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Objective To investigate the effect and mechanism of transplantation of neuregulin1(NRG1)gene-modified bone marrow mesenchymal stem cells(BMSCs)on the repair of hemi-transected spinal cord injury(SCI)in rats.Methods Isolated and cultured rat BMSCs,followed by transfection with the NRG1 gene.The levels of NRG1 in BMSCs lysate and culture supernatant was deected by ELISA method,and the proliferation activity of the BMSCs was detected by cell counting method.Forty-three healthy 8-week-old SD rats were randomly divided into control group(n=10),SCI model group(n=10),BMSCs group(n=10),and NRG1-BMSCs group(n=13).After establishing the spinal cord hemisection model,animals received in-situ transplantation of BMSCs or NRG1-BMSCs.On the 1,7,14,21,and 28 days after transplantation,the hind limb motor function was evaluated using BBB score and inclined plate test;on the 7th day after transplantation,the migration and distribution of transplanted cells was monitored using a fluorescence microscope;on the 28th day after transplantation,the pathological changes of rat spinal cord tissues was examined using HE staining and Nissl staining;cell apoptosis using TUNEL staining,and levels of endoplasmic reticulum stress-related proteins[X-box binding protein 1(XBP1),C/EBP homologous protein(CHOP),activating transcription factor 4(ATF4),ATF6,glucose-regulated protein 78(GRP78)]and apoptosis-related proteins[B-cell lymphoma-2(Bcl-2)and Bcl-2-associated protein X(Bax)]in rat spinal cord tissues using Western blotting.Results BMSCs were successfully isolated,cultured,and transfected with the NRG1 gene.ELISA method results showed that the NRG1 contents in the NRG1-BMSCs lysate and culture supernatant were significantly higher than that of BMSCs in a time-dependent manner(P<0.05).The proliferation activity of NRG1-BMSCs was significantly higher than that of BMSCs(P<0.05).On the 21 and 28 days after transplantation,the BBB score and the slope angle of the inclined plate in NRG1-BMSCs group were higher than those in SCI model group or BMSCs group(P<0.05).However,it did not reverse to the level in control group(P<0.05).On the 28th day after transplantation,compared with the SCI model group and BMSCs group,neuronal pyknosis reduced,the Nissl body density increased,the expression levels of XBP1,CHOP,ATF4,ATF6,GRP78,and Bax,and the rate of TUNEL-positive cells significantly reduced in NRG1-BMSCs group(P<0.05),and the expression level of Bcl-2 significantly increased(P<0.05).Conclusion Transplantation of NRG1 gene-modified BMSCs can alleviate SCI and improve the recovery of motor function in rats.The mechanism may be related to promoting the proliferation activity of BMSCs,inhibiting cell apoptosis,and mitigating endoplasmic reticulum stress.
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Objective To investigate the expression of activating transcription factor 6(ATF6)and interferon α(IFN-α)and their significance in laryngeal squamous cell carcinoma(LSCC)tissue.Methods A total of 100 LSCC patients admitted to Clinical Medical College of Henan University of Science and Technology/the First Affiliated Hospital of Henan University of Science and Technology from March 2015 to March 2020 were selected,and their clinicopathological features such as tumor location,degree of differentiation,and lymph node metastasis were collected and organized.Immunohistochemical method was applied to detect the expression of ATF6 and IFN-α in tissues.Spearman method was used to analyze the correlation between ATF6 and IFN-α expression in LSCC tissue.Kaplan-Meier method was applied to analyze the relationship between ATF6 and IFN-α expression in LSCC tissue and 3-year survival rate of patients.Cox regression was used to analyze the influencing factors of 3-year mortality in LSCC patients.Results The positive rate of ATF6 in LSCC tissue(76.00%)was higher than that in normal tissues adjacent to cancer(13.00%),the positive rate of IFN-α in LSCC tissue(29.00%)was lower than that in normal tissues adjacent to cancer(74.00%),and the difference was statistically significant(χ2=80.352,40.536,all P<0.05).The proportions of ATF6 positive expression in LSCC patients with TNM stage Ⅲ+Ⅳ,deep infiltration depth,and lymph node metastasis were significantly higher than those in LSCC patients with TNM stage Ⅰ+Ⅱ,shallow infiltration depth,and no lymph node metastasis(χ2=7.310,9.223,5.123,all P＜0.05).The proportions of IFN-α negative expression in LSCC patients with TNM stage Ⅲ+Ⅳ,deep infiltration depth,and lymph node metastasis were significantly higher than those in LSCC patients with TNM stage Ⅰ+Ⅱ,shallow infiltration depth,and no lymph node metastasis(χ2=8.564,5.021,5.203,all P＜0.05).There was a negative correlation between ATF6 and IFN-α expression in LSCC tissues(r=-0.415,P<0.05).The 3-year survival rate of LSCC patients in the ATF6 positive expression group(50.00%)was significantly lower than that in the ATF6 negative expression group(83.33%),while the 3-year survival rate of LSCC patients in the IFN-α positive expression group(82.76%)was significantly higher than that in the IFN-α negative expression group(47.89%)(Log rank χ2=8.002,10.854,all P<0.05).ATF6(HR=1.735,95%CI:1.159～2.598)and IFN-α(HR=0.624,95%CI:0.439～0.886)were influencing factors for the mortality of LSCC patients.Conclusion The positive expression rate of ATF6 increased and the positive expression rate of IFN-α decreased in LSCC tissues.They were closely related to the clinical pathological characteristics and prognosis of patients.
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Post-stroke cognitive impairment(PSCI),refers to a range of clinical syndromes of cognitive impairment caused by stroke.Although its specific pathogenesis is still unclear,many studies have confirmed that endoplasmic reticulum-mitochondria interaction has become a key hub for intracellular signal transduction and substance metabolism,and its regulation of various biological processes,such as Ca2+ balance,lipid metabolism,mitochondrial dynamics,autophagy,and neuroinflammation,is closely related to the development of PSCI.There-fore,in this paper,we will review the various functions of endoplasmic reticulum-mitochondrial interactions and explore their specific roles in PSCI,in order to discover new therapeutic targets and provide new theoretical basis and references for the development of PSCI-targeted drugs in the future.
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BACKGROUND:Patients with Alzheimer's disease mainly show cognitive and memory dysfunctions.Aerobic exercise can inhibit endoplasmic reticulum stress and improve cognitive function of the patients.However,whether aerobic exercise can inhibit endoplasmic reticulum stress dependent neuroinflammation is still unclear. OBJECTIVE:To explore the effect of aerobic exercise on neuroinflammation and cognitive impairment in a mouse model of Alzheimer's disease. METHODS:Fifty C57BL/6J wild-type male mouse mice were randomly divided into wild-type control and wild-type exercise groups,while another 50 APP/PS1 double transgenic male mice were randomly divided into Alzheimer's disease group and Alzheimer's disease exercise group,with 25 mice in each group.Mice in the wild-type exercise and Alzheimer's disease exercise groups received aerobic exercise training(treadmill training,45 min/d,12 m/min,5 d/wk,8 weeks in total).Mice in the wild-type control and Alzheimer's disease groups were placed on the quiet running platform.Morris water maze test was used to detect the cognitive ability of mice.Hematoxylin-eosin staining and Nissl staining were used to detect hippocampal tissue damage in mice.Thioflavin-S staining was used to detect β-amyloid content in hippocampal tissue.Immunohistochemistry was used to detect β-amyloid and p-Tau levels in hippocampal tissue.Immunofluorescence staining was used to detect the number of positive cells for neuroinflammation-related factors in hippocampal tissue.Western blot was used to detect p-IRE1,IRE1,p-PERK,PERK,ATF6,GRP78,Bip,Caspase-12,Iba-1,and GFAP protein levels. RESULTS AND CONCLUSION:Compared with the wild-type control group,escape latency was increased,the number of times they reached the previous platform and the time they stayed on the platform were decreased,β-amyloid and Tau levels,p-IRE1/IRE1,p-PERK/PERK,ATF6,GRP78,Bip,Caspase-12,Iba-1,and GFAP protein levels,Iba-1+,Iba-1+TNF-α+,Iba-1+IL-6+,Iba-1+IL-1β+,GFAP+,GFAP+TNF-α+,GFAP+IL-6+,GFAP+IL-1β+ positive cells in hippocampal tissue were increased,and Iba-1+IL-4+,Iba-1+IL-10+,GFAP+IL-4+,GFAP+IL-10+ positive cells were decreased in the Alzheimer's disease group(P＜0.05).Compared with Alzheimer's disease group,escape latency was decreased,the number of times they reached the previous platform and the time they stayed on the platform were increased,β-amyloid and Tau levels,p-IRE1/IRE1,p-PERK/PERK,ATF6,GRP78,Bip,Caspase-12,Iba-1,GFAP protein levels,Iba-1+,Iba-1+TNF-α+,Iba-1+IL-6+,Iba-1+IL-1β+,GFAP+,GFAP+TNF-α+,GFAP+IL-6+,and GFAP+IL-1β+ positive cells in hippocampal tissue were decreased,and Iba-1+IL-4+,Iba-1+IL-10+,GFAP+IL-4+,GFAP+IL-10+ positive cells were increased in the Alzheimer's disease exercise group(P＜0.05).To conclude,aerobic exercise can reduce cognitive impairment in Alzheimer's disease mice by inhibiting endoplasmic reticulum stress and neuroinflammation in hippocampal tissue.
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BACKGROUND:Fluorosis is a disorder of enamel development caused by long-term intake of large amounts of fluoride during enamel development. OBJECTIVE:To further explore the molecular mechanism of dental fluorosis formation by screening the differentially expressed genes associated with calcium homeostasis in ameloblasts by transcriptome sequencing technology. METHODS:LS8 cells were treated with 0,0.4,0.8,1.6,3.2 and 6.4 mmol/L sodium fluoride(NaF)for 24,48 and 72 hours to observe the effects of different concentrations of NaF on the morphology,cell activity and intracellular Ca2+ concentration of LS8 cells.The differentially expressed genes were screened by transcriptome sequencing and validated. RESULTS AND CONCLUSION:After 24 hours of treatment,the cells treated with 0,0.4,and 0.8 mmol/L NaF were in good growth condition,with increased cell number and clear cell outline.When the NaF concentration was≥1.6 mmol/L,the cells were gradually shrunken and became smaller and the number of cells decreased with the increase of NaF concentration.After 48 and 72 hours of treatment,the number of cells increased in the 0,0.4 mmol/L NaF groups,while gradually decreased in the 0.8,1.6,3.2 mmol/L NaF groups,with rounded and smaller cell morphology.The cells in the 6.4 mmol/L NaF group were shrunken,rounded and suspended in the medium,with almost no adherent cells.When treated with the same concentration of NaF,LS8 cells were in optimal growth after 24 hours of treatment.Results from cell counting kit-8 assay showed that when treated with the same concentration of NaF,the cell activity decreased with the increase of treatment time;when the treatment time was the same,the cell activity decreased with the increase of NaF concentration.After 24 hours of treatment,the intracellular Ca2+ concentration increased with the increase of NaF concentration.Transcriptome sequencing analysis identified genes involved in the regulation of cellular calcium homeostasis:Hsp90b1,Canx,Calr,and Hspa5 that were significantly upregulated(P＜0.05)and Cacna1a that was significantly downregulated(P＜0.05).To conclude,the inhibitory effect of NaF on LS8 cell proliferation may be related to the abnormal increase in intracellular Ca2+ concentration,and the mechanism may be caused by the upregulation of the expression of protein processing and synthesis pathways Hsp90b1,Canx,Calr,and Hspa5 and the downregulation of the expression of calcium signaling pathway Cacna1a.
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BACKGROUND:Studies have exhibited that inhibiting apoptosis caused by endoplasmic reticulum stress can save part of nerve function.Epigallocatechin-3-gallate can inhibit endoplasmic reticulum stress,but it has poor bioavailability and is difficult to penetrate the blood-brain barrier.In combination with exosomes targeting spinal cord repair and high-potency drug loading,theoretically,the combination of the two can play a greater role in spinal cord protection. OBJECTIVE:To investigate the effects of epigallocatechin-3-gallate combined with bone marrow mesenchymal stem cell exosomes on endoplasmic reticulum stress and neurological function in rats with spinal cord ischemia/reperfusion injury. METHODS:Fifty SD male rats were randomly divided into sham surgery group,model group,epigallocatechin-3-gallate group,exosome group,and combined treatment group,with 10 rats in each group.The spinal cord ischemia/reperfusion injury model was made in the other four groups except for the sham surgery group.Local injection of physiological saline,exosomes,epigallocatechin-3-gallate,epigallocatechin-3-gallate + bone marrow mesenchymal stem cell exosomes was performed 2 hours after surgery through a caudal vein.Neurological function scores were performed on 7,14 and 28 days after spinal cord injury.14 days after spinal cord injury,hematoxylin-eosin staining,Nissl staining,and immunofluorescence staining of endoplasmic reticulum stress markers such as ATF6 and GADD153 were performed in the spinal cord tissues. RESULTS AND CONCLUSION:(1)Compared with the sham surgery group,neurological function scores of the model group,exosome group,epigallocatechin-3-gallate group and combined treatment group all decreased to different degrees.The neurological function score of combined treatment group was better than that of the epigallocatechin-3-gallate group,exosome group and model group 14 days after surgery(P<0.05).The neurological function score of the combined treatment group was better than that of the model group and epigallocatechin-3-gallate group 28 days after surgery(P<0.05).(2)Hematoxylin-eosin staining and Nissl staining displayed that the number of neurons in the model group decreased,with a large number of cavity necrosis and scar hyperplasia in the spinal cord injury area.The number of neurons and peripheral cavity necrosis improved to varying degrees in the epigallocatechin-3-gallate group,exosome group,and combined treatment group,with the most significant improvement in the combined treatment group.(3)The expression of endoplasmic reticulum stress-related proteins ATF6 and GADD153:14 days postoperatively,the expression of GADD153 in the combined treatment group was lower than that in the model group and epigallocatechin-3-gallate group(P<0.05),and the expression of ATF6 in the combined treatment group was lower than that in the model group,exosome group,and epigallocatechin-3-gallate group(P<0.05).(4)These findings confirm that epigallocatechin-3-gallate combined with bone marrow mesenchymal stem cell exosome can enhance the neurological function in rats with spinal cord ischemia/reperfusionn injury,which may be associated with the inhibition of the expression of endoplasmic reticulum stress-related proteins ATF6 and GADD153.
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BACKGROUND:Icariin,with antiinflammatory,antioxygenatory and immunoregulatory effects,can be a potential drug for preventing and treating acute liver injury. OBJECTIVE:To investigate the protective effect and possible mechanism of icariin in mice with acute liver injury induced by carbon tetrachloride. METHODS:Thirty-two Kunming mice were equally and randomly divided into the following groups:normal,model,low-dose icariin and high-dose icariin groups.The low-and high-dose icariin groups were continuously gavaged with icariin(100 and 200 mg/kg,respectively)once a day for 7 continuous days.The normal group and model group were injected with physiological saline(10 mL/kg)at the same time point.After the last administration,all the groups except for the normal group were injected with carbon tetrachloride to induce acute liver injury.The mice were killed 24 hours later,and the liver index was detected.Serum levels of alanine aminotransferase and aspartate aminotransferase were detected by automated biochemical analysis.Tumor necrosis factor α and interleukin 6 levels in serum were detected using ELISA.The levels of superoxide dismutase,glutathione peroxidase and malondialdehyde in liver tissue were detected through a reagent kit.The histopathology changes of the liver were observed by hematoxylin-eosin staining.TUNEL method was used to detect the apoptosis in hepatocytes.Western blot was performed to detect the expression levels of glucose-regulated protein 78 kDa,endoplasmic reticulum stress-related protein(C/-EBP homologous protein),mixed lineage kinase domain-like protein and Caspase-3 in liver tissue. RESULTS AND CONCLUSION:Compared with the normal group,the liver index and serum levels of alanine aminotransferase,aspartate aminotransferase,tumor necrosis factor α and interleukin 6 were increased in the model group(P＜0.05).Compared with the model group,the above indexes were decreased in the low-dose and high-dose icariin groups(P＜0.05).Compared with the normal group,the activities of superoxide dismutase and glutathione peroxidase in the liver tissue of mice were decreased in the model group(P＜0.05)and the level of malondialdehyde was increased(P＜0.05).Compared with the model group,the activities of superoxide dismutase and glutathione peroxidase were increased in the low-and high-dose icariin groups(P＜0.05)and the level of malondialdehyde was decreased(P＜0.05).Hematoxylin-eosin and TUNEL staining showed that mice in the model group had severe structural destruction of liver tissue,extensive necrosis of hepatocytes and high apoptotic rate of hepatocytes,while the structural destruction of liver tissue and the area of necrosis of hepatocytes in the low-and high-dose icariin groups were significantly milder than those in the model group,and the apoptotic rate of hepatocytes was lower than that in the model group(P＜0.05).Western blot assay showed that the protein expression of glucose-regulated protein 78 kDa,C/-EBP homologous protein,mixed lineage kinase domain-like protein and Caspase-3 in liver tissue of mice in the model group was increased compared with that in the normal group(P＜0.05),while the expression levels of these proteins in liver tissue of mice were significantly reduced after low-and high-dose icariin intervention(P＜0.05).To conclude,icariin can produce a protective effect against carbon tetrachloride-induced acute liver injury,and its mechanism may be related to the regulation of endoplasmic reticulum stress and reduction of programmed necrosis.
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BACKGROUND:Apoptosis is involved in the formation of degenerative joint diseases.Therefore,anti-chondrocyte apoptosis may be an effective way to treat osteoarthritis.Neferine has a wide range of pharmacological activities including anti-inflammatory,anti-tumor and anti-apoptotic activities,and its effect on chondrocyte apoptosis is not clear. OBJECTIVE:To investigate the effect of neferine on chondrocyte apoptosis induced by interleukin-1β and elucidate its possible mechanism. METHODS:(1)The rat chondrocytes at logarithmical stage were taken and intervened in five groups.The control group was cultured routinely.The model group was routinely cultured for 24 hours after treatment with interleukin-1β for 2 hours.The low-,medium-,and high-dose groups were treated with interleukin-1β for 2 hours and then cultured with 5,10,and 20 μmol/L neferine for 24 hours,respectively.At the end of culture,cell apoptosis,chondroglycoprotein 39 and type Ⅱ collagen levels in cell supernatants,mRNA and protein expression of apoptosis-related proteins,and mRNA and protein expression of proteins related to the protein kinase R-like endoplasmic reticulum kinase(PERK)/activating transcription factor 4(ATF4)signaling pathway were detected.(2)Rat chondrocytes at logarithmic growth period were taken and divided into four groups:control group and model group were treated with the same intervention as above,drug group was treated with interleukin-1β for 2 hours and then cultured with 20 μmol/L neferine for 24 hours,and activator group was treated with interleukin-1β for 2 hours and then cultured with 20 μmol/L neferine and CCT020312,an activator of PERK/ATF4 signaling pathway,for 24 hours.At the end of culture,cell proliferation was detected by cell counting kit-8 assay,apoptosis was detected by flow cytometry,and mRNA and protein expressions of apoptosis-related proteins and PERK/ATF4 signaling pathway-related proteins were detected. RESULTS AND CONCLUSION:(1)Compared with the control group,the model group showed increased apoptosis(P<0.05),decreased proliferative activity(P<0.05),increased level of chondroglycoprotein 39(P<0.05),decreased level of type Ⅱ collagen(P<0.05),decreased mRNA and protein expression of Bcl-2 protein(P<0.05),increased mRNA and protein expression of Bax protein,increased caspase-3 mRNA expression,increased Cleaved-caspase-3 protein expression(P<0.05),increased mRNA expression of PERK,ATF4,and C/EBP homologous protein(P<0.05),and increased protein expression of p-PERK,ATF4,and C/EBP homologous protein(P<0.05).Compared with the model group,neferine reversed the above effects of interleukin-1β on chondrocytes in a concentration-dependent manner.(2)Compared with the drug group,the activator group showed increased apoptosis(P<0.05),decreased proliferative activity(P<0.05),elevated mRNA expression of caspase-3,ATF4,and C/EBP homologous protein(P<0.05),and elevated protein expression of Cleaved-caspase-3,ATF4,and C/EBP homologous protein(P<0.05).(3)To conclude,neferine inhibits interleukin-1β-induced chondrocyte apoptosis and enhances cell proliferation activity,and the mechanism of action may be related to blocking the PERK/ATF4 signaling pathway in the endoplasmic reticulum.
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Objective:To study the effects of endoplasmic reticulum stress (ERS) and autophagy mechanisms in the pathogenesis of necrotizing enterocolitis (NEC).Methods:A total of 39 newborn SD rats were randomly assigned into 3 groups: the NEC group (NEC model: artificial feeding+hypoxic stimulation+intragastric injection of lipopolysaccharides), the ERS antagonist group (NEC model+intraperitoneal injection of 4-phenylbutyric acid) and the ERS inducer group (NEC model+intraperitoneal injection of tunicamycin). After successful modeling, the rats were sacrificed and intestinal tissues were obtained. The intestinal pathology was observed using electronic microscope. Intestinal fatty acid binding protein (I-FABP) was detected using ELISA. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to examine mRNA levels of glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150). Western-blot was used to detect p62 and autophagy-related proteins (microtubule-associated protein 1 light chain 3 (LC3)Ⅱ and LC3Ⅰ) and LC3Ⅱ/LC3Ⅰ ratio was calculated.Results:(1) For all 3 groups, the pathological scores of rat intestines were≥2. (2) The ERS inducer group showed significantly higher clinical score, pathological score, I-FABP level, GRP78 and ORP150 mRNA levels and LC3Ⅱ/ LC3Ⅰ ratio than the NEC group and ERS antagonist group, and the NEC group higher than the ERS antagonist group ( P<0.05). The p62 level in the ERS inducer group was significantly lower than the NEC group and the ERS antagonist group, and the NEC group lower than the ERS antagonist group ( P<0.05). (3) The LC3Ⅱ/ LC3Ⅰ ratio was positively correlated with clinical score, pathological score and I-FABP level ( P<0.05). The p62 level was negatively correlated with clinical score, pathological score and I-FABP level ( P<0.05). The mRNA levels of GRP78 and ORP150 were positively correlated with clinical score, pathological score and I-FABP level ( P<0.05). LC3Ⅱ/ LC3Ⅰ ratio was positively correlated with the mRNA levels of GRP78 and ORP150 ( P<0.05). The p62 level was negatively correlated with the mRNA levels of GRP78 and ORP150 ( P<0.05). Conclusions:ERS is associated with the pathogenesis of NEC. Inhibition of ERS can reduce autophagy and improve intestinal barrier function and clinical symptoms of NEC.
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Objective To investigate the effect of inhibiting autophagy induced by endoplasmic reticulum stress(ERS)on necrotiz-ing enterocolitis(NEC)in neonatal rats.Methods First,the NEC model of neonatal rats was established.Then,the intestinal epitheli-al cells were isolated and divided into three groups:control group,inhibition group and induction group.The control group was cultured normally,the inhibition group was added with 4-phenylbutyric acid,and the induction group was added with tunicamycin for 24hours.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of the cellular inflammatory cytokines tumor necrosis factor-α(TNF-α)and intestinal fatty acid binding protein(I-FABP)in each group.Real-time quantitative polymerase chain reac-tion(RT-qPCR)was used to detect the mRNA expression level of the markers of ERS glucose regulated protein 78(GRP78)and oxy-gen-regulated protein 150(ORP150).Western blot was used to detect the expression of autophagy related proteins LC3 Ⅱ/Ⅰ and p62.Results Compared with the control group,the expression of p62 in the inhibition group increased significantly,the expression of TNF-α,I-FABP,GRP78,ORP150,LC3 Ⅱ/Ⅰ in the inhibition group was significantly decreased,while the expression of p62 in the induc-tion group was significantly decreased,the expressions of TNF-α,Ⅰ-FABP,GRP78,ORP150,LC3 Ⅱ/Ⅰ were significantly increased,and the differences were statistically significant(P＜0.05).Conclusion Inhibition of ERS induced autophagy activation can alleviate intestinal mucosal injury and inflammatory response in neonatal rats with NEC and improve intestinal barrier function.
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AIM:To investigate the influence of fat mass and obesity-associated(Fto)gene on the aberrant contraction of aortic smooth muscle in diabetes mellitus(DM)mice,and to explore the mechanism of Fto gene underlying the calcium regulation.METHODS:Smooth muscle-specific Fto gene knockout(FtoSMKO)mice were generated using Cre-loxP technology.The experiment involved 3 groups of mice:wild-type(WT)group,DM model group and FtoSMKO-DM group,with 15 mice in each group.In DM group and FtoSMKO-DM group,type 1 DM was induced by intraperitoneal injec-tion of streptozotocin.The mice in WT group were injected with equal volume of citric acid-sodium citrate buffer solution.The influences of different drugs on the contraction responses of aortic smooth muscle in mice were analyzed using a multi-myograph system.The expression level of FTO protein in the aortic tissues was detected by Western blot.RESULTS:(1)Compared with WT mice,the expression levels of FTO protein in the aortic tissues of DM mice were significantly in-creased(P＜0.01).(2)The expression level of FTO protein in smooth muscle was significantly decreased after knockout of Fto gene(P＜0.01).Compared with WT group,the mice in DM group exhibited a significant decrease in body weight and a marked increase in fasting blood glucose level(P＜0.05).There were no noticeable differences in body weight or fasting blood glucose level between FtoSMKO-DM group and DM group(P＞0.05).(3)The contraction responses of aortic smooth muscle in DM group were substantially increased by phenylephrine compared with WT group.Specifically,vaso-constriction responses mediated by non-L-type calcium channels and store-operated calcium channels(SOCC)were signifi-cantly enhanced in DM group.In addition,the responses mediated by inositol 1,4,5-trisphosphate receptors(IP3R),which facilitate calcium release from the sarcoplasmic reticulum,were significantly enhanced.However,the responses mediated by caffeine-activated ryanodine receptors(RyR),which also facilitate calcium release from the sarcoplasmic re-ticulum,were significantly inhibited(P＜0.05).(4)Compared with DM group,the phenylephrine-induced contraction re-sponses of aortic smooth muscle in FtoSMKO-DM group were greatly weakened(P＜0.05).In particular,the vasoconstriction responses mediated by non-L-type calcium channels and SOCC in FtoSMKO-DM group were greatly suppressed(P＜0.05),while those mediated by caffeine-activated RyR were dramatically boosted(P＜0.05).However,IP3R-mediated responses were not affected(P＞0.05).CONCLUSION:Smooth muscle-specific Fto gene knockout suppresses contractile hyperre-sponsiveness in the aortic smooth muscle of DM mice,which may be attributed to involvement of FTO protein in calcium regulation in the vascular smooth muscle.
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AIM:To study the regulation of endoplasmic reticulum stress(ERS)using the glucose regulated protein 78(GRP78)/CCAAT/enhancer binding protein homologous protein(CHOP)pathway and explore the related mech-anism of Wenyang-Shengji ointment in facilitating the repair of diabetic refractory wounds.METHODS:To establish a rat model of diabetic refractory wound repair,Sprague-Dawley(SD)rats were fed a high-fat diet and intraperitoneally in-jected with streptozotocin.Subsequently,full-thickness skin defects were induced in the dorsal region of the rats.The ex-periment included 4 groups:normal,model(diabetic refractory wounds),Wenyang-Shengji ointment,and Beifuxin(re-combinant bovine basic fibroblast growth factor gel)groups.The normal and model groups were treated with normal saline after disinfection.In the Wenyang-Shengji ointment and Beifuxin groups,the wounds were topically treated with the re-spective ointments once daily.After 14 d of treatment,wound healing was assessed and quantified using the wound healing rate.Hematoxylin-eosin(HE)staining was employed to examine the micromorphology of the wound tissue.Western blot analysis was performed to measure GRP78,CHOP and caspase-12 levels in the wound tissue.Immunohistochemical analy-sis was used to detect the expression and distribution patterns of GRP78,CHOP and caspase-12 in the wounds.Transmis-sion electron microscopy was used to observe reticulum numbers and swelling.Enzyme-linked immunosorbent assay was used to determine interleukin-1β(IL-1β)level as a pro-inflammatory factor within the wound.RESULTS:Indexes of each group were assessed 14 d after the corresponding intervention.Compared with normal group,the rats in model group exhibited a significant decrease in the wound healing rate(P＜0.01),accompanied by increased inflammatory exudation and poor granulation tissue growth.Additionally,there were increases in the expression levels of GRP78,CHOP and cas-pase-12 proteins(P＜0.01),as well as a significant elevation in the content of inflammatory factor IL-1β(P＜0.01).In contrast,compared with model group,treatment with Wenyang-Shengji ointment resulted in a significant improvement in wound healing rate(P＜0.01),reduction in inflammatory exudation,and enhanced granulation tissue growth(P＜0.01).Furthermore,there was a notable decrease in the protein expression of GRP78/CHOP/caspase-12 within the wound tissue following treatment with Wenyang-Shengji ointment(P＜0.01).The levels of inflammatory factor IL-1β also showed a sig-nificant decrease(P＜0.01).CONCLUSION:Wenyang-Shengji promotes the healing of diabetic refractory wounds,which may be associated with the downregulation of the GRP78/CHOP pathway,inhibition of excessive ERS,and reduc-tion in the level of wound cell apoptosis.
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Endoplasmic reticulum stress(ERS)is a subcellular pathological state formed by imbalance of environment in endoplasmic reticulum,and is regarded as an important pathway that mediates apoptosis.Recent studies have found that after ERS occurs in various immune tissues and cells,immunosuppressive effect that produced is involved in regulating body's immune homeo-stasis,affecting occurrence,outcome and prognosis of many diseases.This article reviews production of ERS,signal regulation and pathophysiological changes in immune organs and immune cells,and its role in development of inflammatory diseases and tumors.
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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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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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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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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.