Prognostic and immunological roles of heat shock protein A4 in lung adenocarcinoma

BACKGROUND Heat shock protein A4(HSPA4)belongs to molecular chaperone protein family which plays important roles within variable cellular activities,including cancer initiation and progression.However,the prognostic and immunological significance of HSPA4 in lung adenocarcinoma(LUAD)has not been revealed yet.AIM To explore the prognostic and immunological roles of HSPA4 to identify a novel prognostic biomarker and therapeutic target for LUAD.METHODS We assessed the prognostic and immunological significance of HSPA4 in LUAD using data from The Cancer Genome Atlas database.The association between HSPA4 expression and clinical-pathological features was assessed through Kruskal-Wallis and Wilcoxon signed-rank test.Univariate/multivariate Cox regression analyses and Kaplan-Meier curves were employed to evaluate prognostic factors,including HSPA4,in LUAD.Gene set enrichment analysis(GSEA)was conducted to identify the key signaling pathways associated with HSPA4.The correlation between HSPA4 expression and cancer immune infiltration was evaluated using single-sample gene set enrichment analysis(ssGSEA).RESULTS Overexpressing HSPA4 was significantly related to advanced pathologic TNM stage,advanced pathologic stage,progression disease status of primary therapy outcome and female subgroups with LUAD.In addition,increased HSPA4 expression was found to be related to worse disease-specific survival and overall survival.GSEA analysis indicated a significant correlation between HSPA4 and cell cycle regulation and immune response,particularly through diminishing the function of cytotoxicity cells and CD8 T cells.The ssGSEA algorithm showed a positive correlation between HSPA4 expression and infiltrating levels of Th2 cells,while a negative correlation was observed with cytotoxic cell infiltration levels.CONCLUSION Our findings indicate HSPA4 is related to prognosis and immune cell infiltrates and may act as a novel prognostic biomarker and therapeutic target for LUAD.

Expression of heat shock proteins (HSP27, HSP60, HSP70, HSP90, GRP78, GRP94) in hepatitis B virus-related hepatocellular carcinomas and dysplastic nodules

AIM: Expression of heat shock proteins (HSPs) is frequently up-regulated in hepatocellular carcinoma (HCC), which evolves from dysplastic nodule (DN) and early HCC to advanced HCC. However, little is known about the differential expression of HSPs in multistep hepatocarcinogenesis. It was the purpose of this study to monitor the expression of HSPs in multistep hepatocarcinogenesis and to evaluate their prognostic significance in hepatitis B virus (HBV)related HCC.METHODS: Thirty-eight HCC and 19 DN samples were obtained from 52 hepatitis B surface antigen-positive Korean patients. Immunohistochemical and dot immunoblot analyses of HSP27, HSP60, HSP70, HSP90, glucoseregulated protein (GRP)78, and GRP94 were performed and their expression at different stages of HCC development was statistically analyzed.RESULTS: Expression of HSP27, HSP70, HSP90, GRP78, and GRP94 increased along with the stepwise progression of hepatocarcinogenesis. Strong correlation was found only in GRP78 (Spearman's r= 0.802). There was a positive correlation between the expressions of GRP78, GRP94, HSP90, or HSP70 and prognostic factors of HCC. Specifically, the expression of GRP78, GRP94, or HSP90 was associated significantly with vascular invasion and intrahepatic metastasis.CONCLUSION: The expressions of HSPs are commonly up-regulated in HBV-related HCCs and GRP78 might play an important role in the stepwise progression of HBVrelated hepatocarcinogenesis. GRP78, GRP94, and HSP90 may be important prognostic markers of HBV-related HCC, strongly suggesting vascular invasion and intrahepatic metastasis.

血清HMGB1、CCL20、HSP27水平与慢性牙周炎患者牙周病变程度的相关性分析

目的探讨血清高迁移率族蛋白1(HMGB1)、CC趋化因子配体20(CCL20)、热休克蛋白27(HSP27)水平与慢性牙周炎(CP)患者牙周病变程度的相关性。方法选取2021年9月至2023年8月在新乡医学院第一附属医院诊治的60例CP患者纳入观察组,根据1∶1原则,另选取同期牙周健康者60例纳入对照组。比较两组及不同牙周病变程度CP患者血清HMGB1、CCL20、HSP27水平,分析各指标水平与CP牙周病变程度的相关性及联合诊断价值,并分析不同血清水平患者发生CP的危险度。结果观察组血清HMGB1、CCL20、HSP27水平高于对照组(P<0.05);不同牙周病变程度CP患者血清HMGB1、CCL20、HSP27水平比较:轻度<中度<重度,且各指标水平与牙周病变程度均呈正相关(P<0.05);入院时HMGB1、CCL20、HSP27水平联合诊断CP的曲线下面积(AUC)为0.905,最佳诊断敏感度为91.67%,特异度为88.33%,约登指数0.800,且各指标高水平患者发生CP的危险度是低水平的1.105倍、1.034倍、1.105倍(P<0.05)。结论HMGB1、CCL20、HSP27在CP患者血清中呈异常高表达,各指标水平与牙周病变程度均呈正相关,且联合检测对CP具有较高诊断价值,可作为临床诊断CP、评估牙周病变程度的有效指标。

血清HSP60、HSP70水平与原发性免疫性血小板减少症患儿Th17/Treg细胞和预后的关系

目的分析血清热休克蛋白60(HSP60)、热休克蛋白70(HSP70)与原发性免疫性血小板减少症(ITP)患儿辅助性T细胞17/调节性T细胞(Th17/Treg)和预后的关系。方法选取2020年6月—2022年6月保山市人民医院儿科诊治的ITP患儿128例作为病例组,另选同期体检健康儿童60例作为健康对照组。检测并比较2组血清HSP60、HSP70与Th17/Treg细胞水平。Pearson法分析血清HSP60、HSP70与Th17/Treg细胞的相关性。ITP患儿出院后随访1年,根据不同预后情况分为预后良好亚组(107例)和预后不良亚组(21例)。收集ITP患儿的临床资料,采用单因素、多因素Logistic回归模型分析ITP患儿预后的影响因素,并构建风险预测列线图模型。建立受试者工作特征(ROC)曲线分析血清HSP60、HSP70与Th17/Treg细胞对ITP患儿预后的预测价值。结果病例组血清HSP60、HSP70水平及Th17/Treg细胞显著高于健康对照组(t/Z/P=20.445/<0.001、17.467/<0.001、5.823/<0.001)。Pearson相关分析显示,血清HSP60、HSP70与Th17/Treg细胞呈正相关(r/P=0.417/<0.001、0.348/<0.001)。多因素Logistic回归分析显示,HSP60高、HSP70高、Th17/Treg高为ITP患儿预后不良的独立危险因素[OR(95%CI)=1.177(1.041~1.331),1.181(1.038~1.343),9.895(2.171~68.177)],初诊时PLT计数偏高则为保护因素[OR(95%CI)=0.848(0.726~0.990)]。ROC曲线分析结果显示,初诊时PLT、HSP60、HSP70、Th17/Treg水平及列线图模型的曲线AUC分别为0.793、0.730、0.787、0.840、0.975;Bootstrap法(B=1000)对列线图模型进行内部验证显示,Bias-corrected预测曲线与Ideal线基本重合,该模型预测能力较好。决策曲线显示,该模型的阈值概率范围为0.01~0.98,其净收益率>0,高于两条无效线。结论IPT患儿血清HSP60、HSP70、Th17/Treg水平均明显升高,血清HSP60、HSP70与Th17/Treg呈显著正相关。初诊时PLT、HSP60、HSP70、Th17/Treg水平是ITP患儿预后不良的影响因素,且基于HSP60、HSP70等独立危险因素构建的列线图模型对ITP患儿预后不良具有较好的预测价值。

超重/肥胖合并2型糖尿病患者血清ANGPTL4、HSP70、IL-34水平与胰岛素抵抗的相关性

目的 探讨超重/肥胖合并2型糖尿病(T2DM)患者血清血管生成素样蛋白4(ANGPTL4)、热休克蛋白(HSP)70、白细胞介素-34(IL-34)水平与胰岛素抵抗的相关性。方法 选取2020年5月—2022年5月保定市第一中心医院T2DM患者182例(T2DM组)。参考相关诊断标准,将T2DM患者分为超重/肥胖T2DM组(90例)和体重正常T2DM组(92例)。另选取同期健康体检者90名作为正常对照组,其中超重/肥胖者40名(超重/肥胖对照组)、体重正常者50名(体重正常对照组)。检测所有研究对象血清ANGPTL4、HSP70、IL-34、胰岛素和血糖水平,计算稳态模型胰岛素抵抗指数(HOMA-IR)。T2DM患者治疗3个月后,再次检测其血清ANGPTL4、HSP70、IL-34水平和HOMA-IR。采用Pearson相关分析评估血清ANGPTL4、HSP70、IL-34与HOMA-IR的相关性。结果 与正常对照组和体重正常T2DM组比较,超重/肥胖T2DM组血清ANGPTL4和HSP70显著降低(P<0.05),血清IL-34和HOMA-IR显著升高(P<0.05)。与正常对照组比较,体重正常T2DM组血清ANGPTL4和HSP70显著降低(P<0.05),血清IL-34和HOMA-IR显著升高(P<0.05)。Pearson相关分析结果显示,血清ANGPTL4、HSP70与HOMA-IR呈负相关(r值分别为-0.733、-0.758,P<0.001),IL-34和HOMA-IR呈正相关(r=0.705,P<0.001)。治疗后,超重/肥胖T2DM组和体重正常T2DM组血清ANGPTL4和HSP70均明显升高,血清IL-34和HOMA-IR明显降低;且相对于超重/肥胖T2DM组,体重正常T2DM组血清ANGPTL4和HSP70升高更显著(P<0.05),血清IL-34和HOMA-IR降低更显著(P<0.05)。结论 超重/肥胖合并T2DM患者ANGPTL4、HSP70和IL-34与胰岛素抵抗显著相关,或可作为超重/肥胖合并T2DM的疗效监测指标。

老年糖尿病合并肺部感染患者病原菌分布及血清HSP70、HMGB1水平与病情严重程度和预后的关系

目的探讨老年糖尿病合并肺部感染患者病原菌分布情况,分析血清热休克蛋白70(HSP70)、高迁移率族蛋白B1(HMGB1)水平与病情严重程度和预后的关系。方法选择2021年1月—2023年11月西安交通大学第一附属医院呼吸内科与危重症医学科收治的2型糖尿病(T2DM)合并肺部感染患者253例为研究对象,根据出院时预后情况分为预后良好组(n=191)和预后不良组(n=62)。鉴定患者痰液中病原菌并检测血清HSP70和HMGB1水平;采用多因素Logistic回归分析老年T2DM合并肺部感染预后的影响因素;受试者工作特征曲线(ROC)分析血清HSP70和HMGB1水平对老年T2DM合并肺部感染预后的预测价值。结果预后不良组患者年龄、空腹血糖(FPG)、高血压、慢性阻塞性肺疾病(COPD)及病情重度占比显著高于预后良好组(t/χ^(2)/P=6.251/<0.001、14.949/<0.001、4.666/0.031、5.827/0.016、16.530/<0.001)。253例患者痰标本中共检出病原菌株298株,其中革兰阴性菌占比65.10%(194/298)、革兰阳性菌占比28.86%(86/298)、真菌占比6.04%(18/298)。预后不良组血清HSP70和HMGB1水平明显高于预后良好组(t=11.672、13.069,P均<0.001)。血清HSP70和HMGB1水平比较,重度>中度>轻度患者(F=54.146、231.257,P均<0.001);多因素Logistic回归显示,年龄大、HSP70高、HMGB1高、FPG高、COPD及严重程度重均为老年T2DM合并肺部感染预后的危险因素[OR(95%CI)=1.322(1.015~1.722)、1.993(1.336~2.973)、1.754(1.302~2.363)、1.876(1.401~2.512)、3.016(1.798~5.060)、3.956(2.208~7.718)];血清HSP70、HMGB1及二者联合预测老年T2DM合并肺部感染预后的曲线下面积(AUC)分别为0.785、0.772、0.897,二者联合优于各自单独预测效能(Z=3.452、3.297,P均<0.001)。结论血清HSP70、HMGB1水平与老年T2DM合并肺部感染严重程度和预后密切相关,二者联合对其预后具有较高预测价值。

维持性腹膜透析患者血清CHI3L1、HSP60与钙磷代谢、冠状动脉钙化的关系

目的探讨维持性腹膜透析患者血清壳多糖酶3样蛋白1(CHI3L1)、热休克蛋白60(HSP60)与钙磷代谢、冠状动脉钙化的关系。方法选取2021年1月至2023年1月在周口市中心医院接受持续性非卧床腹膜透析或日间非卧床腹膜透析治疗>3个月的98例维持性腹膜透析患者作为研究对象,根据是否存在钙磷代谢异常分别分入钙磷正常组与钙磷异常组,根据多层螺旋CT结果参照Agatston评分法计算其冠状动脉钙化积分(CACS),将98例维持性腹膜透析患者再次分入未钙化组与钙化组,检测并对比两组患者的血清CHI3L1、血清HSP60水平。采用Pearson相关分析探讨血清CHI3L1、HSP60与钙磷代谢指标的关系,采用受试者工作特征(ROC)曲线评估血清CHI3L1、HSP60对维持性腹膜透析患者冠状动脉钙化的评估价值。通过多因素logistic逐步回归分析探讨维持性腹膜透析患者冠状动脉钙化的影响因素。结果98例维持性腹膜透析患者分为钙磷正常组(43例)、钙磷异常组(55例),钙磷代谢异常组患者血清CHI3L1、HSP60水平高于钙磷代谢正常组患者(P<0.05)。维持性腹膜透析患者血清CHI3L1、HSP60与钙磷乘积呈正相关(P<0.05)。根据多层螺旋CT结果参照Agatston评分法计算其CACS积分,将98例维持性腹膜透析患者分为未钙化组(37例)、钙化组(61例),钙化组患者血清CHI3L1、HSP60水平高于未钙化组患者(P<0.05)。血清CHI3L1、HSP60评估维持性腹膜透析患者预后的AUC分别为0.741、0.826,两者联合评估的AUC为0.915。钙化组患者低蛋白血症、高磷饮食人数及透析龄高于未钙化组(P<0.05)。多因素logistic回归分析显示:高磷饮食、透析龄、CHI3L1、HSP60是影响维持性腹膜透析患者冠状动脉钙化的危险因素(P<0.05)。结论血清CHI3L1、HSP60在钙磷代谢异常、发生冠状动脉钙化的维持性腹膜透析患者中均呈高表达,及早检测血清CHI3L1、HSP60水平有助于及时防治冠状动脉钙化。

Glutamine supplementation attenuates intestinal apoptosis by inducing heat shock protein 70 in heatstroke rats

BACKGROUND:Heatstroke is the most hazardous heat-related illness and has a high fatality rate.We investigated whether glutamine supplementation could have a protective effect on heatstroke rats.METHODS:Twenty-five 12-week-old male Wistar rats(weight 305±16 g)were randomly divided into a control group(n=5),heatstroke(HS)group(n=10),and heatstroke+glutamine(HSG)group(n=10).Seven days before heat exposure,glutamine(0.4 g/[kg·d])was administered to the rats in the HSG group by gavage every day.Three hours after heat exposure,serum samples were collected to detect white blood cells,coagulation indicators,blood biochemical indicators,and inflammatory cytokines in the rats.The small intestine tissue was stained to analyze pathological structural changes and apoptosis.Finally,immunohistochemistry and Western blotting were used to analyze the expression levels of heat shock protein 70(HSP70).Multiple comparisons were analyzed by using one-way analysis of variance,and the Bonferroni test was conducted for the post hoc comparisons.RESULTS:After heat exposure,the core temperature of the HS group(40.65±0.31°C)was higher than the criterion of heatstroke,whereas the core temperature of the HSG group(39.45±0.14°C)was lower than the criterion.Glutamine supplementation restored the increased white blood cells,coagulation indicators,blood biochemical indicators,and inflammatory cytokines that were induced by heatstroke to normal levels.The intestinal mucosa was injured,and the structure of tight junctions was damaged in the HS group;however,the structure of intestinal mucosal epithelial cells was stable in the HSG group.Glutamine supplementation alleviated intestinal apoptosis and up-regulated HSP70 expression.CONCLUSION:Glutamine supplementation may alleviate intestinal apoptosis by inducing the expression of HSP70 and have a protective effect on heatstroke rats.

Abrogation of heat-shock protein (HSP)70 expression induced cell growth inhibition and apoptosis in human androgen-independent prostate cancer cell line PC-3m

Aim: To investigate the effect of abrogating heat shock protein (HSP) 70 expression by antisense HSP70 oligonucleotides treatment on human androgen-independent prostate cancer cell line PC-3m growth. Methods: PC-3m cells were treated with 0-16 μmol/L antisense HSP70 oligomers for 0-100 hr. Cell growth inhibition was analyzed using a trypan blue dye exclusion test. Apoptotic cells were detected and confirmed by flow cytometric analysis and DNA fragmentation analysis. The protein expression of HSP70 and bcl-2 affected by antisense HSP70 oligomers were determined using Western blot. Results: Antisense HSP70 oligomer induced apoptosis and then inhibited proliferation of PC-3m cells in a dose- and time-dependent manner. Ladder-like patterns of DNA fragments were observed in PC-3m cells treated with 10 μmol/L antisense HSP70 oligomer for 48 hr or 8 μmol/L for 72 hr on agarose gel electrophoresis. Antisense HSP70 oligomer pretreatment enhanced the subsequent induction of apoptosis by heat shock in PC-3m cells. In addition, undetectable HSP70 expression was observed at a concentration of 10 μmol/L antisense HSP70 oligomer treatment for 48 hr or 8 μmol/L for 72 hr in Western blot, which was paralleled by decreased expression levels of anti-apoptotic protein bcl-2. Conclusion: HSP70 antisense oligomer treatment abrogates the expression of HSP70, which may disrupt HSP70-bcl-2-interactions and further down-regulate bcl-2 expression, in turn inducing apoptosis and inhibiting cell growth in PC-3m cells.

老年重症急性胰腺炎继发胰腺感染的病原菌分布及其与血清PCT、PA、FIB、HSP70和HMGB1的相关性

目的探讨老年重症急性胰腺炎继发胰腺感染患者的病原菌分布及其与血清降钙素原(PCT)、前白蛋白(PA)、纤维蛋白原(FIB)、热休克蛋白70(HSP70)、高迁徙率族蛋白1(HMGB1)水平的相关性。方法选择2019年1月至2022年12月东莞东华医院收治的62例老年重症急性胰腺炎患者作为研究对象,根据是否继发胰腺感染分为感染组20例和未感染组42例,另选择同期健康体检的80例健康者作为对照组。记录感染组患者病原菌分布情况,比较三组受试者及感染组不同病原菌感染类型患者的血清PCT、PA、FIB、HSP70和HMGB1水平,并采用Pearson相关性分析法分析血清PCT、PA、FIB、HSP70及HMGB1水平和胰腺感染的相关性。结果感染组患者病原菌感染类型主要为细菌感染,占90.00%,其中细菌感染类型中革兰阴性菌占55.00%,革兰阳性菌占35.00%;感染组患者血清PCT、HSP70、HMGB1水平分别为(2.49±0.53)μg/L、(9.12±1.86)ng/mL、(18.33±2.61)ng/mL,明显高于未感染组的(1.56±0.41)μg/L、(5.73±1.45)ng/mL、(13.62±2.47)ng/mL和对照组的(0.23±0.06)μg/L、(0.97±0.13)ng/mL、(0.73±0.15)ng/mL,PA、FIB分别为(0.17±0.02)g/L、(2.06±0.21)g/L,明显低于未感染组的(0.23±0.04)g/L、(2.75±0.30)g/L和对照组的(0.30±0.03)g/L、(3.23±0.36)g/L,且未感染组患者的血清PCT、HSP70、HMGB1水平均高于对照组,PA、FIB均低于对照组,差异均有统计学意义(P<0.05);感染组细菌感染患者的血清PCT、HSP70、HMGB1水平分别为(2.74±0.31)μg/L、(11.06±1.43)ng/mL、(20.68±2.05)ng/mL,明显高于真菌感染患者的(2.13±0.25)μg/L、(7.93±1.05)ng/mL、(16.27±1.76)ng/mL,PA、FIB水平分别为(0.11±0.02)g/L、(1.91±0.24)g/L,明显低于真菌感染患者的(0.17±0.04)g/L、(2.58±0.34)g/L,差异均有统计学意义(P<0.05);经Pearson相关性分析结果显示,血清PCT、HSP70、HMGB1与胰腺感染呈正相关(r=0.581、0.613、0.567,P<0.05),与PA、FIB呈负相关(r=-0.463、-0.531,P<0.05)。结论老年重症急性胰腺炎继发胰腺感染患者的病原菌类型主要为细菌感染,患者血清PCT、HSP70、HMGB1水平明显升高,PA、FIB明显降低,且与胰腺感染的发生有明显相关性,临床上应予以密切关注。

Potential involvement of heat shock proteins in pancreaticduodenal homeobox-1-mediated effects on the genesis of gastric cancer: A 2D gel-based proteomic study

AIM To identify functional proteins involved in pancreaticduodenal homeobox-1(PDX1)-mediated effects on gastric carcinogenesis.METHODS A PDX1-overexpressed model was established by transfecting gastric cancer cell line SGC7901 with pcDNA3.1(+)-PDX1 vector(SGC-PDX1). Transfection with empty pcDNA3.1 vector(SGC-pcDNA) served as control. Comparative protein profiles of the two groups were analyzed by two-dimensional electrophoresis basedproteomics(2 DE gel-based proteomics). The differential proteins identified by 2 DE were further validated by qRTPCR and immunoblotting. Finally, co-immunoprecipitation was used to determine any direct interactions between PDX1 and the differential proteins.RESULTS2 DE gel proteomics identified seven differential proteins in SGC-PDX1 when compared with those in SGC-pcDNA. These included four heat shock proteins(HSPs; HSP70 p1 B, HSP70 p8, HSP60, HSP27) and three other proteins(ER60, laminin receptor 1, similar to epsilon isoform of 14-3-3 protein). Immunoblotting validated the expression of the HSPs(HSP70, HSP60, HSP27). Furthermore, their expressions were lowered to 80%, 20% and 24%, respectively, in SGC-PDX1, while PDX1 exhibited a 9-fold increase, compared to SGC-pcDNA. However, qRT-PCR analysis revealed that mRNA levels of the HSP s were increased in SGC-PDX1, suggesting that the expression of the HSP s was post-translationally regulated by the PDX1 protein. Finally, co-immunoprecipitation failed to identify any direct interaction between PDX1 and HSP70 proteins.CONCLUSION This study demonstrates the potential involvement of HSPs in PDX1-mediated effects on the genesis of gastric cancer.

Preparation and application of a novel monoclonal antibody specific for the heat shock protein 60 of Lawsonia intracellularis

Porcine proliferative enteropathy(PPE),an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis,is commonly associated with diarrhea and reduced weight gain in growing pigs widespread.An accurate method for detecting L.intracellularis is particularly important for preventing and controlling PPE.Heat shock protein 60(Hsp60)is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms.Thus,the purpose of the current investigation was to produce a novel L.intracellularis Hsp60 monoclonal antibody(mAb)useful for immunodiagnostics.Three hybridomas secreted anti-Hsp60 termed 3E5,4E2,and 9G6 were generated,and the titers of ascitic fluids of 3E5,4E2,9G6 were 1:1024000,1:2048000 and 1:2048000,respectively.The Western blotting analysis demonstrated that recombinant Hsp60(rHsp60)was recognized by mAbs 3E5,4E2 and 9G6.Subsequently,analyses of specificity showed all the mAbs were highly specific to L.intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs,such as Escherichia coli,Salmonella Choleraesuis,Salmonella Typhimurium,and Brachyspira hyodysenteriae.Furthermore,the mAbs were useful for detecting L.intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs.Our research will provide a foundation for the development of immunological diagnostic tests.

Effects of heat shock on change of HSC70/HSP68,acid and alkaline phosphatases before and after rat partial hepatectomy

INTRODUCTIONOnly the liver has the great capability ofregeneration in mammal.Few hepatocytes are inthe phase of division in the normal liver of an adultmammal (including human beings),but theremaining hepatocytes can be induced to proliferatequickly by partial hepatectomy (PH),and,to somedegree,they stop dividing and re-differentiate intocells functioning as hepatocytes.This shows

牛HSPA6蛋白特性分析及蛋白互作网络构建

通过构建牛热休克蛋白A6(heat shock protein A6,HSPA6)序列与其他生物的系统进化树,以及运用生物信息学方法分析牛HSPA6蛋白的基本理化性质、亲疏水性等,并结合蛋白互作网络,探究牛HSPA6基因编码蛋白的结构和功能特性。结果显示,牛HSPA6蛋白与羊、长江江豚等哺乳动物的氨基酸序列相似性较高;牛HSPA6蛋白分子质量为70 570.64 u,理论等电点为5.66,为酸性亲水性蛋白,无跨膜结构和信号肽;可能存在11个得分>0.900的磷酸化位点,与N-糖基化激活位点可能位于后端碱基;牛HSPA6蛋白是一种主要由40.38%的α-螺旋和33.65%的无规卷曲组成的二级结构相对稳定的蛋白质,包含N-端核苷酸结合域和C-端多肽结合域两个主要的结构域,主要在细胞质中发挥作用;蛋白质互作网络构建结果显示,牛HSPA6蛋白主要与BAG1、DNAJA4、DNAJB1、DNAJC2等蛋白发生互作,参与腺苷酸交换因子活性、ATP酶调节活性、伴侣绑定等,表明牛HSPA6蛋白在牛机体能量代谢等过程中发挥潜在生物学功能。这些多重生物信息学分析为深入探讨牛HSPA6蛋白对肉品质的影响机制提供了理论依据。

Dorsomorphin induces cancer cell apoptosis and sensitizes cancer cells to HSP90 and proteasome inhibitors by reducing nuclear heat shock factor 1 levels

Objective: Heat shock factor 1(HSF1), a transcriptional regulator of heat shock proteins(HSPs), is an attractive therapeutic target for cancer. However, only a few HSF1 inhibitors have been identified so far.Methods: The mRNA and protein levels of HSF1, HSPs, cleaved PARP, and phosphorylated HSF1 were examined by real-time PCR and Western blot. Forced expression, RNA interference, and immunofluorescence assay were used for mechanistic studies.Cell viability and apoptosis were measured by WST-8 assay and flow cytometry, respectively. Xenograft studies were performed in nude mice to evaluate the effect of dorsomorphin and an HSP90 inhibitor on tumor growth.Results: Dorsomorphin suppressed multiple stimuli-induced and constitutive HSPs expression in cancer cells. Mechanistic studies revealed that dorsomorphin reduced heat-induced HSP expression independent of adenosine monophosphate activated protein kinase. Dorsomorphin reduced heat-stimulated HSF1 Ser320 phosphorylation and nuclear translocation, as well as resting nuclear HSF1 levels in cancer cells. Dorsomorphin induced cancer cell apoptosis by inhibiting HSF1 expression. A structure-activity study revealed that the 4-pyridyl at the 3-site of the pyrazolo [1, 5-a]pyrimidine ring is critical for the anti-HSF1 activities of dorsomorphin. Dorsomorphin sensitized cancer cells to HSP90 and proteasome inhibitors and inhibited HSP70 expression induced by these inhibitors in vitro. In tumor-bearing nude mice, dorsomorphin enhanced HSP90 inhibitor-induced cancer cell apoptosis, tumor growth inhibition, and HSP70 expression.Conclusions: Dorsomorphin is an HSF1 inhibitor. It induces cancer cell apoptosis, sensitizes cancer cells to both HSP90 and proteasome inhibitors, and suppresses HSP upregulation by these drugs, which may prevent the development of drug resistance.Hence, dorsomorphin and its derivates may serve as potential precursors for developing drugs against cancer.

小地老虎AiHSP19.3基因鉴定及其对高温和杀虫剂胁迫的响应

小地老虎Agrotis ipsilon是一种重要的农业害虫。小分子热激蛋白(sHSP)是细胞抵御外界胁迫的关键蛋白,本文旨在探索sHSP基因在小地老虎不同发育阶段以及在高温和杀虫剂胁迫下的响应模式。使用同源检索方法从小地老虎转录组中鉴定到了一个sHSP基因的cDNA序列,命名为AiHSP 19.3。通过RT-PCR技术克隆该序列,利用实时荧光定量PCR技术分析AiHSP 19.3基因在小地老虎不同发育阶段和不同组织中的转录模式,并分析了该基因在高温和杀虫剂胁迫下的转录水平。结果表明,小地老虎AiHSP 19.3基因的开放阅读框为516 bp,编码含171个氨基酸,分子量为19.3 kD的蛋白,具有sHSP典型的α-晶状体结构域(ACD)。在供试小地老虎的不同发育阶段和3龄幼虫组织中均能检测到AiHSP 19.3的转录,其中在6龄幼虫和3龄幼虫脂肪体内转录水平最高。小地老虎幼虫经高温和3种杀虫剂(高效氯氟氰菊酯、氯虫苯甲酰胺及辛硫磷)处理后AiHSP 19.3的转录水平显著提高,表明该基因可能在响应高温及杀虫剂胁迫中起到重要作用。

Effectiveness of conjunctival bleb scarring by knockdown of heat shock protein 47 in rat model

AIM:To evaluate the effectiveness of knock-down of heat shock protein 47(HSP47)on conjunctival bleb scarring in a rat model and its possible mechanism.METHODS:Male Sprague–Dawley rats were used for glaucoma filtration surgery(GFS)and were treated with either phosphate buffered solution,shControl,mitomycin C,or sh-HSP47 using a microsyringe immediately after GFS.The morphology of filtering blebs was observed postoperatively.The levels of HSP47 were analyzed at 2,5,8,and 11d after GFS via real‑time quantitative polymerase chain reaction(PCR)and Western blot.The silencing effect of HSP47,the expression of collagen I and III,and the potential signaling pathways of HSP47 during scarification were explored 11d post GFS.The protein levels of transforming growth factor-β1(TGF-β1),phospho-Smad2(pSmad2),phospho-Smad3(p-Smad3),and phospho-p38(p-p38)were also analyzed using Western blot.RESULTS:Sh-HSP47 treatment significantly prolonged the functional filtration bleb retention.The levels of HSP47 were increased significantly at 5,8,and 11d postoperatively compared to the control group(P<0.05,P<0.01,and P<0.001).The levels of HSP47 protein at day 11 postoperatively were significantly down-regulated after HSP47 silencing using sh-HSP47 adenovirus transfection(P<0.01).Expression levels of collagen I and III within the blebs were significantly reduced in the absence of HSP47(P<0.01).Moreover,the protein levels of TGF-β1,p-Smad2/3,and p-p38 were dramatically inhibited after treatment with sh-HSP47(P<0.01).CONCLUSION:The inhibitory effects of HSP47 knockdown on scarring after GFS have the potential to be an efficacious therapeutic option for the treatment of conjunctival bleb scarring.

Association between heat shock factor protein 4 methylation and colorectal cancer risk and potential molecular mechanisms:A bioinformatics study

BACKGROUND We previously demonstrated that heat shock factor protein 4(HSF4)facilitates colorectal cancer(CRC)progression.DNA methylation,a major modifier of gene expression and stability,is involved in CRC development and outcome.AIM To investigate the correlation between HSF4 methylation and CRC risk,and to uncover the underlying molecular mechanisms.METHODS Differences in β values of HSF4 methylation loci in multiple malignancies and their correlation with HSF4 mRNA expression were analyzed based on Shiny Methylation Analysis Resource Tool.HSF4 methylation-related genes were identified by LinkedOmics in CRC,and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed.Protein-protein interaction network of HSF4 methylation-related genes was constructed by String database and MCODE algorithm.RESULTS A total of 19 CpG methylation loci were identified in HSF4,and their β values were significantly increased in CRC tissues and exhibited a positive correlation with HSF4 mRNA expression.Unfortunately,the prognostic and diagnostic performance of these CpG loci in CRC patients was mediocre.In CRC,there were 1694 HSF4 methylation-related genes;1468 of which displayed positive and 226 negative associations,and they were involved in regulating phenotypes such as immune,inflammatory,and metabolic reprogramming.EGFR,RELA,STAT3,FCGR3A,POLR2K,and AXIN1 are hub genes among the HSF4 methylation-related genes.CONCLUSION HSF4 is highly methylated in CRC,but there is no significant correlation between it and the prognosis and diagnosis of CRC.HSF4 methylation may serve as one of the ways in which HSF4 mediates the CRC process.

基于HSP72探讨通络明目方对青光眼模型大鼠RGC和视神经的保护作用

目的探讨通络明目方调控热休克蛋白72(HSP72)对青光眼模型大鼠视网膜神经节细胞(RGC)和视神经的保护作用。方法采用Shareef-Sharma法将40只大鼠建立青光眼模型,随机分为模型组(MG)和通络明目方组(TM),各20只,另将进行假手术的24只大鼠设为对照组(CG)。TM组大鼠每日通络明目方8.61 g/kg灌胃,CG、MG组大鼠每日等体积0.9%氯化钠注射液灌胃,连续给药21 d。造模后第1、7、14、21天时,使用眼压计测量大鼠眼压,采集大鼠造模对侧眼眼眶血0.5 mL,酶联免疫吸附法(ELISA)检测大鼠血清HSP72水平。给药完成后,苏木精-伊红(HE)染色观察大鼠视网膜形态结构,原位末端转移酶标记技术(TUNEL)检测大鼠RGC细胞数量及凋亡率,测定视网膜厚度;免疫荧光和免疫组织化学法检测RGC细胞HSP72表达。结果(1)眼压:MG组大鼠造模后1、7、14、21 d眼压均高于CG组(t_(1 d)=7.758、t_(7 d)=10.913、t_(14 d)=17.353、t_(21 d)=7.955,均P=0.000),TM组大鼠造模后7、14、21 d眼压均低于MG组(t_(7 d)=-2.604,P=0.021;t_(14 d)=-8.815,P=0.000;t_(21 d)=-4.163,P=0.001),差异均有统计学意义。其他时间比较,差异均无统计学意义(P>0.05)。(2)血清HSP72:MG组大鼠造模后1、7、14、21 d血清HSP72水平均高于CG组(t_(1 d)=16.450、t_(7 d)=23.576、t_(14 d)=13.206、t_(21 d)=8.934,均P=0.000),TM组大鼠造模后21 d血清HSP72水平高于MG组(t=5.936,P=0.000),差异均有统计学意义;其他时间比较,差异均无统计学意义(P>0.05)。(3)视网膜形态和厚度:MG组大鼠RGC出现数目减少和空泡化现象,内核层细胞开始排列紊乱、疏松,神经纤维层水肿,内核层疏松,厚度较CG组薄;而TM组大鼠视网膜RGC数目轻度减少、空泡化、内核层细胞状态与MG相比均较轻,厚度较MG组厚。(4)RGC数量和凋亡率:MG组大鼠造模后14、21 d视网膜RGC数量低于CG组(t_(14 d)=-5.886,P=0.000;t_(21 d)=-4.424,P=0.001),1、7、14、21 d RGC凋亡率高于CG组(t_(1 d)=22.165、t_(7 d)=36.737、t_(14 d)=20.285、t_(21 d)=17.176,均P=0.000);TM组大鼠造模后14、21 d视网膜RGC数量高于MG组(t_(14 d)=3.014,P=0.010;t_(21 d)=3.444,P=0.004),7、14、21 d RGC凋亡率低于MG组(t_(7 d)=-9.762、t_(14 d)=-8.639、t_(21 d)=-9.674,均P=0.000),差异均有统计学意义。其他时间比较,差异均无统计学意义(P>0.05)。(5)视网膜HSP72:CG组大鼠视网膜HSP72几乎无表达,MG、TM组HSP72随时间表达增加;相同时间点比较,TM组HSP72表达高于MG组。结论HSP72对青光眼模型大鼠RGC具有保护效应,而通络明目方可能是通过增强并维持HSP72阳性表达,以增加RGC存活率,从而保护青光眼视神经。

Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a Th1 polarizing adjuvant

Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8 kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and the chemokines IP-10, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and normal T cell expressed and secreted (RANTES). The induction of interferon-gamma-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbumin(OVA)(257-264) induces an OVA(257-264)-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVA tumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.