Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron-and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfor...Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron-and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species(ROS) and inducing ferroptosis in activated hepatic stellate cells(HSCs). By using activity-based protein profiling(ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay(CETSA), we show that celastrol directly binds to peroxiredoxins(PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6,through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1(HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2,PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.展开更多
This study observes the therapeutic detoxification of quercetin, a well-known flavonoid, against carbon tetrachlodde （CCI4） induced acute liver injury in vivo and explores its mechanism. QuerceUn decreased CCI4-incr...This study observes the therapeutic detoxification of quercetin, a well-known flavonoid, against carbon tetrachlodde （CCI4） induced acute liver injury in vivo and explores its mechanism. QuerceUn decreased CCI4-increased serum activities of alanine and aspartate aminotransferases （ALT/AST） when orally taken 30 min after CCI4 intoxica- tion. The results of a histological evaluation further evidenced the ability of quercetin to protect against CCI4-induced liver injury. Quercetin decreased the CCI4-increased malondialdehyde （MDA） and reduced the glutathione （GSH） amounts in the liver. It also reduced the enhanced immunohistochemical staining of the 4-hydroxynonenal （4-HNE） in the liver induced by CCI4. Peroxiredoxin （Prx） 1, 2, 3, 5, 6, thioredoxin reductase 1 and 2 （TrxRl/2）, thioredoxin 1 and 2 （Trxl/2）, nuclear factor erythroid 2-related factor 2 （Nrf2）, and heme oxygenase-1 （HO-1） all play critical roles in maintaining cellular redox homeostasis. Real-time polymerase chain reaction （PCR） results demonstrated that quercetin reversed the decreased mRNA expression of all those genes induced by CCI4. In conclusion, our results demonstrate that quercetin ameliorates CCI4-induced acute liver injury in vivo via alleviating oxidative stress injuries when orally taken after CCI4 intoxication. This protection may be caused by the elevation of the antioxidant capacity induced by quercetin.展开更多
目的探讨细胞外信号调节激酶1/2(ERK1/2)在转化生长因子-β1(TGF-β1)诱导的肺成纤维细胞合成Ⅰ、Ⅲ型胶原蛋白中的作用,及新型过氧化物酶Peroxiredoxin-1(Prx-1)对该作用的影响。方法体外培养肺成纤维细胞随机分为4组:对照组(0.4%血清)...目的探讨细胞外信号调节激酶1/2(ERK1/2)在转化生长因子-β1(TGF-β1)诱导的肺成纤维细胞合成Ⅰ、Ⅲ型胶原蛋白中的作用,及新型过氧化物酶Peroxiredoxin-1(Prx-1)对该作用的影响。方法体外培养肺成纤维细胞随机分为4组:对照组(0.4%血清)、TGF-β1组(5μg/L)、阴性转染组(TGF-β1+阴性对照si RNA)和Prx-1 si RNA转染组(TGF-β1+Prx-1 si RNA)。采用脂质体转染法转染si RNA,实时定量逆转录-聚合酶链反应(RT-PCR)检测转染后Prx-1 m RNA表达;Western blot检测Ⅰ和Ⅲ型胶原蛋白、ERK1/2及Prx-1表达;2,7-二氯荧光素二乙酸(DCFH-DA)检测活性氧(ROS)水平。结果 Prx-1 si RNA转染肺成纤维细胞后,Prx-1 m RNA表达明显降低,最大抑制率为92%。与对照组比较,TGF-β1组的Ⅰ和Ⅲ型胶原蛋白、ROS、磷酸化ERK1/2(p-ERK1/2)及Prx-1蛋白的表达水平均明显提高。与TGF-β1组比较,阴性转染组中的上述观察指标无明显变化,但Prx-1转染组的Ⅰ和Ⅲ型胶原蛋白、ROS、p-ERK1/2水平进一步提高,而Prx-1蛋白的表达被抑制。结论 TGF-β1能够诱导肺成纤维细胞生成ROS,并促进ERK1/2通路的激活,导致Ⅰ、Ⅲ型胶原蛋白合成增加,而Prx-1 si RNA可通过提高ROS水平进一步促进TGF-β1该作用。展开更多
AIM: To investigate the expression characteristics of peroxiredoxin 1(PRDX1) m RNA and protein in liver cancer cell lines and tissues.METHODS: The RNA sequencing data from 374 patients with liver cancer were obtained ...AIM: To investigate the expression characteristics of peroxiredoxin 1(PRDX1) m RNA and protein in liver cancer cell lines and tissues.METHODS: The RNA sequencing data from 374 patients with liver cancer were obtained from The Cancer Genome Atlas. The expression and clinical characteristics of PRDX1 m RNA were analyzed in this dataset. The Kaplan-Meier and Cox regression survival analysis was performed to determine the relationship between PRDX1 levels and patient survival. Subcellular fractionation and Western blotting were used to demonstrate the expression of PRDX1 protein in six liver cancer cell lines and 29 paired fresh tissue specimens. After bioinformatics prediction,a putative posttranslational modification form of PRDX1 was observed using immunofluorescence under confocal microscopy and immunoprecipitation analysis in liver cancer cells.RESULTS: The m RNA of PRDX1 gene was upregulated about 1.3-fold in tumor tissue compared with the adjacent non-tumor control(P = 0.005). Its abundance was significantly higher in men than women(P < 0.001). High levels of PRDX1 m RNA were associated with a shorter overall survival time(P =0.04) but not with recurrence-free survival. The Cox regression analysis demonstrated that patients with high PRDX1 m RNA showed about 1.9-fold increase of risk for death(P = 0.03). In liver cancer cells,PRDX1 protein was strongly expressed with multiple different bands. PRDX1 in the cytosol fraction existed near the theoretical molecular weight,whereas two higher molecular weight bands were present in the membrane/organelle and nuclear fractions. Importantly,the theoretical PRDX1 band was increased,whereas the high molecular weight form was decreased in tumor tissues. Subsequent experiments revealed that the high molecular weight bands of PRDX1 might result from the post-translational modification by small ubiquitin-like modifier-1(SUMO1).CONCLUSION: PRDX1 was overexpressed in the tumor tissues of liver cancer and served as an independent poor prognostic factor for overall survival. PRDX展开更多
BACKGROUND In the present study, we investigated a suppressive role of microRNA-596(miR-596) in gastric cancer(GC). Moreover, the downregulation of miR-596 in GC cell lines was associated with an increase of miR-596 p...BACKGROUND In the present study, we investigated a suppressive role of microRNA-596(miR-596) in gastric cancer(GC). Moreover, the downregulation of miR-596 in GC cell lines was associated with an increase of miR-596 promoter methylation. We also established that miR-596 controls the expression of peroxiredoxin 1(PRDX1),which has never been reported before, suggesting that this interaction could play an important role in GC progression.AIM To study the potential role and possible regulatory mechanism of miR-596 in GC.METHODS The expression levels of miR-596 and PRDX1 in gastric cancer tissues and cell lines were detected by quantitative real-time PCR(qRT-PCR). Western blot and luciferase reporter assay were used to detect the effect of miR-596 on PRDX1 expression. Then, the proliferation, metastasis, and invasion of GC cell lines transfected with miR-596 mimics were analyzed, respectively, by Cell Counting Kit-8 proliferation assay, wound healing assay, and transwell invasion assay.Meanwhile, the methylation status of the promoter CpG islands of miR-596 in GC cell lines was detected by methylation-specific PCR(MSP).RESULTS Expression of miR-596 was decreased and PRDX1 was upregulated in GC tissues and cell lines. Overexpression of miR-596 decreased the expression of PRDX1 and luciferase reporter assays detected the direct binding of miR-596 to the 3'-untranslated region(UTR) of PRDX1 transcripts. Furthermore, we found that overexpression of miR-596 remarkably suppressed cell proliferation, migration,and invasion in GC cells. We further analyzed miR-596 promoter methylation by MSP and qRT-PCR, and found the downregulation of miR-596 was associated with promoter methylation status in GC cell lines. Moreover, DNA demethylation and reactivation of miR-596 after treatment with 5-Aza-2'-deoxycytidine inhibited the proliferative ability of GC cells.CONCLUSION MiR-596 has a tumor suppressive role in GC and is downregulated partly due to promoter hypermethylation. Furthermore, PRDX1 is one of the putative target genes of m展开更多
基金supported by the National Key Research and Development Program of China (2020YFA0908000)the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-C-202002,China)+1 种基金the National Natural Science Foundation of China(81903588,81803456,82074098 and 81841001,China)the Fundamental Research Funds for the Central Public Welfare Research Institutes (ZXKT18003 and ZZ15-YQ-063,China)。
文摘Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron-and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species(ROS) and inducing ferroptosis in activated hepatic stellate cells(HSCs). By using activity-based protein profiling(ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay(CETSA), we show that celastrol directly binds to peroxiredoxins(PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6,through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1(HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2,PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.
基金Project supported by the "Shu Guang" Project from Shanghai Municipal Education Commission and Shanghai Education Development Foundation(No.13SG43)the National Natural Science Foundation of China(No.81322053)the Program for New Century Excellent Talents in University(No.NCET-11-1054),China
文摘This study observes the therapeutic detoxification of quercetin, a well-known flavonoid, against carbon tetrachlodde （CCI4） induced acute liver injury in vivo and explores its mechanism. QuerceUn decreased CCI4-increased serum activities of alanine and aspartate aminotransferases （ALT/AST） when orally taken 30 min after CCI4 intoxica- tion. The results of a histological evaluation further evidenced the ability of quercetin to protect against CCI4-induced liver injury. Quercetin decreased the CCI4-increased malondialdehyde （MDA） and reduced the glutathione （GSH） amounts in the liver. It also reduced the enhanced immunohistochemical staining of the 4-hydroxynonenal （4-HNE） in the liver induced by CCI4. Peroxiredoxin （Prx） 1, 2, 3, 5, 6, thioredoxin reductase 1 and 2 （TrxRl/2）, thioredoxin 1 and 2 （Trxl/2）, nuclear factor erythroid 2-related factor 2 （Nrf2）, and heme oxygenase-1 （HO-1） all play critical roles in maintaining cellular redox homeostasis. Real-time polymerase chain reaction （PCR） results demonstrated that quercetin reversed the decreased mRNA expression of all those genes induced by CCI4. In conclusion, our results demonstrate that quercetin ameliorates CCI4-induced acute liver injury in vivo via alleviating oxidative stress injuries when orally taken after CCI4 intoxication. This protection may be caused by the elevation of the antioxidant capacity induced by quercetin.
文摘目的探讨细胞外信号调节激酶1/2(ERK1/2)在转化生长因子-β1(TGF-β1)诱导的肺成纤维细胞合成Ⅰ、Ⅲ型胶原蛋白中的作用,及新型过氧化物酶Peroxiredoxin-1(Prx-1)对该作用的影响。方法体外培养肺成纤维细胞随机分为4组:对照组(0.4%血清)、TGF-β1组(5μg/L)、阴性转染组(TGF-β1+阴性对照si RNA)和Prx-1 si RNA转染组(TGF-β1+Prx-1 si RNA)。采用脂质体转染法转染si RNA,实时定量逆转录-聚合酶链反应(RT-PCR)检测转染后Prx-1 m RNA表达;Western blot检测Ⅰ和Ⅲ型胶原蛋白、ERK1/2及Prx-1表达;2,7-二氯荧光素二乙酸(DCFH-DA)检测活性氧(ROS)水平。结果 Prx-1 si RNA转染肺成纤维细胞后,Prx-1 m RNA表达明显降低,最大抑制率为92%。与对照组比较,TGF-β1组的Ⅰ和Ⅲ型胶原蛋白、ROS、磷酸化ERK1/2(p-ERK1/2)及Prx-1蛋白的表达水平均明显提高。与TGF-β1组比较,阴性转染组中的上述观察指标无明显变化,但Prx-1转染组的Ⅰ和Ⅲ型胶原蛋白、ROS、p-ERK1/2水平进一步提高,而Prx-1蛋白的表达被抑制。结论 TGF-β1能够诱导肺成纤维细胞生成ROS,并促进ERK1/2通路的激活,导致Ⅰ、Ⅲ型胶原蛋白合成增加,而Prx-1 si RNA可通过提高ROS水平进一步促进TGF-β1该作用。
基金Supported by State Key Project for Infectious Diseases,No.2013ZX10002009 and No.2012ZX10002-017State Key Project for Basic Research,No.2014CBA02001 and No.2014CBA02002+1 种基金National High-tech R and D Program,No.2012AA020206Natural Science Foundation of China,No.81071789 and No.81321091
文摘AIM: To investigate the expression characteristics of peroxiredoxin 1(PRDX1) m RNA and protein in liver cancer cell lines and tissues.METHODS: The RNA sequencing data from 374 patients with liver cancer were obtained from The Cancer Genome Atlas. The expression and clinical characteristics of PRDX1 m RNA were analyzed in this dataset. The Kaplan-Meier and Cox regression survival analysis was performed to determine the relationship between PRDX1 levels and patient survival. Subcellular fractionation and Western blotting were used to demonstrate the expression of PRDX1 protein in six liver cancer cell lines and 29 paired fresh tissue specimens. After bioinformatics prediction,a putative posttranslational modification form of PRDX1 was observed using immunofluorescence under confocal microscopy and immunoprecipitation analysis in liver cancer cells.RESULTS: The m RNA of PRDX1 gene was upregulated about 1.3-fold in tumor tissue compared with the adjacent non-tumor control(P = 0.005). Its abundance was significantly higher in men than women(P < 0.001). High levels of PRDX1 m RNA were associated with a shorter overall survival time(P =0.04) but not with recurrence-free survival. The Cox regression analysis demonstrated that patients with high PRDX1 m RNA showed about 1.9-fold increase of risk for death(P = 0.03). In liver cancer cells,PRDX1 protein was strongly expressed with multiple different bands. PRDX1 in the cytosol fraction existed near the theoretical molecular weight,whereas two higher molecular weight bands were present in the membrane/organelle and nuclear fractions. Importantly,the theoretical PRDX1 band was increased,whereas the high molecular weight form was decreased in tumor tissues. Subsequent experiments revealed that the high molecular weight bands of PRDX1 might result from the post-translational modification by small ubiquitin-like modifier-1(SUMO1).CONCLUSION: PRDX1 was overexpressed in the tumor tissues of liver cancer and served as an independent poor prognostic factor for overall survival. PRDX
基金National Natural Science Foundation of China,No.30572162Natural Science Foundation of Liaoning Province,No.201602817
文摘BACKGROUND In the present study, we investigated a suppressive role of microRNA-596(miR-596) in gastric cancer(GC). Moreover, the downregulation of miR-596 in GC cell lines was associated with an increase of miR-596 promoter methylation. We also established that miR-596 controls the expression of peroxiredoxin 1(PRDX1),which has never been reported before, suggesting that this interaction could play an important role in GC progression.AIM To study the potential role and possible regulatory mechanism of miR-596 in GC.METHODS The expression levels of miR-596 and PRDX1 in gastric cancer tissues and cell lines were detected by quantitative real-time PCR(qRT-PCR). Western blot and luciferase reporter assay were used to detect the effect of miR-596 on PRDX1 expression. Then, the proliferation, metastasis, and invasion of GC cell lines transfected with miR-596 mimics were analyzed, respectively, by Cell Counting Kit-8 proliferation assay, wound healing assay, and transwell invasion assay.Meanwhile, the methylation status of the promoter CpG islands of miR-596 in GC cell lines was detected by methylation-specific PCR(MSP).RESULTS Expression of miR-596 was decreased and PRDX1 was upregulated in GC tissues and cell lines. Overexpression of miR-596 decreased the expression of PRDX1 and luciferase reporter assays detected the direct binding of miR-596 to the 3'-untranslated region(UTR) of PRDX1 transcripts. Furthermore, we found that overexpression of miR-596 remarkably suppressed cell proliferation, migration,and invasion in GC cells. We further analyzed miR-596 promoter methylation by MSP and qRT-PCR, and found the downregulation of miR-596 was associated with promoter methylation status in GC cell lines. Moreover, DNA demethylation and reactivation of miR-596 after treatment with 5-Aza-2'-deoxycytidine inhibited the proliferative ability of GC cells.CONCLUSION MiR-596 has a tumor suppressive role in GC and is downregulated partly due to promoter hypermethylation. Furthermore, PRDX1 is one of the putative target genes of m
