA played an anti-inflammatory role in the F-2 toxin-induced ovarian impairment by decreasing interleukin-1β(IL-1β).IL-6,and tumor necrosis factor-α(TNF-α)mRNA expression,as well as inhibiting p38 protein expression.These data demonstrated that BA exerts its protective effect on F-2 toxin-induced ovarian oxidative impairment and inflammation by inhibiting p38 expression,which implies a natural product-based medicine to ameliorate F-2 toxin-caused female reproductive toxicity and provides a detoxifying method for food contaminated by mycotoxin.

胃复康颗粒对Hp VG湿热证患者胃黏膜p38MAPK及ATF2蛋白表达的影响

目的:探讨胃复康颗粒对幽门螺杆菌相关性疣状胃炎(Hp VG)湿热证患者胃黏膜病理及p38MAPK、ATF2蛋白表达的影响。方法:将Hp VG湿热证患者135例随机分为中药组、联合组及西药组各45例,中药组予胃复康颗粒1袋/次,2次/d;西药组予泮托拉唑钠肠溶片每次1片,1次/d。联合组同时服用中药及西药。3组疗程均为6周。采用定位活检法取得治疗前后胃黏膜病变标本,用HE染色观察胃黏膜病理组织学变化情况;免疫组化Western blot法检测p38MAPK及ATF2蛋白表达情况。结果:3组患者胃黏膜病理改善总有效率中药组为82.2%、联合组为88.9%、西药组为73.3%,中药组、联合组改善优于西药组(P<0.05),中药组与联合组间比较差异无统计学意义(P>0.05)。治疗后3组患者胃黏膜p38MAPK均较治疗前降低(P<0.05),联合组较西药组降低明显(P<0.05),与中药组比较效果相当(P>0.05)。中药组、联合组ATF2蛋白表达较治疗前及西药组降低(P<0.05),西药组较治疗前变化不明显(P>0.05);联合组与中药组比较效果相当(P>0.05)。结论:胃复康颗粒联合西药可能通过下调p38MAPK及ATF2蛋白表达,抑制p38MAPK/ATF2通路,减少下游炎症因子释放,起到减轻Hp VG湿热证患者炎症反应的作用。

To investigate the effects of butylphthalide on reducing neuronal apoptosis in rats with cerebral infarction by inhibiting the JNK/P38 MAPK signaling pathway

Objective:To investigate the effects of butylphthalide on reducing neuronal apoptosis in rats with cerebral infarction by inhibiting the JNK/P38 MAPK signaling pathway.Methods:Forty-eight SD male rats were divided into DZ group(control group),CI group(model group)and NBP group(butylphthalide group).Rats in CI group and NBP group were used to establish cerebral infarction models.NBP group used NBP.The solution(80 mg/(kg?d))was administered orally,and the remaining two groups were administered with the same volume of peanut oil.After 14 consecutive days of treatment,the Zea Longa score was used to evaluate the neurological function of DZ,CI and NBP rats.Scoring,TTC staining was used to observe the cerebral infarction volume of rats in DZ group,CI group and NBP group,HE staining was used to observe the pathological morphology of brain tissue in DZ group,CI group and NBP group.Neuronal apoptosis,Western blot was used to detect the expression of p-JNK and p-p38MAPK in brain tissues of DZ group,CI group and NBP group.Results:The neurological function of the rats in the CI group was higher than that in the DZ group,and the difference was statistically significant(P<0.05).The neurological function score of the rats in the NBP group was reduced compared with the CI group,and the difference was statistically significant(P<0.05).The cerebral infarction volume in the group was 35.56%higher than that in the DZ group,and the difference was statistically significant(P<0.05).The minor infarct volume in the NBP group was 21.59%,which was less than that in the CI group,and the difference was statistically significant(P<0.05).Nerve cells are neatly sorted,with a large number.The gap between blood vessels and interstitial tissue in the CI group is enlarged,the cells are severely contracted,and the neuron structure is incomplete.Compared with the CI group,the NBP group has reduced neuron contraction and increased number;The dead nerve cells were brown.The apoptosis rate of nerve cells in the CI group was 79.65%higher than that in the DZ group was 5.82%.The difference was statistically significant(P<0.05).The nerve cell apoptosis rate in the NBP group was 30.23%.Compared with CI group,the difference was statistically significant(P<0.05);Western blot results showed that p-JNK and p-p38MAPK protein expression in CI group was higher than that in DZ group,and the difference was statistically significant(P<0.05).The levels of p-JNK and p-p38MAPK proteins in the NBP group were lower than those in the CI group.There was statistically significant(P<0.05).Conclusion:Butylphthalide can improve neurological damage,reduce apoptotic nerve cells,and reduce infarct volume in rats with cerebral infarction,which is related to the inhibition of JNK/P38 MAPK pathway expression.

Promotion effect of FOXCUT as a microRNA sponge for miR-24-3p on progression in triple-negative breast cancer through the p38 MAPK signaling pathway

Background:Triple-negative breast cancer(TNBC)is a type of highly invasive breast cancer with a poor prognosis.According to new research,long noncoding RNAs(lncRNAs)play a significant role in the progression of cancer.Although the role of lncRNAs in breast cancer has been well reported,few studies have focused on TNBC.This study aimed to explore the biological function and clinical significance of forkhead box C1 promoter upstream transcript(FOXCUT)in triple-negative breast cancer.Methods:Based on a bioinformatic analysis of the cancer genome atlas(TCGA)database,we detected that the lncRNA FOXCUT was overexpressed in TNBC tissues,which was further validated in an external cohort of tissues from the General Surgery Department of the First Affiliated Hospital of Nanjing Medical University.The functions of FOXCUT in proliferation,migration,and invasion were detected in vitro or in vivo.Luciferase assays and RNA immunoprecipitation(RIP)were performed to reveal that FOXCUT acted as a competitive endogenous RNA(ceRNA)for the microRNA miR-24-3p and consequently inhibited the degradation of p38.Results:lncRNA FOXCUT was markedly highly expressed in breast cancer,which was associated with poor prognosis in some cases.Knockdown of FOXCUT significantly inhibited cancer growth and metastasis in vitro or in vivo.Mechanistically,FOXCUT competitively bounded to miR-24-3p to prevent the degradation of p38,which might act as an oncogene in breast cancer.Conclusion:Collectively,this research revealed a novel FOXCUT/miR-24-3p/p38 axis that affected breast cancer progression and suggested that the lncRNA FOXCUT could be a diagnostic marker and therapeutic target for breast cancer.

Mannitol inhibits the proliferation of neural stem cell by a p38 mitogen-activated protein kinase-dependent signaling pathway

Purpose:Mannitol is one of the first-line drugs for reducing cerebral edema through increasing the extracellular osmotic pressure.However,long-term administration of mannitol in the treatment of cerebral edema triggers damage to neurons and astrocytes.Given that neural stem cell(NSC)is a subpopulation of main regenerative cells in the central nervous system after injury,the effect of mannitol on NSC is still elusive.The present study aims to elucidate the role of mannitol in NSC proliferation.Methods:C57 mice were derived from the animal house of Zunyi Medical University.A total of 15 pregnant mice were employed for the purpose of isolating NSCs in this investigation.Initially,mouse primary NSCs were isolated from the embryonic cortex of mice and subsequently identified through immunofluorescence staining.In order to investigate the impact of mannitol on NSC proliferation,both cell counting kit-8 assays and neurospheres formation assays were conducted.Thein vitro effects of mannitol were examined at various doses and time points.In order to elucidate the role of Aquaporin 4(AQP4)in the suppressive effect of mannitol on NSC proliferation,various assays including reverse transcription polymerase chain reaction,western blotting,and immunocytochemistry were conducted on control and mannitol-treated groups.Additionally,the phosphorylated p38(p-p38)was examined to explore the potential mechanism underlying the inhibitory effect of mannitol on NSC proliferation.Finally,to further confirm the involvement of the p38 mitogen-activated protein kinase-dependent(MAPK)signaling pathway in the observed inhibition of NSC proliferation by mannitol,SB203580 was employed.All data were analyzed using SPSS 20.0 software(SPSS,Inc.,Chicago,IL).The statistical analysis among multiple comparisons was performed using one-way analysis of variance(ANOVA),followed by Turkey’’s post hoc test in case of the data following a normal distribution using a Shapiro-Wilk normality test.Comparisons between 2 groups were determined using Student’s t-test,if the data exhibited a normal distribution using a Shapiro-Wilk normality test.Meanwhile,data were shown as median and interquartile range and analyzed using the Mann-WhitneyU test,if the data failed the normality test.A p<0.05 was considered as significant difference.Results:Primary NSC were isolated from the mice,and the characteristics were identified using immunostaining analysis.Thereafter,the results indicated that mannitol held the capability of inhibiting NSC proliferation in a dose-dependent and time-dependent manner using cell counting kit-8,neurospheres formation,and immunostaining of Nestin and Ki67 assays.During the process of mannitol suppressing NSC proliferation,the expression of AQP4 mRNA and protein was downregulated,while the gene expression of p-p38 was elevated by reverse transcription polymerase chain reaction,immunostaining,and western blotting assays.Subsequently,the administration of SB203580,one of the p38 MAPK signaling pathway inhibitors,partially abrogated this inhibitory effect resulting from mannitol,supporting the fact that the p38 MAPK signaling pathway participated in curbing NSC proliferation induced by mannitol.Conclusions:Mannitol inhibits NSC proliferation through downregulating AQP4,while upregulating the expression of p-p38 MAPK.

ANTI-OXIDATIVE MECHANISMS OF PRAVASTATIN PREVENTING AORTIC ATHEROSCLEROSIS IN apoE KNOCKOUT MICE:ROLE OF p38 MAPK PATHWAY

Objective To determine whether pravastatin exerts anti-oxidative effects on preventing aortic＂ atherosclerosis via modulating p38 MAPK pathway. Methods Male 8-week-old apoE^-/- mice fed a diet containing 1.25% cholesterol （wt/wt） were divided into pravastatin group administered with pravastatin （80 mg. kg ^-1· d^-1 ） and atherosclerosis group administered with PBS; and male 8-week-old C57BL/6J mice fed a normal diet were as control group （ n = 12 ）. In thoracoabdominal aortas of mice, levels of Malondialdehyde （ MDA ） and activities of superoxide dismutase （ SOD ） were measured and expression of phosphorylated p38 MAPK （ p-p38 MAPK） and phosphorylated signal transducer and activator of transcr（ption 1 （pSTAT1） were examined by Western blotting. Results After eight weeks, atherosclerosis in aortic root was significantly prevented by pravastatin. In aortic atherosclerosis lesion, the level of MDA was significantly reduced; adversely the activity, of SOD was increased. Expressions of p-p38 MAPK and pSTAT1 were significantly decreased in aortic atherosclerosis lesion. Conclusion Our results suggests that anti-oxidative mechanisms of pravastatin preventing aortic atherosclerosis may partially depend on modulating p38 MAPK signal pathway.

The p38/MAPK pathway as a therapeutic target to prevent therapeutic escape of breast cancer stem cells

Cancer stem cells(CSCs)play an important role in metastasis development,tumor recurrence,and treatment resistance,and are essential for the eradication of cancer.Currently,therapies fail to eradicate CSCs due to their therapeutic stress-induced cellular escape,which leads to enhanced aggressive behaviors compared with CSCs that have never been treated.However,the underlying mechanisms regulating the therapeutic escape remain unknown.To this end,we established a model to isolate the therapeutic escaped CSCs(TSCSCs)from breast CSCs and performed the transcription profile to reveal the mechanism.Mechanistically,we demonstrated that the behavior of therapeutic escape was regulated through the p38/MAPK signaling pathway,resulting in TSCSCs exhibiting enhanced motility and metastasis.Notably,blocking the p38/MAPK signaling pathway effectively reduced motility and metastasis ability both in vitro and in vivo,which were further supported by downregulated motility-related genes and epithelial-mesenchymal transition(EMT)-related proteins vimentin and N-cadherin.The obtained findings reveal the p38/MAPK pathway as a potential therapeutic target for TSCSCs and would provide profound implications for cancer therapy.

Polysaccharides from Agrocybe cylindracea residue alleviate type 2-diabetes-induced liver and colon injuries by p38 MAPK signaling pathway

In this experiment,we investigated the possible mechanism of polysaccharides from Agrocybe cylindracea residue (PACR) on ameliorating the type-2-diabetes-induced liver and colon injuries.Animal experiments have proved that PACR could reduce the oxidative damage and inflammatory response.Meanwhile,the PACR could restore lipid levels,decrease the level of liver and colon lesions in injured mice,and finally play a role in protecting liver and colon.The results showed that PACR could be used as a supplement to decrease blood glucose and relieve T2DM and reduce oxidative stress and inflammatory response by inhibiting the activation of p38 MAPK signaling pathway.

Cigarette smoke-induced cell cycle arrest in spermatocytes [GC-2spd(ts)] is mediated through crosstalk between Ahr–Nrf2 pathway and MAPK signaling

Our earlier studies have demonstrated that the cigarette smoke in the form of cigarette smoke condensate(CSC)causes growth arrest of a mouse spermatocyte cell line[GC-2spd(ts)]through activation of the AHR–NRF2 pathway.The present study demonstrates the CSC-activated p38 and ERK MAPK signaling in GC-2spd(ts)via arylhydrocarbon receptor(AHR).Pharmacological inhibition by using AHR-antagonist,or p38 MAPK and ERK(MEK1)inhibitors significantly abrogates CSC-induced growth arrest by AHR and MAPK inactivation.QRT-PCR,western blot,and immunofluorescence of Ahr-target of Nrf2,and stress-inducible growth suppressive Atf3 and E2f4 following treatments indicate a crosstalk among these pathways.Regulation of Atf3 by Nrf2 and Ahr through RNA interference suggests the existence of a cross-regulatory loop between the targets.CSC induction of E2f4 via Atf3 and its regulation by pharmacological inhibitors reveal a possible regulatory mechanism of growth inhibitory CSC.SiRNA silencing of Ahr,Nrf2,Atf3,and E2f4 genes and downregulation of cyclins by CSC corroborate the growth inhibitory effect of cigarette smoke.Thus,the data obtained suggest that the CSC-mediated MAPKs and AHR–NRF2 crosstalks lay the molecular basis for the growth arrest and cell death of spermatocytes.

清下化瘀方改善重症急性胰腺炎大鼠肠黏膜屏障功能及对p38MAPK/ATF2信号通路的影响

目的探讨清下化瘀方通过改善肠屏障功能治疗急性胰腺炎的作用机制。方法雄性SD大鼠50只按随机数字表法分为5组:假手术组、模型组、清下化瘀方组(简称中药组)、醋酸奥曲肽注射液(善宁)组、空白组,每组10只。采用逆胆胰管注射牛磺胆酸钠的方法建立重症急性胰腺炎大鼠模型,分别予清下化瘀方及善宁干预后取材,观察胰腺病理损伤程度及检测大鼠肠黏膜屏障功能改变(肠黏膜上皮损伤程度),并分析p38MAPK/ATF2表达的变化。结果(1)胰腺组织病理损伤:与空白组及假手术组比较,模型组大鼠胰腺病理总积分明显升高(P<0.01),提示造模成功。与模型组比较,清下化瘀方中药组及善宁组胰腺病理变化半定量积分明显降低(P<0.01)。(2)肠上皮损伤程度比较:与空白组及假手术组比较,模型组肠黏膜损伤明显增加(P<0.01),而中药组及善宁组对肠黏膜损伤有改善作用(P<0.01)。中药组与善宁组比较差异无统计学意义(P>0.05)。(3)肠屏障功能变化:空白组及假手术组肠黏膜上皮细胞表面微绒毛排列整齐,实验各组回肠出现不同程度的线粒体肿胀,嵴不清,内质网肿胀,上皮细胞微绒毛脱落,上皮细胞连接增宽。(4)p38MAPK/ATF2表达变化:模型组大鼠胰腺组织p38MAPK、ATF2阳性表达较空白组及假手术组明显升高(P<0.01),中药及善宁组表达较模型组显著降低(P<0.01)。结论清下化瘀方作用于SAP的途径可能与减轻重症急性胰腺炎大鼠的肠黏膜屏障的损伤,从而调控p38MAPK/ATF2表达,减轻机体炎症反应有关。

Galectin-9 Promotes Human Trophoblast Cell Invasion through Matrix Metalloproteinase-2 and p38 Signaling Pathway

Objective:Adequate extravillous trophoblast(EVT)invasion plays a crucial role in the establishment of successful pregnancy.Insufficient trophoblast migration and invasion can result in defective placentation,which is associated with a number of clinical pathological conditions of pregnancy including spontaneous abortion and preeclampsia.Galectin-9(Gal-9)has a wide variety of regulatory functions in innate and adaptive immunity during infection,tumor growth,and organ transplantation.Methods:We utilized immortalized human first-trimester EVT cells(HTR8/SVneo)for our functional study.We examined the effects of Gal-9 on viability,proliferation,and invasion of HTR8/SVneo cells,as well as on matrix metalloproteinase-2(MMP-2)production in HTR8/SVneo cells.Furthermore,we observed the effects of different MAPK-signaling pathway inhibitors on the stimulatory functions of Gal-9 on HTR8/SVneo cells’invasion.Results:We verified the secretion of Gal-9 by trophoblasts and detected a correlation between low levels of Gal-9 and spontaneous abortion.Gal-9 promoted the invasion of HTR8/SVneo cells through its interaction with Tim-3,not CD44,and subsequently increased MMP-2 production.Blockade of p38 signaling pathway inhibited Gal-9 activities in HTR8/SVneo cells.Conclusion:Gal-9 promotes human trophoblast cell invasion through MMP-2 and p38 signaling pathway in a Tim-3-dependent manner.

二甲双胍抑制p38MAPK/ATF2信号通路改善糖尿病大鼠认知功能障碍

目的探索二甲双胍对糖尿病大鼠认知功能障碍的保护作用,探讨p38 MAPK/ATF2信号通路的作用。方法30只SD大鼠随机分成空白组、模型组及二甲双胍组,每组10只。除空白组外均给予高糖高脂饲料,50 d后腹腔注射链脲佐菌素(STZ,30 mg/kg)建立2型糖尿病脑病模型。模型制备成功后,二甲双胍组大鼠腹腔注射100 mg/kg二甲双胍,连续给药6周,空白组和模型组给予等体积蒸馏水。Morris水迷宫检测各组大鼠认知能力;HE染色观察海马区形态学变化;TUNEL染色检测神经元凋亡;Western blot检测凋亡相关蛋白Bax、Bcl-2、Cleaved-caspase3以及p38 MAPK、p-p38 MAPK、ATF2蛋白的表达情况。结果二甲双胍组大鼠逃避潜伏期明显缩短,目标象限停留时间延长,穿越平台次数相应增加,海马区神经细胞结构改善、神经元凋亡数量显著降低,Bax/Bcl-2比值和Cleaved-caspase3蛋白表达水平显著降低,二甲双胍还能够抑制p-p38 MAPK、ATF2蛋白的表达。结论二甲双胍改善糖尿病大鼠认知功能,与抑制p38 MAPK/ATF2信号通路有关。

The EGFR-P38 MAPK axis up-regulates PD-L1 through miR-675-5p and down-regulates HLA-ABC via hexokinase-2 in hepatocellular carcinoma cells

Background:Immunotherapy has been shown to be a promising strategy against human cancers.A better understanding of the immune regulation in hepatocellular carcinoma(HCC)could help the development of immunotherapy against HCC.The epidermal growth factor receptor(EGFR)signaling is frequently activated in HCC and plays important roles in tumorigenesis.However,its role in HCC immunity is still largely unknown.This study aimed to investigate the impact of EGFR signaling on programmed death-ligand 1(PD-L1)and human leukocyte antigen class-I(HLA-I)expression in HCC cells and its underlying mechanisms.Methods:The expression of phosphorylated EGFR(p-EGFR),PD-L1,and HLAI(HLA-ABC)in HCC specimens was detected by immunohistochemistry,and their correlations were analyzed.PD-L1 and HLA-ABC expression in EGFRactivated HCC cells were detected by quantitative real-time PCR,Western blotting,and flow cytometry,and T cell-mediated lysis was performed to test the immunosuppressive effects of PD-L1 and HLA-ABC alterations in HCC cells.Furthermore,the underlying mechanisms of EGFR activation-induced PD-L1 up-regulation and HLA-ABC down-regulation were explored by animal experiments,luciferase reporter assay,and gene gain-and loss-of-function studies.Results:p-EGFR was positively correlated with PD-L1 and negatively correlated with HLA-ABC expression in HCCs.EGFR activation by its ligand EGF up-regulated PD-L1 and down-regulated HLA-ABC in HCC cells,which was functionally important and could be abolished by the EGFR inhibitor,gefitinib,both in vitro and in vivo.Mechanistically,enhanced P38 mitogenactivated protein kinase(MAPK)activation down-regulated microRNA-675-5p(miR-675-5p)and up-regulated glycolysis-related enzyme hexokinase 2(HK2);miR-675-5p down-regulation enhanced the stability of PD-L1 mRNA probably via the 3’-untranslated region(3’-UTR)of PD-L1 and thereby caused PD-L1 accumulation,and HK2 up-regulation enhanced aerobic glycolysis and mediated a decrease in HLA-ABC.Conclusions:The EGFR-P38 MAPK axis could up-regulate PD-L1 through miR-675-5p and down-regulate HLA-ABC via HK2 in HCC cells.Our study reveals a novel signaling network that may cause immune suppression in HCC and suggests that EGFR signaling can be targeted for HCC immunotherapy.

香菇多糖通过调控SIRT1表达对急性呼吸窘迫综合征小鼠肺损伤的改善作用

目的探究香菇多糖通过调节SIRT1表达调控p38 MAPK/ATF2信号通路对急性呼吸窘迫综合征(ARDS)小鼠肺损伤的改善作用。方法小鼠随机分为对照组、模型组和香菇多糖低、高剂量组(50、100 mg/kg),每组15只。通过鼻腔内滴入6 mg/kg LPS至肺构建ARDS模型,在第0、24小时腹腔注射相应剂量药物,第48小时检测各组小鼠动脉血气指标、肺干重比、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)水平、SIRT1 mRNA和蛋白表达、p38 MAPK和ATF2磷酸化水平。人肺上皮BEAS-2B细胞分为阴性对照组、LPS组、香菇多糖组、香菇多糖+siSIRT1组,通过转染siSIRT1抑制细胞中SIRT1蛋白表达,检测细胞SIRT1蛋白表达、p38 MAPK和ATF2磷酸化水平。结果与对照组比较,模型组小鼠PaO_(2)/FiO_(2)值、SIRT1 mRNA和蛋白表达降低(P<0.05),肺湿干重比、肺组织损伤评分、血清IL-6和TNF-α水平、肺组织p-p38 MAPK/p38 MAPK和p-ATF/ATF蛋白表达升高(P<0.05);与模型组比较,香菇多糖各剂量组上述指标均有改善(P<0.05)。与阴性对照组比较,LPS组细胞SIRT1蛋白表达降低(P<0.05),p-p38 MAPK/p38 MAPK和p-ATF/ATF蛋白表达升高(P<0.05);与LPS组比较,香菇多糖组细胞SIRT1蛋白表达升高(P<0.05),p-p38 MAPK/p38 MAPK和p-ATF/ATF蛋白表达降低(P<0.05),而siSIRT1能逆转香菇多糖的作用(P<0.05)。结论香菇多糖可促进SIRT1蛋白表达抑制p38 MAPK/ATF2信号通路的激活,从而抑制炎性反应,并改善ARDS小鼠肺功能。

The functional analysis of transiently upregulated miR-101 suggests a “braking” regulatory mechanism during myogenesis

Skeletal muscle differentiation is a highly coordinated process that involves many cellular signaling pathways and microRNAs(miRNAs).A group of muscle-specific miRNAs has been reported to promote myogenesis by suppressing key signaling pathways for cell growth.However,the functional role and regulatory mechanism of most non-muscle-specific miRNAs with stage-specific changes during differentiation are largely unclear.Here,we describe the functional characterization of miR-101a/b,a pair of non-muscle-specific miRNAs that show the largest change among a group of transiently upregulated miRNAs during myogenesis in C2C12 cells.The overexpression of miR-101a/b inhibits myoblast differentiation by suppressing the p38/MAPK,Interferon Gamma,and Wnt pathways and enhancing the C/EBP pathway.Mef2a,a key protein in the p38/MAPK pathway,was identified as a direct target of miR-101a/b.Interestingly,we found that the long non-coding RNA(lncRNA)Malat1,which promotes muscle differentiation,interacts with miR-101a/b,and this interaction competes with Mef2a mRNA to relieve the inhibition of the p38/MAPK pathway during myogenesis.These results uncovered a“braking”role in differentiation of transiently upregulated miRNAs and provided new insights into the competing endogenous RNA(ceRNA)regulatory mechanism in myoblast differentiation and myogenesis.