Mechanism of Yishen Tongluo Prescription in Inhibiting Endoplasmic Reticulum Stress and Improving Apoptosis of Renal Tubular Epithelial Cells Based on PERK/ATF4/CHOP / 中国实验方剂学杂志

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.

Effects and mechanisms of total flavones of Abelmoschus manihot in attenuating diabetic tubulopathy by targeting endoplasmic reticulum stress-induced cell apoptosis / 中国中药杂志

Renal tubular injury in patients with diabetic kidney disease(DKD) may be accompanied by glomerular and microvascular diseases. It plays a critical role in the progression of renal damage in DKD, and is now known as diabetic tubulopathy(DT). To explore the multi-targeted therapeutic effects and pharmacological mechanisms in vivo of total flavones of Abelmoschus manihot(TFA), an extract from traditional Chinese medicine for treating kidney disease, in attenuating DT, the authors randomly divided all rats into four groups: a normal control group(normal group), a DT model group(model group), a DT model+TFA-treated group(TFA group) and a DT model+rosiglitazone(ROS)-treated group(ROS group). The DT rat model was established based on the DKD rat model by means of integrated measures. After successful modeling, the rats in the four groups were continuously given double-distilled water, TFA suspension, and ROS suspension, respectively by gavage every day. After 6 weeks of treatment, all rats were sacrificed, and the samples of their urine, blood, and kidneys were collected. The effects of TFA and ROS on various indicators related to urine and blood biochemistry, renal tubular injury, renal tubular epithelial cell apoptosis and endoplasmic reticulum stress(ERS), as well as the activation of the protein kinase R-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP) signaling pathway in the kidney of the DT model rats were investigated. The results indicated that hypertrophy of renal tubular epithelial cells, renal tubular hyperplasia and occlusion, as well as interstitial extracellular matrix and collagen deposition occurred in the DT model rats. Moreover, significant changes were found in the expression degree and the protein expression level of renal tubular injury markers. In addition, there was an abnormal increase in tubular urine proteins. After TFA or ROS treatment, urine protein, the characteristics of renal tubular injury, renal tubular epithelial cell apoptosis and ERS, as well as the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney of the DT model rats were improved to varying degrees. Therein, TFA was superior to ROS in affecting the pathological changes in renal tubule/interstitium. In short, with the DT model rats, this study demonstrated that TFA could attenuate DT by multiple targets through inhibiting renal tubular ERS-induced cell apoptosis in vivo, and its effect and mechanism were related to suppressing the activation of the PERK-eIF2α-ATF4-CHOP signaling pathway in the kidney. These findings provided preliminary pharmacological evidence for the application of TFA in the clinical treatment of DT.

Augmented reality in interventional radiology education: a systematic review of randomized controlled trials

ABSTRACT BACKGROUND: Augmented reality (AR) involves digitally overlapping virtual objects onto physical objects in real space so that individuals can interact with both at the same time. AR in medical education seeks to reduce surgical complications through high-quality education. There is uncertainty in the use of AR as a learning tool for interventional radiology procedures. OBJECTIVE: To compare AR with other learning methods in interventional radiology. DESIGN AND SETTING: Systematic review of comparative studies on teaching techniques. METHODS: We searched the Cochrane Library, MEDLINE, Embase, Tripdatabase, ERIC, CINAHL, SciELO and LILACS electronic databases for studies comparing AR simulation with other teaching methods in interventional radiology. This systematic review was performed in accordance with PRISMA and the BEME Collaboration. Eligible studies were evaluated using the quality indicators provided in the BEME Collaboration Guide no. 11, and the Kirkpatrick model. RESULTS: Four randomized clinical trials were included in this review. The level of educational evidence found among all the papers was 2B, according to the Kirkpatrick model. The Cochrane Collaboration tool was applied to assess the risk of bias for individual studies and across studies. Three studies showed an improvement in teaching of the proposed procedure through AR; one study showed that the participants took longer to perform the procedure through AR. CONCLUSION: AR, as a complementary teaching tool, can provide learners with additional skills, but there is still a lack of studies with a higher evidence level according to the Kirkpatrick model. SYSTEMATIC REVIEW REGISTRATION NUMBER: DOI 10.17605/OSF.IO/ACZBM in the Open Science Framework database.

Catalpol improves non-alcoholic fatty liver and lipid-lowering mechanism through endoplasmic reticulum stress-mediated PERK-eIF2α signaling pathway / 中华内分泌代谢杂志

Objective:To investigate effect and underlying lipid-lowering mechanisms of catalpol in non-alcoholic fatty liver disease(NAFLD).Methods:In vivo model of NAFLD was established with high-fat diet-fed ICR mice for 8 weeks. Low(50 mg/kg), medium(150 mg/kg), and high(300 mg/kg) doses of catalpol were administered, and the body weight, liver weight, hepatic index, and biochemical parameters of the mice were analyzed. Free fatty acid-induced LO2 in human hepatocytes to establish NAFLD cell model. Quantitative realtime PCR reaction to detect fatty acid synthesis-related gene levels. Western blotting assay was adopted to analyze proteins in the endoplasmic reticulum stress(ERS)-mediated protein kinase RNA-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α) signaling pathway. Results:Compared with model mice, body weight [(39.43±1.84)g, (34.01±1.83)g, (32.28±1.11)g vs(42.17±1.37)g, all P<0.001], liver weight [(1.03±0.06)g, (0.79±0.05)g, (0.64±0.04)g vs(1.30±0.13)g, P<0.01 or P<0.001], and liver index [(2.60±0.09)%, (2.32±0.09)%, (1.99±0.11)% vs(3.07±0.30)%, P<0.05 or P<0.001] were reduced in low, medium, and high doses of catapol model. Medium and high doses of catalpol diminished total cholesterol, triglyceride, low density lipoprotein-cholesterol, aspartate aminotransferase, and alanine aminotransferase( P<0.01 or P<0.001), increased high density lipoprotein-cholesterol( P<0.01 or P<0.001). In the cell model, elevated levels of both fatty acid synthesis genes and PERK-eIF2α pathway proteins were attenuated by catalase, and this attenuation was reversed by signaling pathway agonists. Conclusion:The Chinese herb catalpol may play a role in improving NALFD by regulating the ERS-mediated PERK-eIF2α signaling pathway.

Effects of Ziziphi Spinosae Semen and Albiziae Flos on Behavior and Endoplasmic Reticulum Stress PERK/ATF4/CHOP Pathway in Depression Model Rats / 中国实验方剂学杂志

Objective:To investigate the effect of Ziziphi Spinosae Semen （ZSS） and Albiziae Flos （AF） on behavior and endoplasmic reticulum stress endoplasmic reticulum stress protein kinase R-like endoplasmic reticulum kinase （PERK）/activated transcription factor 4 （ATF4）/CCAAT enhancer binding protein （CHOP） pathway in depression model rats， and to explore its antidepressant mechanism. Method:The male SD rats were divided into normal group， model group， ZSS-AF high dose， middle dose and low dose groups （16， 8， 4 g·kg^-1） and Venlafaxine group （0.008 g·kg^-1）， <italic>n</italic>=15 in each group. Except the normal group， the depression model was established in the rats of other 5 groups by the method of chronic unpredictable mild stress （CUMS） combined with isolated feeding. The normal group and model group were given with distilled water by gavage when modeling， while other groups received corresponding drug by intragastric administration for 21 days. Behavior changes of rats in each group were observed by the open field test and sugar water consumption test on 1^thand 21^thday of the experiment. The protein expressions of PERK， CHOP， B-cell lymphoma-2 associated X protein （Bax） and cysteine-containing aspartate-specific proteases-3（Caspase-3） were detected by Western blot（WB）， the ultrastructural changes of the hippocampus were observed by transmission electron microscope， the apoptosis of hippocampal neurons was observed by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL） method. Result:Compared with the normal group， the scores of open field test and sugar water consumption rate in model group rats decreased （<italic>P</italic><0.01）. Compared with the model group， the scores of open field test and water consumption rate increased （<italic>P</italic><0.01） in ZSS-AF groups and Venlafaxine group. Transmission electron microscope showed that the changes of neuronal damage in hippocampal were revealed in the model group， whereas those neuronal damages were relieved in ZSS-AF groups and Venlafaxine group. TUNEL method showed that the number of apoptotic neurons in hippocampal increased in the model group （<italic>P</italic><0.01）， but decreased in ZSS-AFgroups and Venlafaxine group （<italic>P</italic><0.01）. WB results showed that as compared with the normal group， protein expressions of PERK， CHOP， Bax and Caspase-3 were up-regulated significantly in the model group （<italic>P</italic><0.01）， whereas those were down-regulated in ZSS-AF groups and Venlafaxine group （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:The antidepressant effect of ZSS-AF herbal pair may be correlated with the regulation of endoplasmic reticulum stress PERK/ATF4/-CHOP pathway.

Protective effect of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress / 中国中药杂志

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P<0.05 or P<0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P<0.05 or P<0.01), while Bcl-2 was significantly up-regulated(P<0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P<0.01), whereas Bcl-2 was significantly up-regulated(P<0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.

Spinal protein kinase A and phosphorylated extracellular signal-regulated kinase signaling are involved in the antinociceptive effect of phytohormone abscisic acid in rats / A proteína quinase A da medula espinhal e a sinalização da quinase fosforilada regulada por sinal extracelular estão envolvidas no efeito antinociceptivo do ácido fito-hormônio abscísico em ratos

Abstract Objective: The phytohormone abscisic acid (ABA) as a signaling molecule exists in various types of organisms from early multicellular to animal cells and tissues. It has been demonstrated that ABA has an antinociceptive effect in rodents. The present study was designed to assess the possible role of PKA and phosphorylated ERK (p-ERK) on the antinociceptive effects of intrathecal (i.t.) ABA in male Wistar rats. Methods: The animals were cannulated intrathecally and divided into different experimental groups (n=6‒7): Control (no surgery), vehicle (received ABA vehicle), ABA-treated groups (received ABA in doses of 10 or 20 µg/rat), ABA plus H.89 (PKA inhibitor)-treated group which received the inhibitor 15 min prior to the ABA injection. Tail-flick and hot-plate tests were used as acute nociceptive stimulators to assess ABA analgesic effects. p-ERK was evaluated in the dorsal portion of the spinal cord using immunoblotting. Results: Data showed that a microinjection of ABA (10 and 20 µg/rat, i.t.) significantly increased the nociceptive threshold in tail flick and hot plate tests. The application of PKA inhibitor (H.89, 100 nM/rat) significantly inhibited ABA-induced analgesic effects. Expression of p-ERK was significantly decreased in ABA-injected animals, which were not observed in the ABA+H.89-treated group. Conclusions: Overall, i.t. administration of ABA (10 µg/rat) induced analgesia and p-ERK down-expression likely by involving the PKA-dependent mechanism.

Resumo Objetivo: O ácido fito-hormônio abscísico (ABA) existe como molécula sinalizadora em vários tipos de organismos, de multicelulares a células e tecidos animais. Foi demonstrado que o ABA tem efeito antinociceptivo em roedores. O presente estudo foi desenhado para avaliar o possível papel da PKA e da ERK fosforilada (p-ERK) nos efeitos antinociceptivos do ABA intratecal (i.t.) em ratos Wistar machos. Métodos: Os animais foram canulados por via i.t. e divididos em diferentes grupos experimentais (n=6‒7): controle (sem cirurgia), veículo (veículo ABA recebido), grupos tratados com ABA (recebeu ABA em doses de 10 ou 20 µg/rato), grupo tratado com ABA mais H.89 (inibidor de PKA) que recebeu o inibidor 15 minutos antes da injeção de ABA. Os testes de movimento da cauda e placa quente foram utilizados como estimuladores nociceptivos agudos para avaliar os efeitos analgésicos da ABA. A p-ERK foi avaliada na porção dorsal da medula espinhal por imunotransferência. Resultados: A microinjeção de ABA (10 e 20 µg/rato, i.t.) aumentou significativamente o limiar nociceptivo nos testes de movimento da cauda e placa quente. A aplicação de inibidor de PKA (H.89, 100 nM/rato) inibiu significativamente os efeitos analgésicos induzidos por ABA. A expressão de p-ERK diminuiu significativamente em animais injetados com ABA que não foram observados no grupo tratado com ABA+H.89. Conclusões: No geral, a administração i.t. de ABA (10 µg/rato) induziu a analgesia e expressão negativa de p-ERK provavelmente envolvendo mecanismo dependente de PKA.

Effect of nickel sulfate on cell survival rate and related apoptotic proteins in human normal hepatocytes / 中国职业医学

OBJECTIVE: To investigate the effect of nickel sulfate on cell survival rate and apoptosis of normal human liver L02 cells. METHODS: i) L02 cells in logarithmic growth phase were divided into 9 groups, each with 6 wells. L02 cells in each group were treated with 0, 100, 200, 300, 400, 500, 600, 700 and 800 μmol/L nickel sulfate. The survival rate of L02 cells was determined by CCK-8 assay after cells were treated for 0, 6, 12, 24, 48 and 72 hours. The nickel sulfate exposure dose and exposure time for subsequent experiments were selected based on the results of CCK-8 assay. ii) L02 cells in logarithmic growth phase were divided into control group, 100 and 300 μmol/L dose groups, and were exposed to 0, 100 and 300 μmol/L nickel sulfate for 12 hours, respectively. Western blot was used to detect the relative protein expression of B cell lymphoma/leukemia 2(BCL-2), Bcl-2 related protein X(BAX), caspase-3, phosphorylated RNA-dependent protein kinase-like endoplasmic reticulum kinase(p-PERK), phosphorylated eukaryotic translation initiation factor 2α(p-eIF2α), CCAAT/enhancer-binding protein homologous protein(CHOP) and glucose regulatory protein 78(GRP78). RESULTS: i) After treatment with nickel sulfate, the survival rate of cells decreased with the increase of dose and the prolongation of exposure time(all P values were <0.01). According to the half inhibitory concentration of nickel sulfate on L02 cells, the nickel sulfate exposure time in subsequent experiments was selected as 12 hours, and the exposure concentration was 100 and 300 μmol/L. ii) Compared with the control group, the relative expression of BCL-2 protein in L02 cells in the 100 and 300 μmol/L dose groups decreased(all P values were <0.05), while the relative protein expression of BAX, caspase-3 protein and ratio BAX/BCL-2 increased(all P values were <0.05). Compared with 100 μmol/L dose group, the relative expression of BCL-2 protein in L02 cells of 300 μmol/L dose group decreased(P<0.05), while the relative expression of BAX and caspase-3 protein and the ratio of BAX/BCL-2 increased(all P values were <0.05). Compared with the control group, the relative expression levels of p-PERK, p-eIF2α, CHOP and GRP78 protein in L02 cells were increased in 100 and 300 μmol/L dose groups(all P values were P<0.05). Compared with 100 μmol/L dose group, the relative expression levels of p-eIF2α, CHOP and GRP78 protein in 300 μmol/L dose group were increased(all P values were<0.05).CONCLUSION: Nickel sulfate can regulate the expression of apoptosis related proteins and PERK signaling pathway related proteins in L02 cells, aggravate apoptosis of L02 cells and decrease the cell survival rate.

Effect of periapical inflammation on calcium binding proteins and ERK in the trigeminal nucleus / Efecto de la inflamación periapical sobre las proteínas fijadoras de calcio y ERK en el núcleo trigeminal

Peripheral inflammation induces plastic changes in neurons and glia which are regulated by free calcium and calcium binding proteins (CaBP). One of the mechanisms associated with the regulation of intracellular calcium is linked to ERK (Extracellular Signal-Regulated Kinase) and its phosphorylated condition (pERK). ERK phosphorylation is important for intracellular signal transduction and participates in regulating neuroplasticity and inflammatory responses. The aim of this study is to analyse the expression of two CaBPs and pERK in astrocytes and neurons in rat trigeminal subnucleus caudalis (Vc) after experimental periapical inflammation on the left mandibular first molar. At seven days post-treatment, the periapical inflammatory stimulus induces an increase in pERK expression both in S100b positive astrocytes and Calbindin D28k positive neurons, in the ipsilateral Vc with respect to the contralateral side and control group. pERK was observed coexpressing with S100b in astrocytes and in fusiform Calbindin D28k neurons in lamina I. These results could indicate that neural plasticity and pain sensitization could be maintained by ERK activation in projection neurons at 7 days after the periapical inflammation.

La inflamación periférica induce cambios plásticos en las neuronas y en la glía, los cuales están regulados por el calcio libre y las proteínas fijadoras calcio (CaBP). Uno de los mecanismos asociados con la regulación del calcio intrace-lular está vinculado con la fosforilación de la pro teína quinasa ERK. Asimismo, ERK fosforilado es importante para la trans-ducción de señales intracelulares y participa en la regulación de la neuroplasticidad y las respuestas inflamatorias. El objetivo de este estudio es analizar la expresión de dos CaBPs y pERK en astrocitos y neuronas del subnúcleo caudal del trigémino (Vc) después de una inflamación periapical experimental en el primer molar inferior izquierdo en ratas. A los siete días posteriores al tratamiento, el estímulo inflamatorio periapical induce un aumento en la expresión de pERK, en el número de astrocitos positivos para la proteína marcadora astroglial S100b y en neuronas positivas para Calbindina D28k, en el Vc ipsilateral respecto del lado contralateral y el grupo de control. Además, se observó coexpresión de pERK tanto en astrocitos S100b positivos, como en neuronas fusiformes Calbindin D28k positivas, de la lámina I. Estas observaciones podrían indicar que la neuroplasticidad y la sensibilización al dolor podrían mantenerse mediante la activación de ERK en las neuronas de proyección a los 7 días de la inflamación periapical.

Effect of Duhuo Jisheng Tang on PERK/Bip Signaling Pathway in Knee Osteoarthritis Model Rats / 中国实验方剂学杂志

Objective: To explore the possible mechanism of Duhuo Jisheng Tang in relieving knee osteoarthritis based on protein kinase R-like endoplasmic reticulum kinase (PERK)/immunoglobulin-binding protein (Bip) signaling pathway.Method: A model of knee osteoarthritis was established by cold stimulation.Rats were randomly divided into blank group,model group,celecoxib group (0.021 g·kg-1),low,medium and high-dose Duhuo Jisheng Tang groups (8.37,16.72,33.48 g·kg-1).Blank group and model group were given equal volume of physiological saline.The changes of knee joint diameter were recorded.The pathological changes of rat articular cartilage were observed by hematoxylin-eosin (HE) staining.The expressions of tumor necrosis factor-alpha (TNF-α),interleukin-1β(IL-1β) and hyaluronic acid (HA) in serum were detected by enzyme-linked immunosorbent assay (ELISA).The mRNA and protein expression levels of PERK,Bip and cysteinyl as parates pecific protein-9(Caspase-9) in cartilage were detected by Real-time PCR and Western blot.Result: The knee joint redn ess and the joint diameter of celecoxib group and high-dose Duhuo Jisheng Tang group were improved,and the joint diameter was reduced significantly (Pα,IL-1β and HA were increased in model group (PPPα,IL-1β and HA in serum of celecoxib group and high-dose Duhuo Jisheng Tang group were decreased (PPPPConclusion: Duhuo Jisheng Tang can alleviate the symptoms of knee osteoarthritis model rats,and its mechanism may be related to the regulation of PERK/Bip signaling pathway in rat cartilage.

Induction of Endoplasmic Reticulum Stress by Cadmium and Its Regulation on Nrf2 Signaling Pathway in Kidneys of Rats / 生物医学与环境科学（英文）

OBJECTIVE@#This study was conducted to investigate the regulation of endoplasmic reticulum stress on Nrf2 signaling pathway in the kidneys of rats.@*METHODS@#Rats were divided into twelve groups of six animals each. Some groups were pre-administered with bacitracin or tauroursodeoxycholic acid (TUDCA), and all of them were treated with 5-20 μmol/kg cadmium (Cd) for 48 h. The oxidative stress levels were analyzed using kits. The mRNA and protein expression levels of endoplasmic reticulum stress-related factors and Nrf2 signaling pathway-related factors were determined using RT-PCR and western blot.@*RESULTS@#Cd exposure resulted in oxidative stress in the kidneys of rats and upregulated the expression of endoplasmic reticulum stress (ERS)-related factors and Nrf2 signaling pathway-related factors, especially at doses of 10 and 20 μmol/kg Cd, and the expression changes were particularly obvious. Moreover, after pretreatment with bacitracin, Cd upregulated the expression of ERS-related factors to a certain extent and, at higher doses, increased the mRNA expression of Nrf2. After pretreatment with TUDCA, Cd reduced the level of ERS to a certain extent; however, at these doses, there were no significant changes in the expression of Nrf2.@*CONCLUSION@#Cadmium can result in ERS and oxidative stress in the kidneys of rats, activate Nrf2, and upregulate the transcriptional expression of phase II detoxification enzymes under these experimental conditions. ERS has a positive regulation effect on Nrf2 signaling pathway but has little effect on the negative regulation of Nrf2 signaling pathway in cadmium toxicity.

Effects of Salvianolic acid on endoplasmic reticulum stress pathway in brain hippocampus of PAP mice / 中国中西医结合急救杂志

Objective To investigate the effect of Salvianolic acid on endoplasmic reticulum stress (ERS) pathway in brain hippocampus of PAP mice. Methods Twenty PAP dual transgenic male mice were selected, they were randomly divided into a PAP mice model group and a Salvianolic acid group, 10 mice in each group; another 10 SPF grade C57BL/6J male mice were selected as a normal control group. In the Salvianolic acid group, 0.9% normal saline solution of Salvianolic lyophilized injection (400 g/L) of dosage 21 mg·kg-1·d-1 was injected intravenously through a tail vein of mice; the PAP mice model and normal control groups were given the same amount of 0.9% normal saline, and the therapeutic course was consecutive 4 weeks in the three groups. At the end of the 4th week, the Morris water maze test was carried out to observe the changes of escape latency, the third quadrant residence time (RTQ), entry angle into water and cross-platform times of mice in each group; amyloid precursor protein (APP) positive cell expression in cerebral hippocampus of mice were detected by immunohistochemistry; Western Blot was used to detect the expression level of PER like endoplasmic reticulum kinase-eukaryon initiation factor 2α-C/EBP homogenous protein (PERK-eIF2α-CHOP) pathway related proteins in hippocampus of mice. Results The escape latency of the PAP mice model group on the 1st to 5th day were significantly longer than those of the normal control group, although a downward trend was observed on the 5th day, it was still significantly longer than that of the model group (seconds: 58.41±2.36 vs. 28.60±10.15); compared with the PAP mice model group, the escape latency of Salvianolic acid group was shorter at each time point, and reached the shortest level on the 5th day (seconds: 31.97±8.36 vs. 58.41±2.36). In the PAP mice model group, the RTQ and the number of crossing platforms were significantly lower than those in the normal control group [RTQ (seconds): 8.27±2.95 vs. 15.97±7.33, numbers of crossing platforms (frequency/90 s): 0.70±0.95 vs. 2.70±0.48]; the entry angle was obviously greater than that of the normal control group [(47.94±4.68)°vs. (32.66±2.55)°, P < 0.05]. Compared with PAP mice model group, in Salvianolic acid group, the RTQ and number of crossing platform were significantly higher [RTQ (seconds): 13.57±1.86 vs. 8.27±2.95, number of crossing platforms (frequency/90 s):1.60±0.97 vs. 0.70±0.47], the entry angle was markedly smaller [(35.46±6.79)°vs. (47.94±4.68)°,P < 0.05]. The positive expression rate of APP and the protein expressions of CHOP, p-eIF2α in PAP mice model group were significantly higher than those in the normal control group [the positive rate of APP: (60.44±6.19)% vs. (21.05±5.87)%, CHOP protein expression (gray value): 3.09±0.07 vs. 1.46±0.09, p-eIF2αprotein expression (gray value): 0.98±0.09 vs. 0.47±0.06, all P < 0.01], the expression of PERK and p-PERK were lower than those in normal control group [PERK (gray value): 0.42±0.06 vs. 0.82±0.11, p-PERK protein expression (gray value): 0.98±0.09 vs. 0.64±0.10, both P < 0.01]; the positive expression rate of APP and protein expressions of CHOP, p-eIF2α in Salvianolic acid group were significantly lower than those in PAP mice model group [positive expression rate of APP: (33.09±10.33)% vs. (60.44±6.19)%, CHOP protein expression (gray value): 1.57±0.12 vs. 3.09±0.07, p-eIF2α protein expression (gray value): 0.80±0.07 vs. 0.98±0.09, all P < 0.01], while PERK and p-PERK expression were significantly higher than those in the model group [PERK (gray value): 0.89±0.12 vs. 0.42±0.06, p-PERK (gray value): 0.78±0.08 vs. 0.98±0.09, both P < 0.01]. Conclusion Salvianolic acid might work through the PERK-eIF2α-CHOP pathway to reduce the retention of APP in the hippocampus tissue of PAP dual-transgenic mice, thereby the learning ability of the mice is improved, and the progression of brain injury delayed.

Effect of sulindac derivative K-80003 in combination with MEK inhibitor cobimetinib in breast cancer cells / 中国药理学通报

Aim: To determine whether the inhibition of K-80003-activated p-ERK could potentiate the anticancer effect of K-80003 in vitro and in vivo. Methods: The effects of K-80003 in combination with the MEK inhibitor cobimetinib on ERK activation and tumor cell apoptosis in breast cancer cells were detected by Western blot and immunohistochemical staining in MCF-7 breast cancer cells and MMTV-PyMT mammary transgenic mice. Results: K-80003 activation of ERK in MCF-7 breast cancer cells and in MMTV-PyMT mammary transgenic mice was strongly inhibited by co-treatment with cobimetinib. The co-treatment also resulted in a strong induction of apoptosis and inhibition of the growth of tumor cells in vitro and in animals, as compared with K-80003 alone. It was detected that K-80003 in combination with cobimetinib synergistically inhibited the growth of MMTV-PyMT tumor strongly, suggesting that K-80003 activation of ERK serves as an escape mechanism by which tumor cells develop resistance to K-80003 treatment. Conclusion: An attractive approach is identified to enhance the therapeutic effect of K-80003 and to overcome potential resistance associated with the K-80003 therapy.

Effect of endoplasmic reticulum stress on the osteogenic differentiation of periodontal ligament cells under continuous static pressure / 口腔疾病防治

Objective@#To study the effect of continuous static pressure on the endoplasmic reticulum of human periodontal ligament cells (hPDLCs) and the mechanism of osteogenic differentiation.@*Methods@#hPDLCs cultured in vitro were subjected to 1 g/cm 2 of continuous compressive pressure (CCP) by custom-made, round, glass panes for 0, 2, 4, and 6 h, respectively. Alkaline phosphatase staining was used to detect osteogenic differentiation, and real-time quantitative PCR was used to detect the expression of protein kinase receptor-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), and transcription activation factor 4 (ATF-4). The 0 h loading group was the control group.@*Results@#After CCP treatment, the alkaline phosphatase staining of hPDLCs was blue-violet and significantly stronger than that of cells in the control group. The expression levels of PERK and ATF4 in the hPDLCs after CCP treatment were higher than those of cells in the control group (P < 0.05) and increased over time (P < 0.05). The expression of eIF2α was lower in the experimental groups than in the control group (P < 0.05) and decreased over time (P < 0.05).@*Conclusion @#Mechanical stimulation can activate ERS in hPDLCs, leading to enhanced PERK-eIF2α-ATF4 signaling and inducing osteogenic differentiation.

Effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism / 中国应用生理学杂志

OBJECTIVE@#To explore the effect of oxidative stress on periprosthetic osteolysis induced by TCP wear particles in mouse calvaria and its mechanism.@*METHODS@#Thirty-six male ICR mice were randomly divided into three groups (=12):sham group, TCP wear particles (TCP) group and N-acetyl-L-cysteine (NAC) group. Aperiprosthetic osteolysis model in mouse was established by implanting 30 mg of TCP wear particles onto the surface of bilateral parietal bones following removal of the periosteum. On the 2nd day post-operation, NAC (1.0 mg/kg) was locally injected to the calvarium under the periosteum every other day for 2 weeks. Then, all the mice were sacrificed to obtain blood and the calvaria. Periprosthetic osteolysis in the mouse calvaria was observed by tartrate resistant acid phosphatase (TRAP) staining; serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), interleukin-6 (IL-6); total anti-oxidation capacity (T-AOC) and superoxide dismutase (SOD) activity were examined by ELISA and chemical colorimetry, respectively; protein levels of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α) and phospho-eIF2α (p-eIF2α) in periprosthetic bone tissue were detected by Western blot.@*RESULTS@#Compared with sham group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were increased obviously in TCP group (<0.05), and serum level of T-AOC and SOD activity were decreased significantly in TCP group (<0.05), GRP78 expression, the ratio of p-PERK and PERK, p-eIF2α and eIF2α in the mouse calvaria of TCP group were up-regulated markedly. Compared with TCP group, serum levels of TNF-α, IL-1β and IL-6, and osteolysis area were decreased markedly in NAC group (<0.05), serum level of T-AOC and SOD activity were increased obviously in NAC group (<0.05), and GRP78 expression, the ratio of p-PERK/PERK and p-eIF2α/eIF2α were obviously down-regulated.@*CONCLUSIONS@#Inhibition of oxidative stress can prevent periprosthetic osteolysis induced by TCP wear particles, which may be mediated by inactivation of PERK/eIF2α signaling pathway.

Effect of allicin on proliferation and apoptosis of KG-1 cells and its molecular mechanism / 中国中药杂志

Allicin is one of the main bioactive substances in garlic, with antibacterial, hypolipidemic and other pharmacological effects. In this study, apoptosis-related indicators were detected to explore the molecular mechanism of allicin on KG-1 cell proliferation inhibition. The apoptosis rate of KG-1 cells induced by allicin was detected by flow cytometry. The effect of allicin on the expressions of Bax, Bcl-2, survivin and ERK mRNA in KG-1 cells was detected by RT-qPCR. Western blot was used to detect the expressions of caspase 3, cleaved caspase 3, ERK1/2, p-ERK1/2 and survivin protein in KG-1 cells. According to the findings, compared with the control group, allicin could significantly inhibit the proliferation activity of KG-1 cells in a concentration-dependent and time-dependent manner. Flow cytometry showed that allicin could induce the apoptosis of KG-1 cells, which was mainly late apoptosis. The results of RT-qPCR showed that the expressions of Bax mRNA, Bcl-2, survivin and ERK mRNA in KG-1 cells increased after treatment with allicin. The results of Western-blot showed that after KG-1 cells were treated with allicin, the expressions of caspase 3 and its active form cleaved caspase 3 increased, the expressions of survivin, ERK1/2 and its active form p-ERK1/2 were decreased, of which p-ERK1/2 was down-regulated in a dose-dependent manner. The above results suggest that allicin inhibited the proliferation of KG-1 cells primarily by inducing late apoptosis; the execution of apoptosis involved cleaved caspase 3; the induction of apoptosis involved the protein expression, the decrease of ERK1/2andexpression of survivin and the dose-dependent decrease of p-ERK1/2; the mRNA expression involved the increase of Bax, and the down-regulation of survivin, Bcl-2 and ERK1/2.

Effect of PERK signaling pathway on osteoblast differentiation of female rats with experimental postmenopausal osteoporosis / 吉林大学学报(医学版)

Objective:To investigate the changes of PERK,Runx2,osterix,RANKL and OPG in bone tissue of the female rats with experimental postmenopausal osteoporosis(PMOP)before and after treatment,and to elucidate the role of PERK signaling pathway in PMOP.Methods:The ovariectomized rats were reproduced to osteoporosis models.A total of 45 rats were divided into normal control group(the rats didn't receive any treatment,n=15),osteoporosis group(the rats were ovariectomized,n=15)and osteoporosis treatment group (the ovariectomized rats were injected with estrogen through caudal vein,n=15).The changes of serum collagenⅠ(Col Ⅰ),alkaline phosphatase(ALP)and osteocalcin(OCN)of the rats in various groups were observed. Three months after feeding,the femoral shaft of the rats in various groups were taken for pathological section.The gene expression levels of PERK,ATF4,Runx2,osterix,RANKL and OPG in bone tissue of the rats in various groups were detected by RT-PCR;the protein expression levels of PERK,ATF4,Runx2,osterix,RANKL and OPG were detected by Western blotting method.Results:Compared with control group,the levels of serum Col Ⅰ,ALP and OCN in the rats in osteoporosis group were significantly decreased(P<0.05 or P<0.01);compared with osteoporosis group,the levels of serum ColⅠ,ALP and OCN of the rats in osteoporosis treatment group were significantly increased(P<0.01).Compared with control group,the gene expression levels of PERK, ATF4,Runx2 and osterix in bone tissue of the rats in osteoporosis group were significantly decreased(P<0.01), and the gene expression level of RANKL was increased(P<0.01);compared with osteoporosis group,the gene expression levels of PERK,ATF4 Runx2 and osterix in bone tissue of the rats in osteoporosis treatment group were significantly increased(P<0.01),and the gene expression level of RANKL was significantly decreased(P<0.01).Compared with control group,the protein expression levels of PERK,Runx2 and osterix in bone tissue of the rats in osteoporosis group were significantly decreased(P<0.05 or P<0.01),and the protein expression level of RANKL were increased(P<0.05 or P<0.01);compared with osteoporosis group,the protein expression levels of PERK,Runx2 and osterix in bone tissue of the rats in osteoporosis treatment group were significantly increased(P<0.05 or P<0.01),and the protein expression level of RANKL was significantly decreased(P<0.01).The HE staining results showed that compared with control group,the bone resorption pits in bone tissue of the rats in osteoporosis group became large with the increased bone absorption,which caused bone loss;compared with osteoporosis group,the resorption in bone tissue of the rats in osteoporosis treatment group was decreased,and the bone structure returned to normal.Conclusion:After the female rats are ovariectomized and injected with estrogen,the expression trends of PERK and osteoblast transcription factors Runx2 and osterix are consistent,in contrast with the osteoclast transcription factor RANKL expression,suggesting that the osteoblast function is reduced and it is related to the decreased expression of PERK in PMOP onset.

Effect of PERK signaling pathway on osteoblast differentiation of female rats with experimental postmenopausal osteoporosis / 吉林大学学报(医学版)

Objective: To investigate the changes of PERK, Runx2, osterix, RANKL and OPG in bone tissue of the female rats with experimental postmenopausal osteoporosis (PMOP) before and after treatment, and to elucidate the role of PERK signaling pathway in PMOP. Methods: The ovariectomized rats were reproduced to osteoporosis models. A total of 45 rats were divided into normal control group (the rats didn' t receive any treatment, n=15), osteoporosis group (the rats were ovariectomized, n=15) and osteoporosis treatment group (the ovariectomized rats were injected with estrogen through caudal vein, n=15). The changes of serum collagen I (Col I), alkaline phosphatase (ALP) and osteocalcin (OCN) of the rats in various groups were observed. Three months after feeding, the femoral shaft of the rats in various groups were taken for pathological section. The gene expression levels of PERK, ATF4, Runx2, osterix, RANKL and OPG in bone tissue of the rats in various groups were detected by RT-PCR; the protein expression levels of PERK, ATF4, Runx2, osterix, RANKL and OPG were detected by Western blotting method. Results: Compared with control group, the levels of serum Col I, ALP and OCN in the rats in osteoporosis group were significantly decreased (P<0.05 or P<0.01); compared with osteoporosis group, the levels of serum Col I, ALP and OCN of the rats in osteoporosis treatment group were significantly increased (P<0.01). Compared with control group, the gene expression levels of PERK, ATF4, Runx2 and osterix in bone tissue of the rats in osteoporosis group were significantly decreased (P<0.01), and the gene expression level of RANKL was increased (P<0.01); compared with osteoporosis group, the gene expression levels of PERK, ATF4 Runx2 and osterix in bone tissue of the rats in osteoporosis treatment group were significantly increased (P<0.01), and the gene expression level of RANKL was significantly decreased (P< 0.01). Compared with control group, the protein expression levels of PERK, Runx2 and osterix in bone tissue of the rats in osteoporosis group were significantly decreased (P<0.05 or P<0.01), and the protein expression level of RANKL were increased (P<0.05 or P<0.01); compared with osteoporosis group, the protein expression levels of PERK, Runx2 and osterix in bone tissue of the rats in osteoporosis treatment group were significantly increased (P<0.05 or P<0.01), and the protein expression level of RANKL was significantly decreased (P< 0.01). The HE staining results showed that compared with control group, the bone resorption pits in bone tissue of the rats in osteoporosis group became large with the increased bone absorption, which caused bone loss; compared with osteoporosis group, the resorption in bone tissue of the rats in osteoporosis treatment group was decreased, and the bone structure returned to normal. Conclusion: After the female rats are ovariectomized and injected with estrogen, the expression trends of PERK and osteoblast transcription factors Runx2 and osterix are consistent, in contrast with the osteoclast transcription factor RANKL expression, suggesting that the osteoblast function is reduced and it is related to the decreased expression of PERK in PMOP onset.

Expression of endoplasmic reticulum-associated apoptosis protein CHOP in lung tissues of COPD model rats / 中国病理生理杂志

AIM:To investigate whether cigarette smoke(CS)promotes the expression of endoplasmic reticu-lum-associated apoptosis protein CCAAT/enhancer-binding protein homologous protein(CHOP)in rat lung tissues. METHODS:Adult male Wistar rats(n=40)were randomly divided into 4 groups with 10 rats in each group: control group,CS-2 group(exposed to CS for 2 months),CS-4 group(exposed to CS for 4 months)and ex-smoking(Ex-S)group (exposed to CS for 4 months and then quit smoking for 1 month).The percentage of forced expiratory volume in 0.3 second to forced vital capacity(FEV0.3/FVC)and peak expiratory flow(PEF)were measured.TUNEL assay was used to detect the apoptotic cells.In situ hybridization and RT-PCR were used to determine the mRNA expression of CHOP.The methods of immunohistochemistry and Western blot were used to determine the protein expression of CHOP.Western blot was also used to determine the protein levels of protein kinase R-like endoplasmic reticulum kinase(PERK),p-PERK,eukaryotic initiation factor(eIF)2αand p-eIF2α.RESULTS:The pulmonary function greatly decreased in the rats exposed to CS for 2 months in comparison with control group(P<0.05),markedly decreased in the rats exposed to CS for 4 months as com-pared with the rats after exposure to CS for 2 months(P<0.05),and was improved little in ex-smoking rats(P>0.05). The structural destruction of the lung was observed in the rats exposed to CS for 2 months,and more obvious changes were found in the rats exposed to CS for 4 months.However,the structural destruction of the lung remained obvious in ex-smok-ing rats.The apoptotic cells were markedly increased in the rats exposed to CS for 2 months and were even more in the rats exposed to CS for 4 months.The apoptotic cells were alveolar epithelial cell I(ACE I),ACE II,vascular endothelial cells and bronchial epithelial cells.The protein levels of p-PERK,p-eIF2αand CHOP were remarkably increased in the rats af-ter exposure to CS for 2 months compared with the control rats(P<0.05),significantly elevated in the rats exposed to CS for 4 months compared with the rats exposed to CS for 2 months(P<0.05),and slightly decreased in ex-smoking rats in comparison with the rats after exposure to CS for 4 months(P>0.05).The total protein levels of PERK and eIF2αdid not change between the control rats and those exposed to CS.CONCLUSION: CS promotes the development of chronic ob-structive pulmonary disease(COPD)by inducing the expression of endoplasmic reticulum-associated apoptosis protein CHOP via PERK/eIF2α/CHOP signaling pathway.

Effects of 1,25-(OH)2D3on endoplasmic reticulum stress and ATG12 expression in pulmonary fibrosis / 医学研究生学报

Objective It is not yet clear whether 1,25-(OH)2D3acts on endoplasmic reticulum stress(ERS)and autoph-agy in pulmonary fibrosis(PF).This study aimed to investigate the roles of ERS and autophagy in the development and progression of pulmonary fibrosis in rats and the effects of 1,25-(OH)2D3on the expressions of ERS-related molecules and autophagy-related gene 12 (ATG12). Methods Ninety male SD rats were randomly divided into a control, a PF model and a treatment group of equal number.Bleomycin was injected into the tracheas of the latter two groups of rats to induce PF.On the second day after modeling, the rats of the treatment group were injected intraperitoneally with 1,25-(OH)2D3at 2 μg/kg,those of the PF model group with 1,25-(OH)2D3solvent at 200 μL,and those of the control group with iso-tonic saline at 200 μL,all once 2 days.Then the mRNA expressions of PERK,ATF4 and ATG12 were measured by real-time PCR and the protein expressions of PERK and ATF4 detected by immunohistochemistry. Results At 14, 21 and 28 days after treat-ment,the expression levels of PERK were significantly higher in the PF model group(2.30±0.19, 3.59±0.27, and 4.63±0.19) and treatment group(1.44±0.34,1.92±0.17,and 2.52±0.15)than in the control(1.01±0.23,1.05±0.09,and 1.04±0.08)(P<0.05), and so were the expression levels of ATF4 in the PF models(2.10±0.12, 3.91±0.14, and 6.20±0.28)and treated rats (1.49±0.27,2.52±0.42, and 4.02±0.31)than in the controls(1.04±0.07,1.05±0.08,and 1.03±0.10)(P<0.05).Compared with the control group,the PF model and treatment groups showed markedly increased expression levels of ATG 12 mRNA at 14 days (P<0.05), but decreased at 21 and 28 days(P<0.05).Both the expressions of PERK and ATF4 proteins were remarkably higher in the model and treatment groups than in the control at 14,21 and 28 days(P<0.05), increasing in a time-dependent manner. Conclusion By suppressing the PERK -eIF2α-ATF4 signaling pathway,1,25-(OH)2D3inhibits the development and progression of pulmonary fibrosis.