Effect of low concentration of sodium fluoride on osteogenic/odontogenic differentiation of human dental pulp cells / 口腔疾病防治

Objective@#To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells (hDPCs) in vitro.@*Methods@#This study was reviewed and approved by the Ethics Committee. hDPCs were cultured using a modified tissue explant technique in vitro. The effects of different concentrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium (MTT) assay. Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium, and the cells were induced in vitro. Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells, and the mRNA expression of the key differentiation factors was detected by RT-qPCR. Moreover, the expression of key molecules of endoplasmic reticulum stress (ERS) was detected by RT-qPCR and Western blot. The data were analyzed with the SPSS 18.0 software package.@*Results@#Low concentration of NaF (0.1 mmol/L) could stimulate cell proliferation in vitro, while a high concentration (5-10 mmol/L) could inhibit cell proliferation (P<0.05). According to the literature and the experimental data, 0.1 mmol/L NaF was selected as the following experimental concentration. The levels of alizarin red S staining were increased after NaF induction of mixed osteogenic/odontogenic differentiation in vitro. The mRNA expression levels of key molecules for osteogenic/odontogenic differentiation, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP) and osteocalcin (OCN), were increased (P<0.05). The mRNA levels of ERS markers (splicing x-box binding protein-1 (sXBP1), glucose-regulated protein 78 (GRP78) and activating transcription Factor 4 (ATF4) were increased in NaF-treated cells. The protein expression levels of key ER stress molecules (phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α) and ATF4) were higher in NaF-treated cells.@*Conclusion@#A low concentration of NaF promotes the osteogenic/odontogenic differentiation of hDPCs and increases the level of ER stress.

The proliferation promoting effect of eIF2α on fibroblasts of rheumatoid arthritis synovium / 中华风湿病学杂志

Objective:To observe the expression characteristics of eukaryotic translation initiation factor 2α(eIF2α), and analyze its proliferation regulation effect on fibroblasts of rheumatoid arthritis synovium.Methods:The synovial tissues were collected in patients with rheumatoid arthritis(RA)(40 cases) and osteoarthritis(OA)(40 cases). EIF2α and proliferating cell nuclear antigen(PCNA) were detected by immunohistochemistry method. Fibroblast cell line of rheumatoid arthritis synovium(MH7A) were cultured to establish si-eIF2α group(siRNA-eIF2α plasmid transfection), vector transfection group and blank control group in vitro. PCNA was detected by Western blot method, cell proliferation activity was detected by CCK-8 method. χ2 test was performed on count data, two-sample t-test was performed on quantitative data, one-way analysis of variance (ANOVA) was performed to compare the means of more than two groups, regression equation was calculated by correlation regression analysis. Results:The positive rate of eIF2α was significantly higher in RA synovial fibroblasts than that of OA [52.5%(21/4) vs 20.00%(8/20), χ2=9.14, P=0.003]. Positive correlation was found between eIF2α and PCNA in RA ( Y=0.366 X+2.220, P=0.001) . Compared with blank control group and vector transfection group, cell proliferation activity decreased significantly in si-eIF2α group of MH7A cell line at 72 h [(0.65±0.08) vs (0.96±0.12) vs (1.09±0.06), F=4.52, P=0.022] and 96 h [(1.13±0.14) vs (1.42±0.97) vs (1.56±0.12), F=9.87, P=0.001) , PCNA expression decreased significantly [(0.84±0.15) vs (1.32±0.18) vs (1.28±0.14), F=5.22, P=0.012) . Conclusion:High expression of eIF2α can promote the proliferation of fibroblasts of RA synovium.

Study of endoplasmic reticulum stress in human dental pulp cells induced by oxygen-glucose deprivation culture in vitro / 口腔疾病防治

Objective@#Oxygen-glucose deprivation (OGD) is used to mimic ischemia in vitro to observe whether endoplasmic reticulum (ER) stress is involved in human dental pulp cells (hDPCs) after OGD and to better understand the regulatory mechanism of hDPCs in ischemia.@*Methods@# hDPCs were cultured in glucose-free DMEM and hypoxia (volume fraction 2% O2) to establish an hDPCs OGD model in vitro, which mimics hDPCs ischemia in vitro. hDPCs were divided into a control group (normal culture) and an experimental group (OGD 0 h, 2 h, 4 h and 8 h groups). After pretreatment with OGD for 0, 2, 4 and 8 h, hDPC viability was measured by methylthiazol tetrazolium (MTT) assay. qRT-PCR was used to detect the mRNA expression of ER stress markers [splicing x-box binding protein1 (sXBP1), activating transcription Factor 4 (ATF4) and C/EBP homologous protein (chop)]. Western blot was used to detect the protein expression of ER stress markers [phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-perk) and phosphorylated eukaryotic initiation factor-2α (p-eIF2α)]. @*Results@#Compared with OGD 0 h group, cell viability of hDPCs decreased when exposed to OGD treatment for 2 h, 4 h and 8 h. Compared with the control group, mRNA expressions of ER stress makers (sXBP1, ATF4 and chop) and the protein expressions of ER stress protein markers (p-perk andp-eIF2α) increased in OGD treatment cells after 4 h were higher in OGD cells. The differences were statistically significant (P<0.05).@*Conclusion@#The results indicate that ER stress response is involved in hDPCs in OGD treatment.

Pollen Typhae Total Flavone Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis in Human Aortic-Vascular Smooth Muscle Cells through Down-Regulating PERK-eIF2α-ATF4-CHOP Pathway / 中国结合医学杂志

OBJECTIVE@#To test the hypothesis that the inhibition of endoplasmic reticulum (ER) stress-induced apoptosis in oxidized low-density lipoproteins (ox-LDL)-induced human aortic-vascular smooth muscle cells (HA-VSMCs) was associated with suppression of the protein kinase RNA-like ER kinase (PERK)-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) signaling pathway by Pollen Typhae total flavone (PTF).@*METHODS@#Primary HA-VSMCs were cultured and identified. The cultured HA-VSMCs were randomized into 5 groups, including a normal control group, an ox-LDL group (70 μg/mL high ox-LDL), an HPTF group (70 μg/mL high ox-LDL+500 μg/mL PTF), an MPTF group (70 μg/mL high ox-LDL+250 μg/mL PTF), and a LPTF group (70 μg/mL high ox-LDL+100 μg/mL PTF) in the first part; and a normal control group, an ox-LDL group (70 μg/mL high ox-LDL), an MPTF group (70 μg/mL high ox-LDL+250 μg/mL PTF), a shRNA group (transducted with PERK shRNA lentiviral particles), a scramble shRNA group (transducted with control shRNA lentiviral particles), an MPTF+ox-LDL+shRNA group (250 μg/mL PTF+70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) and an ox-LDL+shRNA group (70 μg/mL high ox-LDL+PERK shRNA lentiviral particles) in the second part. The protein expression levels of ER-associated apoptosis proteins were detected by Western blot, and their mRNA expression levels were detected by quantitative real-time reverse transcription-polymerase chain reaction. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to test cell viability, and the level of apoptosis was monitored by flow cytometry.@*RESULTS@#The MTT assay and flow cytometry showed that the ox-LDL group had a significant increase in apoptosis, which was attenuated in PTF treatment groups and shRNA groups. Moreover, the ox-LDL group had increased protein and mRNA levels of binding immunoglobulin protein and ER-associated apoptosis proteins, such as PERK, eIF2α, ATF4 and CHOP, which were attenuated in PTF treatment groups and shRNA groups.@*CONCLUSIONS@#The apoptosis induced by ox-LDL had a strong relation to ER stress. The protective effect of PTF on ER stressinduced apoptosis was associated with inhibition of the PERK-eIF2α-ATF4-CHOP pathway, which might be a potential therapeutic strategy for enhancing the stability of atherosclerotic plaques.

Effect of electroacupuncture of "Fengfu" (GV16) and "Taichong" (LR3) on EIF2α-ATF4-GRP78/Bip signaling in substantia nigra of rats with Parkinson's disease / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) on expression of eukaryotic initiation factor 2α (EIF2α), activating transcription factor 4 (ATF4), glucose regulator protein-78 / immunoglobulin heavy-chain-binding protein (GRP78/Bip) in the substantia nigra (SN) in rats with Parkinson's disease (PD), so as to explore its mechanism underlying improvement of PD. METHODS: Forty-eight male SD rats were randomly divided into control, sham-operation, model and EA groups (n=12 in each group). The PD model was established by 28-day consecutive subcutaneous injection of rotenone (1 mg/kg dissolved in dimethyl sulfoxide and normal saline) at the back shoulder. EA (2 Hz, 1 mA) was applied to "Fengfu" (GV16) and "Taichong" (LR3) for 30 min, once daily for 2 weeks. The behavio-ral changes of rats in each group were measured and scored at 28th day and 44th day, respectively. The expressions of tyrosine hydroxylase (TH) and α-synuclein (α-syn) in the SN were observed by immunohistochemistry, and the expressions of EIF2α, ATF4 and GRP78/Bip were detected by Western blot. RESULTS: Following modeling and compared with the control and sham-operation groups, the behavioral scores of rats in the model group were elevated (P<0.01), which were significantly decreased by EA intervention (P<0.01). The expression of TH decreased whereas the α-syn, EIF2α, ATF4 and GRP78/Bip increased in the rats of model group, and EA intervention reversed these changes (all P<0.01). CONCLUSION: EA at GV16 and LR3 can improve PD rats' behavioral changes, which is probably related with its effects in up-regulating the expression of TH in the SN and down-regulating the expression of α-syn and EIF2α-ATF4-GRP78/Bip signaling.

Celastrol inhibits multiple myeloma cell growth by activating eukaryotic initiation factor 2α in unfolded protein response / 第二军医大学学报

Objective: To investigate the relationship between celastrol inhibition against multiple myeloma cell growth and unfolded protein response (UPR) and the related molecular mechanism, so as to provide new drug targets for multiple myeloma treatment. Methods: Four multiple myeloma cell lines RPMI 8226, U266, SKO and KMS-11 were treated with different concentrations (proliferation: 0.0-10.0 μmol/L; apoptosis: 0.0-4.0 μmol/L; cell cycle: 0.0-1.5 μmol/L) of celastrol for different periods (proliferation: 1-3 d; apoptosis: 1 d; cell cycle: 1 d), and cell proliferation, apoptosis and cell cycle were examined. Western blotting analysis was used to detect the main molecules in the inositol-requiring enzyme 1 (IRE1), PRKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) signaling pathways of UPR, which included glucose-regulated protein 78 (GRP78), ATF6, PERK, eukaryotic initiation factor 2α (eIF2α), phosphorylated-eIF2α (p-eIF2α), C/EBP homologous protein (CHOP), IRE1 and phosphorylated-IRE1 (p-IRE1). After the lentivirus vector containing short hairpin RNA (shRNA) was used to interfere with eIF2α expression in RPMI 8226 cells, the effects of celastrol were detected on signaling molecule expression, apoptosis and cell cycle. Results: Celastrol inhibited the proliferation of 4 multiple myeloma cells, induced apoptosis, and arrested the cell cycle at G0/G1 phase in dose- and time-dependent manners. RPMI 8226 cells were most sensitive to celastrol. For RPMI 8226 cells, when 0.5-2.0 μmol/L of celastrol was used for 30 min to 24 h, the p-eIF2α levels in the PERK signaling pathway of UPR were significantly increased (P<0.05 or P<0.01), and the downstream CHOP expression was risen correspondingly (P<0.05 or P<0.01), but the other two pathways ATF6 and IRE1 were not affected obviously. After interference of eIF2α expression with lentivirus vector containing shRNA, the effects of celastrol to increase CHOP expression, induce apoptosis and arrest cell cycle were significantly attenuated or disappeared. Conclusion: Celastrol can inhibit the growth of a variety of multiple myeloma cells, and the activated eIF2α molecule in the UPR PERK signaling pathway may be one of the mechanisms.

The effects of mechanical loading on high-fat induced obesity and non-alcoholic fatty liver disease / 天津医药

Objective To investigate the therapeutic effect of mechanical loading on obesity and non-alcoholic fatty liver disease. Methods Thirty 6-week-old female C57BL/6 mice (body weight 18 g) were randomly assigned into three groups: normal control group (NC group, n=10), high-fat diet group (HF group, n=10) and high-fat diet with mechanical loading treatment group (HF+L group, n=10). All mice except for NC group were fed with high-fat diet for 12 weeks. After 6 weeks of high-fat diet, mice of HF+L group received 6-week mechanical loading. The whole body composition was analyzed to detect the total body fat content. The mesenteric fat, perirenal fat, inguinal fat, periuterine fat and the liver were collected and weighed. A portion of the liver sample was isolated for histological analysis (Oil red O staining and HE staining) to observe pathologic changes, while the other was used for Western blot assay to detect the expression of eIF2α, p-eIF2α and ATF4, which were the marker proteins of endoplasmic reticulum stress. Results Compared with the NC group, high-fat diet resulted in a significant increase in body weight and body fat (P＜0.05). After mechanical loading treatment, the body weight and body fat were significantly decreased in the HF+L group compared with those of HF group (P＜0.05). Hepatic histological analysis showed that high-fat diet induced hepatic steatosis, which was effectively alleviated by mechanical loading treatment (P＜0.05). Western blot analysis indicated that high-fat diet led to higher expression levels of p-eIF2α and ATF4 in liver, and mechanical loading was effective in inhibiting the increased expressions of p-eIF2α and ATF4. Conclusion Mechanical loading can effectively alleviate obesity and non-alcoholic fatty liver disease caused by high-fat diet, and its effects may be associated with endoplasmic reticulum stress in liver.

Effect of p53 on Protein Kinase R and Biological Behavior of Cervical Cancer HeLa Cells / 天津医药

Objective To investigate the effects of p53 on expression and activity of protein kinase R (PKR) as well as biological characters of HeLa cells from cervical carcinoma patients. Methods Recombinant plasmid vector pEGFP-C1/p53 was constructed to over-express p53 then it was transfected into HeLa cells. Transcription levels of p53 and PKR mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) among pEGFP-C1/p53 transfection group, pEGFP-C1 transfection group and blank control group(only transfection reagent was added);Protein expression lev?els of p53, PKR, phosphated PKR(p-PKR)and phosphatedαsubunit of eukaryotic initiation factor 2(p-eIF2α)which is the downstream substrate of PKR were detected by Western Blot among three groups;Proliferation of HeLa cell were deter?mined by methyl thiazolyl tetrazolium(MTT)assay;Invasion of HeLa cell were determined by Transwell cell assay. Re?sults Recombinant plasmid vector pEGFP-C1/p53 was successfully constructed to overexpress p53;Transcription level of p53 and PKR mRNA in pEGFP-C1/p53 transfection group were higher than those in pEGFP-C1 transfection group and in blank control group (P<0.05),and there were no significant difference between their levels in pEGFP-C1 transfection group and in blank control group;Protein expression levels of p53, PKR, p-PKR andp-eIF2α in pEGFP-C1/p53 transfection group were higher than those in pEGFP-C1 transfection group and in blank control group (P<0.05),and there were no sig?nificant difference between those expression levels in pEGFP-C1 transfection group and in blank control group;MTT and Transwell cell results showed that proliferation and invasion of HeLa cells in pEGFP-C1/p53 transfection group were weaker than those in pEGFP-C1 transfection group and in blank control group (P<0.05),and there were no significant difference between proliferation and invasion of HeLa cells in pEGFP-C1 transfection group and in blank control group. Conclu?sion p53 can up-regulate the expression and activity of PKR, promote activation of PKR/eIF2αsignal transduction pas?sage and restrain cell proliferation and invasion of HeLa cells.

Influences of Radix salvia miltiorrhiza preconditioning on the expression of phosphorylated eukaryotic initiation factor 2α and caspase12 in hepatic ischemia reperfusion in rats / 中华消化外科杂志

Objective To investigate the influences of Radix salvia miltiorrhiza (RSM) preconditioning on the expressions of phosphorylated eukaryotic initiation factor 2α (p-eIF-2ot) and caspase12 in hepatic ischemia reperfusion in rats.Methods Eighty Wistar rats were randomly divided into the control group (5 rats),sham operation group (25 rats),ischemia reperfusion (IR) group (25 rats) and RSM pretreated group (25 rats).Rats in the sham operation group,IR group and RSM pretreated group were subdivided into 5 groups at different time intervals (0,3,12,24 and 72 hours).Mter midline laparotomy,all structures in the hepatic portal were clamped for 45 minutes followed by different periods of reperfusion.Rats in the control group did not receive any treatment; rats in the sham operation group only received anatomy of the hepatic portal without clamping; rats in the RSM pretreated group received RSM by intravenous injection 30 minutes before ischemia at a dose of 6 ml/kg.Rats in the sham operation group and the IR group received a dose of normal saline as RSM pretreated group.The protein expressions of p-eIF-2α and caspase12 in the hepatic tissues of each group were detected by the Western blot,and the pathological changes of hepatic tissues in each group were detected by hematoxylin and eosin staining.All data were analyzed using the analysis of variance,LSD test or Games-Howell method.Results The relative expression of p-eIF-2α in the control group was 0.296 ± 0.038,and the relative expressions of p-eIF-2α in the sham operation group at different time points were 0.304 ± 0.048,0.298 ± 0.038,0.272 ± 0.042,0.266 ± 0.076 and 0.296 ± 0.043,with no significant difference between the 2 groups (P ＞ 0.05).The relative expression of p-eIF-2α in the IR group at 0 hour was 0.310 ± 0.034,which had no significant difference compared with the control group and the sham operation group (F =0.15,P ＞0.05).The relative expressions of p-eIF-2α in the IR group at 3,12,24 hours were 0.386 ± 0.021,0.710 ± 0.034,0.474 ± 0.017,which had no significant difference compared with the control group and the sham operation group (F =11.90,211.52,25.15,P ＜ 0.05).The relative expression of p-eIF-2α in the IR group at 72 hours was 0.336 ± 0.043,which was back to the level of the control group and the sham operation group (F =1.57,P ＞ 0.05).The relative expressions of p-eIF-2α in the RSM pretreated group at different time intervals were 0.278 ± 0.044,0.800 ± 0.079,1.056 ± 0.125,0.736 ±0.087 and 0.442 ± 0.047,which were significantly lower than the group at 3,12,24,72 hours (P ＜ 0.05).The relative expression of caspase12 in the control group was 0.983 ± 0.003,and the relative expressions of caspase12 in the sham operation group at different time intervals were 0.974 ± 0.004,0.983 ± 0.005,0.985 ± 0.003,0.981 ± 0.004 and 0.978 ± 0.004,with no significant difference between the 2 groups (P ＞ 0.05).The relative expression of caspase12 in the IR group at 0 hour was increased to 1.018 ± 0.076,while it had no significant difference compared with the control group and the sham operation group (F =1.43,P ＞ 0.05).The relative expressions of caspase12 in the IR group began to rise at 3 hours (2.056 ±0.067),and peaked at the 12 hours (2.804 ± 0.050),still at a relative higher level at 24 hours (1.882 ± 0.037),and began to decrease at 72 hours (1.282 ± 0.066),it had significant difference compared with the control group and the sham operation group (F=1290.69,6490.84,2898.71,103.61,P ＜ 0.05).The relative expressions of caspase12 in the RSM pretreated group at different time intervals were 0.998 ± 0.056,1.442 ± 0.066,1.990 ± 0.068,1.364 ± 0.056and 0.962 ±0.054.The relative expressions of caspase12 in the RSM pretreated group at 3,12,24,72 hours were significantly lower than the IR group (P ＜ 0.05).The results of pathomorphological examination showed that the hepatic lobules were integrated and the nucleuses were large and round in the control group an d the sham operation group; deformation of the hepatic lobule,smaller cell volume,nuclear condensation and necrosis were observed in the IR group; cell swelling and slight spotty necrosis were detected in the RSM pretreated group.Conclusions RSM could protect liver from damages during IR through up-regulating the expression of p-eIF-2α,reducing the apoptosis induced by endoplasmic reticulum stress.

Expression and Significance of PKR,p-PKR and p-EIF2α in Human Cervical Lesions / 天津医药

Objective:To identify the relationship between expression of protein kinase R(PKR), phosphating PKR, EIF2α(p-PKR, p-EIF2α) in PKR→EIF2α signal transduction passage and the grades of cervical lesions, the role in generation and progression of cervical tumor and their effects to prognosis of cervical cancer patients. Methods:The expressions of PKR, p-PKR and p-EIF2α in human cervical cancer tissue of 63 cases, cervical intraepithelial neoplasia(CINⅠ-Ⅲ) of 114 cases and normal cervical epithelium of 15 cases were detected by immunohistochemical technique. Results:With the increase in grades of cervical lesions, the positive-expression rate of PKR increased and significantly correlated with the grades of cervical lesions(P < 0.05). With the increase in grades of cervical lesions, the positive-expression rates of p-PKR and p-EIF2α increased firstly, and then decreased. In cervical cancer group, the positive-expression rate of PKR was much higher than that of p-PKR(P < 0.01). The development and progression was quicker in later clinical stages of cervical cancer than that of earlier clinical stages of cervical cancer (P < 0.01). The development and progression of cervical cancer was quicker in patients with negative-expression of p-PKR and p-EIF2α than that in patients with positive-expression of p-PKR and p-EIF2α(P < 0.05). Conclusion:The positive-expression rate of PKR was correlated with the grades of cervical lesions. There are some factors which can impede PKR and EIF2α to be phosphorylated or make p-PKR and p-EIF2α dephosphorylate in high level cervical lesions, which promotes the development and progression of cervical lesions, worsens the prognosis of cervical cancer.