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Objective:To investigate the regulatory role of Wilms tumor 1-associating protein (WTAP) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its molecular mechanism.Methods:① Experiment Ⅰ: H9C2 cardiomyocytes were divided into blank control group and H/R model group. H/R was used to induce myocardial ischemia/reperfusion (I/R) injury model in H9C2 cells. The blank control group was not treated. N6-methyladenosine (m6A) RNA methylation assay kit was used to detect the level of m6A. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the mRNA and protein expression levels of methyltransferases [WTAP, methyltransferase-like proteins (METTL3, METTL14)], respectively. ② Experiment Ⅱ: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, and H/R+sh-WTAP group. sh-WTAP was transfected to knock down the expression of WTAP in H/R+sh-WTAP group, and the model establishment method in the other groups was the same as experimentⅠ. At 48 hours after transfection, the apoptosis rate of cells was detected by flow cytometry. The protein expressions of WTAP, activated caspase-3, activated poly (ADP-ribose) polymerase (PARP), activating transcription factor 4 (ATF4), proline-rich receptor-like protein kinase (PERK), phosphorylated PERK (p-PERK) and CCAAT/enhancer-binding protein homologous protein (CHOP) were detected by Western blotting. The positive expression of ATF4 was observed by immunofluorescence staining. ③ Experiment Ⅲ: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, H/R+sh-WTAP group and H/R+sh-WTAP+ATF4 group. The overexpression plasmid ATF4 was transfected into H9C2 cardiomyocytes, and the modeling method of the other groups were modeled the same as experimentⅡ. The apoptosis rate was detected by flow cytometry. Western blotting was used to detect the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP.Results:① ExperimentⅠ: the methylation level of m6A in the H/R group was significantly higher than that in the blank control group. RT-qPCR results showed that the gene expressions of METTL3, METTL14 and WTAP in the H/R model group were significantly higher than those in the blank control group, and WTAP was the most significantly up-regulated. Western blotting results showed the same trend. These results suggested that the expression level of methyltransferase WTAP is significantly up-regulated in H/R-induced cardiomyocytes. ②Experiment Ⅱ: the apoptosis level in H/R+sh-WTAP group was significantly lower than that in H/R+sh-NC group [(14.16±1.58)% vs. (24.51±2.38)%, P < 0.05]. Western blotting results showed that the protein expressions of WTAP, activated caspase-3, activated PARP, p-PERK, ATF4 and CHOP in the H/R+sh-WTAP group were significantly lower than those in the H/R+sh-NC group. Fluorescence microscopy results showed that the ATF4 positive signal in the H/R+sh-WTAP group was significantly weaker than that in the H/R+sh-NC group [(19.36±1.81)% vs. (32.83±2.69)%, P < 0.01]. The above results suggested that knockdown of WTAP could inhibit H/R-induced cardiomyocyte apoptosis and endoplasmic reticulum stress. ③ Experiment Ⅲ: the apoptosis level of H/R+sh-WTAP+ATF4 group was significantly higher than that of H/R+sh-WTAP group [(26.61±2.76)% vs. (17.14±0.87)%, P < 0.05]. Western blotting results showed that the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP in the H/R+sh-WTAP+ATF4 group were significantly higher than those in the H/R+sh-WTAP group. These results suggested that overexpression of ATF4 reversed the inhibitory effect of sh-WTAP on endoplasmic reticulum stress and apoptosis in H/R-induced cardiomyocytes. Conclusion:Methyltransferase WTAP could regulate ATF4 expression, mediate cell apoptosis and endoplasmic reticulum stress, and promote H/R-induced myocardial cell injury.
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Objective To investigate the binding sites of dihydroartemisinin(DHA)and sarcoplasmic/endoplasmic reticulum calcium ATPase(SERCA)and explore the mechanisms underlying apoptosis induction in HCT-116 colon cancer cells through the mitochondrial pathway.Methods HCT-116 cells were cultured in DHA concentrations of 0,10,20,40 μmol/L.After 24 h of culture,Western blot-ting assessed SERCA concentration,CCK-8 measured cell proliferation,Hoechst nuclear staining examined cell apoptosis,JC-1 probe evaluated mitochondrial membrane potential.LeDock molecular docking predicted DHA and SERCA binding sites.Synthetic SERCA2b mutated and non-mutated proteins at Ile315 and Thr316 sites were combined with small molecule DHA using biofilm interference tech-nology.Results Western blotting revealed a significant decrease in SERCA2 protein levels with increasing DHA concentration.CCK-8 demonstrated a statistically significant decrease in cell proliferation with increasing DHA concentration(P＜0.01).Hoechst nuclear staining illustrated DHA-induced rounding and pyknosis of HCT-116 cell nuclei compared to the control group.DHA gradually decreased mitochondrial membrane potential within the first 4 h of treatment,starting from 5 h,followed by a sustained reduction.Molecular docking predicted a hydrogen bond with Thr316 and a hydrophobic interaction with Ile315.Biofilm interference techniques indicated robust binding of DHA to non-mutated SERCA2b protein,while binding to the SERCA2b mutant protein at Ile315 and Thr316 sites was poor.Conclusion DHA directly binds to the Ile315 and Thr316 sites of SERCA2b,inducing apoptosis in colon cancer cells HCT-116 through the mitochondrial pathway.
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Objective To observe the effects of Gegen Qinlian Decoction on pancreatic endoplasmic reticulum stress in mice with type 2 diabetes mellitus(T2DM);To explore its mechanism of action in the treatment of T2DM.Methods Totally 75 SPF male db/db mice were randomly divided into model group,metformin group,and Gegen Qinlian Decoction high-,medium-,low-dosage groups,with 15 mice in each group.15 db/m mice were set as the blank group.The administration groups received corresponding medicine for gavage for 12 weeks.Body mass,fasting blood glucose(FBG)and glycated hemoglobin(HbA1c)in mice were detected,HE staining was used to observe the pathological changes of pancreatic tissue,the apoptosis of islet cells was determined by TUNEL staining,Western blot was used to detect pancreatic tissue glucose regulatory protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),p-PERK,activated transcription factor 4(ATF4)and C/EBP homologous protein(CHOP)protein expression,RT-PCR was used to detect pancreatic tissue PERK,ATF4,CHOP mRNA expressions.Results Compared with the blank group,the body mass,FBG and HbA1c contents in the model group significantly increased(P<0.01);the pancreatic tissue structure was incomplete,with blurry boundaries and vacuoles inside,leading to a significant increase in pancreatic islet cells apoptosis(P<0.01);the expressions of GRP78,p-PERK,ATF4,and CHOP proteins in pancreatic tissue significantly increased(P<0.01),and the mRNA expressions of PERK,ATF4 and CHOP significantly increased(P<0.01).Compared with the model group,the body mass,FBG and HbA1c contents of mice in each administration group significantly decreased(P<0.05,P<0.01);pathological changes in pancreatic tissue was reduced,and islet cells apoptosis decreased to varying degrees(P<0.05,P<0.01);the expressions of GRP78,p-PERK,ATF4 and CHOP proteins in pancreatic tissue significantly decreased(P<0.01)in Gegen Qinlian Decoction high-and medium-dosage groups and the metformin group,and the expressions of PERK,ATF4 and CHOP mRNA significantly decreased(P<0.05,P<0.01).Conclusion Gegen Qinlian Decoction may decreased pancreatic islet cells apoptosis,protect pancreatic cell function,and delay the progression of T2DM by inhibiting the endoplasmic reticulum stress PERK/ATF4/CHOP signaling pathway,and down-regulating the expressions of related genes and proteins.
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Objective@#To investigate the improvement of endoplasmic reticulum stress mediated by microRNANotchl)signaling axis on hypoxia/reoxygenation(H/R)human(miR)-34a-5p/transmembrane fusion protein 1 (es were randomly divided into Control group, H/R group, mimiccardiomyocytes. @*Methods@#Human cardiomyocytNC group, mimic group, inhibitor NC group andinhibitor group. Except the Control group, H/R injury model wasof miR-34a-5p and Notchl were detected by quantitative real.established in other groups. The expression levesurvival rate was detected by thiazolyl blue ( MTT), cell apopto-time polymerase chain reaction(qRT-PCR), celtargeting relationship between miR-34a-5p and Notchl was detec-sis rate was detected by flow cytometry, and theexpressions of transcriptional activator 6(ATF6), inositol demandted by dual luciferase gene reporting method. Thesmic reticulum kinase (PERK) and glucose regulatory protein 78 (GRP78)were detected by Western blot. @*Results@#miR-34a-5p targeted Notchl(P<0.05);compared with Con-apoptosis rate and protein expressions of ATF6, IREl, PERK andtrol group, the expression level of miR-34a-5pGRP78 in H/R group increased, while the cellsurvival rate and Notchl mRNA and protein expressions decreased(P<0.05). Compared with H/R group and minic NC group, miR-34a-5p expression, apoptosis rate , and proteinexpressions of ATF6, IRE1, PERK and GRP78in mimic group increased, while cell survival rate and Notchl mRNA and protein expressions decreased (P <0. 05).Compared with H/R group and inhibitor NC group, the exprespressions of ATF6 , IREl , PERK and GRP78 decreased in inhibi-sion of miR-34a-5p, apoptosis rate and protein etor group,while cell survival rate and Notch1 mINA and protein expressions increased ( P <0.05). @*Conclusion@#miR-34a-5p can inhibit the apoptosis of H/R human cardiomyocytes, possibly through the targeted inhibition ofNotchl-mediated endoplasmic reticulum stress.
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Objective To evaluate the effect of spliced X-box binding protein 1 (XBP1s) on hypoxia/reoxygenation (H/R) injury of mouse renal tubular epithelial cells and unravel underlying mechanism. Methods Mouse renal tubular epithelial cells were divided into adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), Ad-shNC+H/R group and Ad-shXBP1s+H/R group. The apoptosis level, mitochondrial reactive oxygen activity, mitochondrial membrane potential and mitochondrial calcium ion level were detected in each group. Chromatin immunocoprecipitation followed by sequencing (ChIP-seq) was employed to analyze the binding sites of XBP1s in regulating the inositol 1,4,5-trisphosphate receptor (ITPR) family. The expression levels of XBP1s and ITPR family messenger RNA (mRNA) and protein were determined in each group. Results Compared with the Ad-shNC group, the apoptosis level was higher, mitochondrial reactive oxygen species level was increased, mitochondrial membrane potential was decreased and mitochondrial calcium ion level was elevated in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the apoptosis level was lower, mitochondrial reactive oxygen species level was decreased, mitochondrial membrane potential was elevated, and mitochondrial calcium ion level was decreased in the Ad-shXBP1s+H/R group (all P<0.05). Compared with the Ad-shNC group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 mRNAs and proteins were down-regulated in the Ad-shXBP1s group (all P<0.05). Compared with the Ad-shNC group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 proteins were up-regulated in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 were down-regulated in the Ad-shXBP1s+H/R group (all P<0.05). ChIP-seq results showed that XBP1s could bind to the promoter and exon of ITPR1, the exon of ITPR2, and the exon of ITPR3. Conclusions XBP1s may affect mitochondria-associated endoplasmic reticulum membrane structure and function by directly regulating ITPR transcription and translation. Down-regulating XBP1s may inhibit ITPR expression and mitigate mitochondrial damage.
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Objective Based on the endoplasmic reticulum stress(ERS)inositol-requiring enzyme-1α(IRE1α)/apoptosis signal-regulated kinase 1(ASK1)/c-Jun N-terminal kinase(JNK)pathway to investigate the intervention effect of Ziziphi Spinosae Semen(ZSS)-Albiziae Flos(AF)on the depression model of rats,which were prepared by solitary rearing combined with chronic unpredictable mild stress(CUMS).Methods A total of 144 rats were randomly divided into normal group,model group,high-,medium-and low-dose groups of ZSS-AF,and Venlafaxine group.In addition to the normal group,the rats in other groups were isolated for 21 days combined with CUMS to prepare the depression model.The behavioral changes of rats were observed by open field test and Morris water maze test.The ultrastructural changes of hippocampal neurons were observed by transmission electron microscope.TUNEL staining was used to observe the apoptosis of nerve cells in hippocampus.The protein expression levels of IRE1α,phosphorylated(P)-IRE1α,ASK1,P-ASK1,JNK,P-JNK,B cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)and cysteme aspartate specific protease-3(Caspase-3)in hippocampus were detected by Western Blot.The mRNA expression levels of IRE1α,ASK1,JNK,Bax,Bcl-2 and Caspase-3 in hippocampus were detected by Real-Time PCR.Results Compared with the normal group,the scores of horizontal movement and vertical movement in the open field test of rats in the model group were decreased(P＜0.01).In the water maze test,the escape latency was increased and the number of crossing the original platform was decreased(P＜0.01).The number of hippocampal apoptosis was increased(P＜0.01).The protein expression levels of P-IRE1 α/IRE1 α,P-ASK1/ASK1,P-JNK/JNK,Bax,Caspase-3 and mRNA expressions of IRE1α,ASK1,JNK,Bax,Caspase-3 in hippocampus were increased,while the protein and mRNA expression levels of Bcl-2 were decreased(P＜0.01).Compared with model group,the scores of horizontal movement and vertical movement in the open field test of rats in each dose ZSS-AF groups and Venlafaxine group were increased(P＜0.01).In the water maze test,the escape latency was decreased and the times of crossing the original platform was increased(P＜0.01).The number of hippocampal apoptosis was decreased(P＜0.01).The mRNA expression levels of P-IRE1α/IRE1α,P-ASK1/ASK1,P-JNK/JNK,Bax,Caspase-3 protein and IRE1α,ASK1,JNK,Bax,Caspase-3 in hippocampus were decreased,while the protein and mRNA expression levels of Bcl-2 were increased(P＜0.05,P＜0.01).The effect of medium-dose ZSS-AF group was better than that of high-and low-dose groups.Conclusion ZSS-AF may play an antidepressant role by regulating IRE1α/ASK1/JNK pathway of endoplasmic reticulum stress.
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ObjectiveTo investigate the effect of the protein kinase RNA-like endoplasmic reticulum kinase （PERK）/eukaryotic initiation factor 2α （eIF2α） pathway in endoplasmic reticulum stress on the activation of hepatic stellate cells （HSC）. MethodsPathological sections of normal liver tissue after surgery were collected from 11 patients with hepatic fibrosis （S1-S4） and 9 patients with hepatic hemangioma and hepatic adenoma confirmed by liver biopsy， and immunohistochemistry was used to measure the protein expression levels of PERK， eIF2α， and C/EBP homologous protein （CHOP）. Human HSC-LX2 cells were treated with different concentrations of the endoplasmic reticulum stress inducer thapsigargin （0， 125， 250， 500， and 1 000 nmol/L）， and qRT-PCR was used to measure the mRNA expression level of PERK， while Western blot was used to measure the protein expression levels of PERK， inositol requiring protein 1 （IRE1）， activating transcription factor 6 （ATF6）， CHOP， and α-smooth muscle actin （α-SMA）. The method of lentivirus transfection was used to construct a PERK stable overexpression LX-2 group and a control group； qRT-PCR was used to measure the mRNA expression levels of PERK， eIF2α， and α-SMA， Western blot was used to measure the protein expression levels of PERK， phosphorylated eIF2α （p-eIF2α）， and α-SMA， and immunofluorescence assay was used to measure the expression of collagen type I alpha 1 （COL1A1）. The independent samples t-test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups. ResultsCompared with normal liver tissue， the liver tissue of patients with hepatic fibrosis had significantly higher expression levels of PERK， eIF2α， and CHOP （Z=-3.56， t=-5.75， Z=-3.52， all P<0.001）. Compared with the solvent group， the groups treated with different concentrations of thapsigargin had significant increases in the expression levels of the endoplasmic reticulum-associated proteins PERK， CHOP， IRE1， ATF6， and α-SMA （all P<0.05）. Compared with the control group， the PERK stable overexpression group had significant increases in the mRNA expression levels of PERK， eIF2α， and α-SMA and the protein expression levels of PERK， p-eIF2α， and α-SMA （all P<0.05）， and immunofluorescence assay showed a significant increase in the expression level of COL1A1 in the PERK stable overexpression group （P<0.05）. ConclusionPERK overexpression can induce the expression of α-SMA and COL1A1 in LX-2 cells， suggesting that the PERK signaling pathway might be one of the important mechanisms of HSC activation.
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【Objective】 To explore the effects and mechanism of p38 mitogen activated protein kinase (MAPK) pathway on the formation of calcium oxalate (CaOx) kidney stones in rats,so as to provide new ideas for the treatment of kidney stones. 【Methods】 A total of 40 rats were divided into control, SB203580, CaOx and SB203580+CaOx groups, with 10 rats in each group.Intragastric administration of a mixture of 1% ethylene glycol and 1% ammonium chloride was given to the CaOx and SB203580+CaOx groups to construct CaOx models, while intragastric administration of drinking water was given to the control and SB203580 groups.After molding, SB203580 and SB203580+CaOx groups were injected with 5 mg/kg SB203580 peritoneally once a day for 14 days, while the control and CaOx groups were injected with equal volume of normal saline.The renal mass of rats was measured and the renal coefficient was calculated; the serum levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were measured with an automated biochemical analyzer; the urinary levels of neutrophil gelatinase-associated lipid carrier protein (NGAL) and kidney injury molecule-1 (KIM-1) were determined with enzyme-linked immunosorbent assay (ELISA); the crystal deposition and tissue damage in renal tissues were observed with Von Kossa staining; the apoptosis of renal tubule cells was observed with TUNEL; the expressions of autophagy markers in kidney tissues were detected with immunohistochemical staining; the molecular expressions of autophagy-endoplasmic reticulum stress related pathways in renal tissues were determined with RT-qPCR and Western blot. 【Results】 Compared with the CaOx group, the SB203580+CaOx group had increased body mass after molding (P<0.05); decreased kidney mass, kidney coefficient, BUN, SCr, NGAL and KIM-1 levels (P<0.05); alleviated pathological damage of kidney tissues; significantly reduced black crystal; down-regulated proportion of positive TUNEL cells, positive expression area of LC3B and Beclin-1, mRNA expressions of LC3B, Beclin-1, CHOP and GRP78, protein ratio of LC3-Ⅱ/LC3-Ⅰ, and protein expressions of Beclin-1, CHOP and GRP78 (P<0.05); but up-regulated mRNA and protein expressions of p62 (P<0.05). 【Conclusion】 The p38 MAPK pathway is involved in the formation of CaOx kidney stones in rats.Inhibition of this pathway can reduce the formation of kidney stones, which may be related to the regulation of autophagy and endoplasmic reticulum stress.
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【Objective】 To construct a prognostic model of clear cell renal cell carcinoma (ccRCC) based on endoplasmic reticulum stress (ERS)-related long non-coding ribonucleotides (lncRNA),so as to explore the correlation between immune cell infiltration and prognosis of ccRCC patients,and to search for new drugs for the treatment of ccRCC. 【Methods】 The transcriptome and clinical data of cancerous and paracancerous tissues of ccRCC were obtained from the TCGA database.The ERS-associated gene set was obtained from the MSigDB database.ERS co-expressed lncRNAs were screened with Pearson correlation analysis.ERS-related lncRNA (ERSRL) with prognostic significance were screened with Lasso regression,univariate and multivariate Cox regression analyses,and a prognostic model was constructed.The risk value of each sample was calculated according to the prognostic model formula.The patients were divided into high- risk and low- risk groups for survival difference analysis.The predictive performance of the prognostic model was evaluated with survival curve,receiver operating characteristic (ROC) curve and calibration curve.The infiltration of immune cells in high-and low-risk groups was analyzed with CIBERSORT database.The relationship between ERSRL and drug sensitivity was analyzed with GDSC database to identify drugs with potential efficacy against ccRCC. 【Results】 A total of 9 lncRNAs with independent prognostic significance were screened to construct the prognostic model.Kaplan-Meier analysis showed significant survival differences between the high- and low-risk groups.Univariate and multivariate Cox regression analyses showed that age,grade,stage and risk score could be used as independent prognostic factors.The area under the ROC curve (AUC) of the 1-,3-,and 5-year survival rates of the training set were 0.754 (95%CI:0.659-0.848),0.744 (95%CI:0.667-0.815),and 0.759 (95%CI:0.662-0.820),respectively,and the C-index was 0.777 (95%CI:0.759-0.796).Immune infiltration results showed that plasma cells,activated memory CD4+T cells,regulatory T cells,M0 macrophages,and activated mast cell infiltration levels were higher in the high-risk group than those in the low-risk group.Drug susceptibility analysis identified 12 drugs with potential curative effects on ccRCC,including AZD8055. 【Conclusion】 Based on 9 ERSRLs,a prognostic model for ccRCC patients was constructed,and 12 drugs with potential therapeutic effects were screened,including AZD8055.
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ObjectiveTo explore the molecular mechanism of modified Shengjiangsan in alleviating endoplasmic reticulum （ER） stress and reducing urinary protein in the rat model of diabetic nephropathy （DN）. MethodSeventy-five SD rats were randomized into normal， model， low-， medium-， and high-dose （4.37， 8.73， 17.46 g·kg-1， respectively） modified Shengjiangsan， and irbesartan （0.014 g·kg-1） groups， with 10 rats in each group. Rats were administrated with corresponding doses of medications or distilled water by gavage， once a day， for 8 consecutive weeks. After the last administration， the levels of glucose （GLU） in the blood， 24-hour urinary protein （24 h-UTP）， and superoxide dismutase （SOD）， malondialdehyde （MDA）， and glutathione peroxidase （GSH-Px） in the renal tissue were measured. Hematoxylin-eosin staining， periodic acid-Schiff staining， and transmission electron microscopy were employed to observe the pathological changes in rat kidneys. Immunohistochemistry was employed to measure the expression levels of nephrin， podocin， glucose-regulated protein 78 （GRP78）， C/EBP homologous protein （CHOP）， and activating transcription factor 4 （ATF4） in the kidneys of rats. Western blot was employed to measure the protein levels of silent information regulator 1 （Sirt1）， phosphorylated （p）-protein kinase RNA-like endoplasmic reticulum kinase （PERK）， and p-eukaryotic translation initiation factor 2 alpha （eIF2α） in rat kidneys. ResultCompared with the normal group， the modeling caused pathological damage to the kidneys， elevated the levels of GLU and 24 h-UTP （P<0.05）， up-regulated the protein levels of GRP78， CHOP， ATF4， p-PERK， and p-eIF2α （P<0.05）， and down-regulated the protein level of Sirt1 （P<0.05） in rat kidneys. Compared with the model group， modified Shengjiangsan and irbesartan lowered the GLU and 24 h-UTP levels （P<0.05）， alleviated the pathological damage in the renal tissue， down-regulated the protein levels of GRP78， CHOP， ATF4， p-PERK， and p-eIF2α （P<0.05）， and up-regulated the protein level of Sirt1 （P<0.05）. ConclusionModified Shengjiangsan up-regulates Sirt1 expression and inhibits phosphorylation of proteins in the PERK/eIF2α pathway to reduce ER stress and oxidative stress in the renal tissue， thus alleviating the pathological damage in the renal tissue and reducing urinary protein in DN rats.
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Acute liver failure （ALF） is one of the most critical liver diseases in clinical practice and seriously affects the life and health of Chinese people. Due to its high morbidity and mortality rates， unclear pathogenesis， and limited treatment methods， ALF has become a major problem that needs to be solved urgently in the field of liver diseases. In recent years， more and more studies have shown that endoplasmic reticulum stress is a key biological process in the progression of ALF， and the IRE1α/TRAF2/JNK pathway， as a part of endoplasmic reticulum stress signaling， plays a role in amplifying inflammatory response， promoting hepatocyte apoptosis， and inhibiting liver regeneration ability during the progression of diseases. As a traditional treasure of China， traditional Chinese medicine has become a research hotspot in search for effective prevention and treatment drugs for ALF from monomers of Chinese herbs. This article elaborates on the mechanism of action of the IRE1α/TRAF2/JNK pathway in the progression of ALF and summarizes the potential value of several monomers of Chinese herbs in regulating this pathway， such as salidroside， Fructus Broussonetiae， Fructus Psoraleae+Schisandra chinensis， baicalein， genipin， kaempferol， resveratrol， sea buckthorn polysaccharide extract， and luteol， in order to provide a reference for further research and clinical practice of ALF.
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Objective @#To explore the mechanism of ferroptosis induced by endoplasmic reticulum stress (ERs ) in acute respiratory distress syndrome (ARDS) .@*Methods @#In order to determine the effects of LPS on oxidative stress and Fe2 + level of mouse capillary alveolar epithelial cells (MLE12 cells ) , the cells were treated with LPS (0 , 1 , 2 , 5 μg/ml) for 24 h . To verify the role of ferroptosis in lipopolysaccharide ( LPS)-induced cell death , MLE12 cells were divided into control ( Con ) group , iron removal inhibitor ( Fer-1) group , LPS group and LPS + Fer-1 group . LPS + Fer-1 group was pretreated with 10 μmol/L Fer-1 for 6 h , then the cells were exposed to 5 μg/ml LPS for 24 h . Con group was treated with solvent DMSO for 24 h . Fer-1 group was pretreated with 10 μmol/L Fer-1 for 6 h , and then treated with DMSO for 24 h . The cells in LPS group were exposed to 5 μg/ml LPS for 24 h . The MLE12 cells were divided into three groups : Con + Vector group , Con + sequence similarity family 134 mem ber B ( FAM134B ) group , LPS + Vector group and LPS + FAM134B group . After transfected with vector or FAM134B overexpression plasmid for 48 h , the cells were exposed or not exposed to 5 μg/ml LPS for 24 h . Cell viability was measured by CCK-8 . The levels of malondialdehyde (MDA) , glutathione and iron , the protein levels of ferroptosis markers [ cyclooxygenase 2(PTGS2) , glutathione peroxidase 4(GPX4)] and ERs markers [glucose reg ulatory protein 78( GRP78) , activated transcription factor 4 ( ATF4) and C/EBP homologous protein ( CHOP)] were measured in different groups . In order to further confirm the results of in vitro cell experiments , 40 mice were randomly divided into Con + Vector group , Con + FAM134B group , LPS + Vector group and LPS + FAM134B group , with 10 mice in each group . LPS induced sepsis models were established in LPS + Vector group and LPS + FAM134B group , and the levels of GPX4 and ERs in lung tissue were evaluated by immunofluorescence staining and protein blot. @*Results @#LPS treatment increased the levels of PTGS2 and MDA , and decreased the levels of GPX4 and GSH in MLE12 cells in a dose dependent manner. Compared with LPS group , the cell viability , GPX4 and GSH levels in LPS + Fer-1 group increased significantly (P < 0.05) , while the PTGS2 protein level and MDA level decreased significantly (P < 0.05) . Compared with LPS + Vector group , LPS + FAM134B group significantly increased cell viability (P < 0.05) , decreased PTGS2 protein level (P < 0.05) and increased GPX4 level ( P < 0.05) . At the same time , the level of MDA in LPS + FAM134B group was lower than that in LPS + Vector group (P < 0.05) , and the level of GSH was higher than that in LPS + Vector group (P < 0.05) . In animal experiment , compared with LPS + Vector group , the expression levels of 4 HNE , ATF4 and CHOP in lung tissue of LPS + FAM134B group decreased significantly ( P < 0.05 ) , and the expression levels of GPX4 , FAM134B group in creased significantly (P < 0.05) .@*Conclusion @#LPS induces ferroptosis and ERs in MLE12 cells in a dose depend ent manner. Activating the endoplasmic reticulum autophagy associated FAM134B receptor helps to inhibit ERs and alleviate cell ferroptosis .
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AIM: To investigate the relationship between vascular smooth muscle cell (VSMC) injury, organelle stress response and autophagic cell death (autophagy) and ferroptosis induced by the chemical hypoxia inducer cobalt chloride (CoCl2) through the bioinformatics analysis and in vitro cell experimentation. METHODS: The dataset GSE119226 of VSMC treated with cobalt chloride was acquired from the gene expression database (GEO). The R language was used to investigate the relationship between CoCl2 treatment and organelle stress response (Golgi stress, endoplasmic reticulum stress) and two forms of cell death (ferroptosis and autophagic cell death). With primary cultured rat VSMC (rVSMC) and CoCl2-induced anoxia model, the changes in cell viability were detected by CCK-8 method, and reactive oxygen species (ROS) levels were measured using DCFH-DA method. The expression levels of HIF-1α (a key molecule in hypoxia), Golgi stress markers GM130 and p115, endoplasmic reticulum stress markers GRP78 and CHOP, autophagy markers LC3-II / LC3-I and Beclin1, and ferroptosis markers GPx4 and xCT were detected by Western blot. The effect of inducing or inhibiting organelle stress and cell death on the CoCl2-induced cell damage was also observed. RESULTS: Differentially expressed genes analysis of GSE119226 dataset showed that CoCl2 treatment of VSMCs had significant effects on organelle function and stress response, autophagy and ferroptosis-related genes, in which endoplasmic reticulum stress, protein processing in endoplasmic reticulum, regulation of Golgi to plasma membrane protein transport, autophagy / autophagic cell death, and ferroptosis pathways were remarkably enriched. The results of in vitro experiment showed that compared with normal rVSMC, cell viability was significantly decreased after CoCl2 treatment, as well as HIF-1α protein expression and ROS levels in rVSMCs were increased. In rVSMC treated with Co-Cl2, the expression levels of Golgi structural proteins GM130 and p115 (reflecting the occurrence of Golgi stress) were decreased, while the markers GRP78 and CHOP (reflecting the occurrence of endoplasmic reticulum stress) were increased. At the same time, CoCl2 treatment also reduced the expression of autophagy markers LC3-II/LC3-I and Beclin1 (indicating the decrease levels of autophagy), while the expression of ferroptosis markers GPx4 and xCT were decreased (indicating the occurrence of ferroptosis). Compared with CoCl2 treatment group, induced Golgi stress, endoplasmic reticulum stress, or ferroptosis could further reduce cell viability, while inhibition of these processes could improve cell viability. On the other hand, increasing the level of autophagy can improve the cell viability. CONCLUSION: Hypoxia induced by cobalt chloride can lead to VSMC injury. Golgi stress, endoplasmic reticulum stress, ferroptosis, and the reduction of autophagy level play an important role in it. Inhibition of organelle stress response and ferroptosis, or increase of autophagy level can improve VSMC injury caused by cobalt chloride.
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Non-infectious chronic diseases in human including diabetes, non-alcoholic fatty liver disease (NAFLD), atherosclerosis (AS), neurodegenerative diseases, osteoporosis, as well as malignant tumors may have some common pathogenic mechanisms such as non-resolved inflammation (NRI), gut microbiota dysfunction, endoplasmic reticulum stress, mitochondria dysfunction, and abnormality of the mammalian target of rapamycin (mTOR) pathway. These pathogenic mechanisms could be the basis for "homotherapy for heteropathy" in clinic. Some commonly used clinical drugs, such as metformin, berberine, aspirin, statins, and rapamycin may execute therapeutic effect on their targeted diseases,and also have the effect of "homotherapy for heteropathy". The mechanisms of the above drugs may include anti-inflammation, modulation of gut microbiota, suppression of endoplasmic reticulum stress, improvement of mitochondria function, and inhibition of mTOR. For virus infectious diseases, as some viruses need certain commonly used replicases, the inhibitors of the replicases become examples of "homotherapy for heteropathy" for antiviral therapy in clinic (for example tenofovir for both AIDS and HBV infection). Especially, in case of outbreak of new emerging viruses, these viral enzyme inhibitors such as azvudine and sofibuvir, could be rapidly used in controlling viral epidemic or pandemic, based on the principle of "homotherapy for heteropathy". In this review article, we show the research progress of the biological basis for "homotherapy for heteropathy" and the possible mechanisms of some well-known drugs, in order to provide insights and new references for innovative drug R&D.
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ObjectiveTo investigate the effect of Yishen Tongluo prescription （YSTLP） on apoptosis of renal tubular epithelial cells and explore the mechanism based on endoplasmic reticulum stress pathway of protein kinase R-like endoplasmic reticulum kinase （PERK）/activating transcription factor 4 （ATF4）/transcription factor C/EBP homologous protein （CHOP）. MethodThe db/db mice were randomly divided into model group， valsartan group （10 mg·kg-1）， and low， middle， high-dose YSTLP groups （1， 2.5， 5 g·kg-1）. Samples were collected after eight weeks of drug intervention. In addition， db/m mice in the same litter served as the control group. Human renal tubular epithelial cells （HK-2） were cultured in vitro and divided into the control group， advanced glycated end-product （AGE） group， and AGE + low， middle， and high-dose YSTLP groups （100， 200， 400 mg·L-1）. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect the apoptosis rate of HK-2 cells. Methylthiazolyldiphenyl-tetrazolium bromide （MTT） assay was conducted to detect the viability of HK-2 cells. Calcium fluorescence probe staining and luciferase reporter gene method were adopted to detect the luciferase activity of folded protein response element （UPRE） and endoplasmic reticulum stress. Immunohistochemical （IHC） analysis was carried out to measure the protein expressions of phosphorylated PKR （p-PERK）， CHOP， and ATF4. Real-time polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of CHOP and X-box binding protein 1 （XBP1） in mouse kidney and HK-2 cells. Western blot was used to detect the protein expression level of p-PERK， PERK， CHOP， ATF4， B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， and cleaved Caspase-3 in mouse kidney and HK-2 cells. ResultIn the cellular assay， HK-2 cell viability was significantly reduced， and the apoptosis rate was elevated in the AGE group compared with the control group （P<0.01）. The mRNA and protein expression levels of apoptosis-related factor Bcl-2 were significantly reduced （P<0.01）， and those of Bax were significantly increased （P<0.01）. The protein expression level of cleaved Caspase-3 was significantly increased （P<0.01）. Compared with the AGE group， YSTLP administration treatment resulted in elevated cell viability and reduced apoptosis rate （P<0.01）. The mRNA and protein expression levels of Bcl-2 were significantly elevated in a time- and dose-dependent manner （P<0.01）， and those of Bax were significantly reduced in a time- and dose-dependent manner. The protein expression level of cleaved Caspase-3 was significantly reduced in a time- and dose-dependent manner （P<0.01）. The intracellular Ca2+ imbalance and UPRE luciferase fluorescence intensity were increased in the AGE group compared with the control group （P<0.01）. The mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 were significantly increased （P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the AGE group， YSTLP effectively improved intracellular Ca2+ imbalance in HK-2 cells and decreased UPRE luciferase fluorescence intensity in a dose-dependent manner （P<0.01）. It reduced the mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 （P<0.01） and the protein expression levels of intracellular p-PERK， CHOP， and ATF4 in a dose- and time-dependent manner （P<0.01）. In animal experiments， the protein expression level of Bcl-2 was significantly reduced（P<0.01）， and that of cleaved Caspase-3 and Bax was significantly increased in the model group compared with the control group （P<0.05）. The protein expression level of Bcl-2 was dose-dependently elevated， and that of cleaved Caspase-3 and Bax was dose-dependently decreased in the YSTLP groups compared with the model group （P<0.01）. Compared with the control group， the mRNA expression levels of CHOP and XBP1 were significantly elevated in the model group （P<0.05， P<0.01）， and the protein expression levels of p-PERK， CHOP， and ATF4 were significantly increased （P<0.05）. Compared with the model group， YSTLP significantly decreased the mRNA expression levels of CHOP and XBP1 （P<0.01） and the protein expression levels of p-PERK， CHOP， and ATF4 （P<0.01）. ConclusionYSTLP can effectively inhibit endoplasmic reticulum stress and improve apoptosis of renal tubular epithelial cells， and its mechanism may be related to the regulation of the PERK/AFT4/CHOP pathway.
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Objective@#To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells (hDPCs) in vitro.@*Methods@#This study was reviewed and approved by the Ethics Committee. hDPCs were cultured using a modified tissue explant technique in vitro. The effects of different concentrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium (MTT) assay. Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium, and the cells were induced in vitro. Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells, and the mRNA expression of the key differentiation factors was detected by RT-qPCR. Moreover, the expression of key molecules of endoplasmic reticulum stress (ERS) was detected by RT-qPCR and Western blot. The data were analyzed with the SPSS 18.0 software package.@*Results@#Low concentration of NaF (0.1 mmol/L) could stimulate cell proliferation in vitro, while a high concentration (5-10 mmol/L) could inhibit cell proliferation (P<0.05). According to the literature and the experimental data, 0.1 mmol/L NaF was selected as the following experimental concentration. The levels of alizarin red S staining were increased after NaF induction of mixed osteogenic/odontogenic differentiation in vitro. The mRNA expression levels of key molecules for osteogenic/odontogenic differentiation, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP) and osteocalcin (OCN), were increased (P<0.05). The mRNA levels of ERS markers (splicing x-box binding protein-1 (sXBP1), glucose-regulated protein 78 (GRP78) and activating transcription Factor 4 (ATF4) were increased in NaF-treated cells. The protein expression levels of key ER stress molecules (phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α) and ATF4) were higher in NaF-treated cells.@*Conclusion@#A low concentration of NaF promotes the osteogenic/odontogenic differentiation of hDPCs and increases the level of ER stress.
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Atherosclerosis （AS） is a chronic inflammatory pathological process in which lipid and/or fibrous substances are deposited in the intima of arteries， and it is one of the pathological bases of many cardiovascular and cerebrovascular diseases. Endoplasmic reticulum stress （ERS） is a protective mechanism of cell adaptation. Moderate ERS can reduce abnormal protein aggregation and increase the degradation of misfolded proteins to repair and stabilize the internal environment， while excessive ERS can cause unfolded protein reaction， activate inflammation， oxidative stress， apoptosis， autophagy， and other downstream pathways， and lead to cell damage， or even apoptosis. A large number of studies have shown that ERS mediates a variety of pathological processes related to AS， affects endothelial cells， smooth muscle cells， macrophages， endothelial progenitor cells， and other cell components closely related to its occurrence and development， influences the progress of AS by regulating cell function， and promotes the formation of AS plaque， the transformation of stable plaque to unstable plaque， and the rupture of unstable plaque. Regulation of ERS may be a key target for the prevention and treatment of AS， and it is a research hotspot at present. Traditional Chinese medicine （TCM） believes that the origin of AS is the imbalance of Yin and Yang， the disharmony of Zangfu organs， and the abnormal operation of Qi， blood， and body fluid， which leads to the accumulation of phlegm， blood stasis， and other pathological products in the pulse channels， making the blood flow blocked or misfunction and causing the disease， which belongs to the syndrome of deficiency in origin and excess in superficiality. As the pathogenesis of AS is complex， and the symptoms are diverse， TCM has significant advantages in treating AS because of its multiple targets， multiple pathways， stable efficacy， strong individualization， and high safety. This paper systematically elaborated on the role of ERS in the occurrence and development of AS and summarized the mechanism research on the regulation and control of ERS by Chinese herbal monomer， Chinese herbal extract， Chinese herbal compound， and proprietary medicine， so as to provide a theoretical basis for clinical research and drug development in the prevention and treatment of AS.
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ObjectiveTo explore the expression of transcription factor KLF16 in nonalcoholic fatty liver disease （NAFLD） and its effect on lipid metabolism. MethodsAn animal model of NAFLD was constructed in mice induced by a high-fat diet. The mice were divided into normal diet group （ND） and high fat diet group （HFD）. NAFLD cell model was constructed by primary mouse liver cells induced by oleic acid. The cells were divided into control group （Control group） and oleic acid induction group （OA group）. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blot were used to detect KLF16 expression in NAFLD animal and cell models. In vitro and in vivo models of KLF16 knockdown were constructed by injection of adeno-associated virus （AAV） into mouse tail veins and transient transfection of cell siRNA. Hematoxylin-eosin staining （HE） and other methods were used to detect changes in lipid deposition in NAFLD models before and after KLF16 knockout. RT-qPCR was used to detect the expression of key genes of lipid metabolism in both cellular and animal NAFLD models before and after KLF16 knockdown. Western blot assay was used to detect the expression of endoplasmic reticulum stress protein in NAFLD model before and after KLF16 knockdown. ResultsThe expression level of KLF16 was up-regulated in HFD group and OA group， and lipid deposition was increased in OA group after KLF16 was depressed. There was no change in TC level in hepatocytes between groups （P>0.05）， and TG level was increased in different degrees （P<0.05， P<0.001）. At the same time， the change of KLF16 expression also caused the change of ER stress protein expression in OA group. ConclusionThe transcription factor KLF16 may alleviate lipid deposition in nonalcoholic fatty liver disease by endoplasmic reticulum stress.
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OBJECTIVE@#To investigate the effects of asperuloside on cervical cancer based on endoplasmic reticulum (ER) stress and mitochondrial pathway.@*METHODS@#Different doses (12.5-800 µg/mL) of asperuloside were used to treat cervical cancer cell lines Hela and CaSki to calculate the half maximal inhibitory concentration (IC50) of asperuloside. The cell proliferation was analyzed by clone formation assay. Cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by flow cytometry. The protein expressions of cleaved-caspase-3, Bcl-2, Bax, Cyt-c, cleaved-caspase-4 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot. And the inhibitor of ER stress, 4-phenyl butyric acid (4-PBA) was used to treat cervical cancer cells to further verify the role of ER stress in the apoptosis of cervical cancer cells induced by asperuloside.@*RESULTS@#Asperuloside of 325, 650, and 1300 µg/mL significantly inhibited the proliferation and promoted apoptosis of Hela and CaSki cells (P<0.01). All doses of asperuloside significantly increased intracellular ROS levels, reduced mitochondrial membrane potential, significantly reduced Bcl-2 protein expression level, and increased Bax, Cyt-c, GRP78 and cleaved-caspase-4 expressions (P<0.01). In addition, 10 mmol/L 4-PBA treatment significantly promoted cell proliferation and reduced apoptosis (P<0.05), and 650 µg/mL asperuloside could reverse 4-PBA-induced increased cell proliferation, decreased apoptosis and cleaved-caspase-3, -4 and GRP78 protein expressions (P<0.05).@*CONCLUSION@#Our study revealed the role of asperuloside in cervical cancer, suggesting that asperuloside promotes apoptosis of cervical cancer cells through ER stress-mitochondrial pathway.
Subject(s)
Female , Humans , Uterine Cervical Neoplasms/metabolism , Caspase 3/metabolism , bcl-2-Associated X Protein/metabolism , Reactive Oxygen Species/metabolism , Endoplasmic Reticulum Chaperone BiP , HeLa Cells , Proto-Oncogene Proteins c-bcl-2/metabolism , Apoptosis , Endoplasmic Reticulum Stress , Cell Line, TumorABSTRACT
The biosynthesis and maturation of proteins are primarily regulated by the endoplasmic reticulum in its physiological state. Thus, the disruption of physiological homeostasis initiates the buildup of unfolded and misfolded proteins in the endoplasmic reticulum, resulting in endoplasmic reticulum stress (ERS) and unfolded protein response (UPR). One of the important pathways by which UPR maintains intracellular homeostasis under ERS is activating protein kinase R-like endoplasmic reticulum kinase (PERK). The activation of the PERK pathway stimulates eukaryotic translation initiation factor 2 subunit-α (eIF2α) phosphorylation and the selective translation of active transcription factor 4 (ATF4), and PERK induces cell apoptosis by directly binding to the promoter of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). This signaling pathway is also one of the important mechanisms by which UPR participates in the regulation of hematological malignancies and immune cells in a tumor microenvironment. This article provides an overview of advancements in research into the PERK-eIF2α-ATF4-CHOP signaling pathway in hematological malignancies and the potential therapeutic benefits of targeting this signaling pathway.