Objective To explore the effects of 4-phenylbutyric acid (4-PBA) on idiopathic pulmonary fibrosis (IPF) using a murine model of bleomycin (BLM)-induced pulmonary fibrosis. Methods Pulmonary fibrosis was induced in C57BL/6 mice by intratracheal injection of BLM. A total of 120 mice were randomly allocated into three groups: BLM group, BLM+4-PBA group, and control group. Pathology of lung tissue was analyzed to evaluate the degree of pulmonary fibrosis, and the survival of the mice was noted. The expression levels of the endoplasmic reticulum stress markers, activating transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP), were analyzed in lung tissues from mice. Results BLM induced significant collagen deposition in the lungs of the mice, which was alleviated by 4-PBA. 4-PBA also dramatically improved the pulmonary function and increased the survival rate in the BLM+4-PBA group compared with that in the BLM group. Both the mRNA and protein expression levels of ATF6 and CHOP were significantly reduced in mouse lung tissue after 2 weeks of 4-PBA treatment. Conclusions 4-PBA treatment could alleviate BLM-induced pulmonary fibrosis in mice via the attenuation of endoplasmic reticulum stress.
ObjectiveTo explore the mechanism of renal tubular epithelial cell apoptosis induced by endoplasmic reticulum stress in rats with intermittent hypoxia (IH) and the intervention effect of losartan.MethodsSixty SPF grade healthy male SD rats were randomly divided into four groups (15 rats in each group), namely as group A (control group), group B (IH group), group C (IH+losartan group), and group D (IH+saline group). The group C and D were intraperitoneally injected with losartan 30 mg/kg and the same dose of saline 30 minutes daily before the experiment, and then the group B, C and D were placed in the intermittent hypoxia chamber. After 6 weeks of modeling, serum of the rats was sampled to detect the renal function. Hematoxylin-eosin staining was used to observe histomorphological changes of the kidney; transmission electron microscopy was used to observe ultrastructural changes of the kidney; TUNEL was used to detect apoptotic index of the renal tubular epithelial cells; and RT-PCR method was used to detect expressions of caspase-12, JNK and CHOP mRNA in the kidney.ResultsThe differences of renal function among these four groups were statistically significant (all P<0.05). Hematoxylin-eosin staining and transmission electron microscopy showed the histomorphological and ultrastructural changes of the kidneys in group B, C and D compared with group A, and the damages in group B and D were more significant. TUNEL results showed that the apoptotic index of renal tubular epithelial cells in group B and D was significantly higher than that in group A (P<0.01), while that in group C was significantly lower than that in group B and D (all P<0.01). RT-PCR results showed that caspase-12, JNK and CHOP mRNA expressions were significantly higher in group B and D than those in group A (all P<0.01); caspase-12 mRNA expression was significantly lower in group C than that in group B and D (P<0.01; P<0.05); and CHOP mRNA expression was significantly lower in group C than that in group B and D (all P<0.01).ConclusionsIH may induce apoptosis of renal tubular epithelial cells by activating endoplasmic reticulum stress through caspase-12, JNK and CHOP. Losartan has protective effects on the kidney of rats with intermittent hypoxia. Its mechanism may be related to the inhibition of apoptotic pathways mediated by endoplasmic reticulum stress.
ObjectiveTo investigate the effects of phenethyl isothiocyanate (PEITC) on apoptosis and proliferation of breast cancer SK-BR-3 cells. MethodsSK-BR-3 cells were treated with different concentrations (0, 10, 30, 50 μmol/L) of PEITC respectively. The proliferation capacity of SK-BR-3 cells was detected by MTT and BrdU staining methods. The cell apoptosis was detected by TUNEL and flow cytometry methods. The protein and mRNA expressions levels of indexes related apoptosis such as Bcl-2, Bax, and MCL-1 and indexes related endoplasmic reticulum stress (ERS) such as PERK, eIF2α, CHOP, IRE1α, ATF6α were detected by Western blot and quantitative real-time PCR (qRT-PCR), respectively. ResultsCompared with the control group (0 μmol/L PEITC treatment group), the results of MTT and BrdU staining methods showed that the proliferations of SK-BR-3 cells in the 10, 30 and 50 μmol/L PEITC treatment group were decreased in turn with the increase of concentration. The results of TUNEL and flow cytometry methods showed that the apoptosis rates of SK-BR-3 cells in the 10, 30 and 50 μmol/L PEITC treatment group were increased in turn with the increase of concentration. The results of Western blot and qRT-PCR methods showed that the protein and mRNA expression levels of anti-apoptotic indexes (Bcl-2, MCL-1) were decreased with the increase of concentration, while the expression levels of protein and mRNA of the pro-apoptotic index (Bax) and ERS-related indexes (PERK, eIF2α, CHOP, IRE1α, ATF6α) increased with the increase of concentration. ConclusionFrom the preliminary results of this study, PEITC can promote the apoptosis of breast cancer SK-BR-3 cells and inhibit cell proliferation, which might be achieved by regulating the expression levels of indexes related apoptosis and ERS.
Objective To investigate whether cigarette smoke promote endoplasmic reticulum associated apoptosis gene Caspase-12 expression. Methods Forty adult male Wistar rats were randomly divided into four groups, ie. group A ( control group) , group B ( exposed to cigarette smoke for two months) ,group C ( exposed to cigarette smoke for four months) , and group D ( exposed to cigarette smoke for four months, then quit smoking for one month) . The COPD rat model was established with passive smoking.Percentage of forced expiratory volume in first 0. 3 second to forced vital capacity ( FEV0. 3 /FVC) and peak expiratory flow ( PEF) were measured. Reverse transcriptase-polymerase chain reaction ( RT-PCR) was used to determine the mRNA expression of Caspase-12. Immunohistochemistry and Western blot were used todetermine the protein expression of Caspase-12. Caspase-12-fluorometric-assay-kit was used to determine Caspase-12 activity. Results The pulmonary function decreased ( P lt; 0. 05) and the lung structure was damaged in the group B compared with the group A. The lung function markedly decreased( P lt; 0. 05) andthe lung structure was obviously damaged in the group C compared with the group B. The pulmonary function had minor improvement( P gt; 0. 05) , and the lung structure injury was also significant in the group D in contrast with the group C. The expression and activity of Caspase-12 were remarkably increased in the group B compared with the group A( P lt; 0. 05) , elevated significantly in the group C compared with the group B ( P lt; 0. 05) , decreased slightly in the group D compared with the group C ( P gt; 0. 05 ) . Conclusion Cigarette smoke promotes the development of COPD by inducing the endoplasmic reticulum associated apoptosis gene Caspase-12 expression.
Objective To realize the research progress of regulating the endoplasmic reticulum stress response to inhibit autophagy in tumor cells. Method The literatures about regulating the endoplasmic reticulum stress response to inhibit autophagy in tumor cells were reviewed. Result In the endoplasmic reticulum stress response induced by the release of calcium and accumulation of unfolded proteins, autophagy can be activated by several pathways, and to regulate physiological and pathological processes. Conclusion Further research about the endoplasmic reticulum stress response in tumor cells need to be done to regulate the response factors to inhibit autophagy.
ObjectiveTo investigate the role of endoplasmic reticulum stress in liver regeneration after associating liver partition and portal vein ligation for staged hepatectomy (ALPPS).MethodsSeventy-two C57bl/6 mice were randomly divided into ALPPS group, portal vein ligation group (PVL group), and sham operation group (Sham group), 24 mice in each group. And then one-stage ALPPS operation, simple PVL, and sham operation will be performed. Six mice were randomized selected of the three groups on the 1st, 2nd, 4th, and 7th day after surgery, respectively, the liver weight to body weight ratio (FLR/BW) of each group was measured, and the liver tissues were taken for immunohistochemical staining to calculate the proportion of Ki-67 positive cells, Western blot was used to detect the expression levels of X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1α (IRE1α) proteins.Results① FLR/BW: On the 4th day and the 7th day after operation, the FLR/BW of the Sham group, PVL group, and ALPPS group increased in sequence at the same time, and the difference between the three groups was statistically significant (P<0.05). ② Ki-67 positive cell ratio: On the 2nd day after operation, the ratio of Ki-67 positive cells in the Sham group, PVL group, and ALPPS group increased sequentially, and the difference between the two groups was statistically significant (P<0.05). On the 4th day after operation, the ratio of Ki-67 positive cells in the PVL group and the ALPPS group were still higher than that of the Sham group (P<0.05). ③ Expression levels of XBP1 and IRE1α: On the 2nd and 4th postoperative day, the expression levels of XBP1 and IRE1α in the ALPPS group were higher than those in the Sham group and the PVL group (P<0.05). On the 7th day after surgery, the expression levels of XBP1 and IRE1α in the ALPPS group were higher than those in the Sham group (P<0.05), while compared with the PVL group, the expression level of XBP1 in the ALPPS group was still higher (P<0.05).ConclusionsALPPS-induced liver regeneration is more advantageous than traditional PVL in mice. It may be attributed to the obvious endoplasmic reticulum stress activation after ALPPS leading to the up-regulation of IRE1α-XBP1 expression, which is involved in the regulation of hepatocyte cell cycle and promotes hepatocyte proliferation, thus promoting rapid liver regeneration.
ObjectiveTo investigate the endoplasmic reticulum stress associated apoptosis gene Caspase-12 expression in paraquat-induced pulmonary fibrosis. Methods30 adult healthy Sprague-Dawley(SD) rats were randomly divided into a nomal control group,two pulmonary fibrosis model groups (intragastrically administered paraquat for 14 days and 28 days,respectively).The model of pulmonary fibrosis was established through intragastrically administering paraquat at the dose of 30 mg/kg.RT-PCR was used to determine the mRNA expression of Caspase-12.Immunohistochemistry was used to determine the protein expression of Caspase-12.HE staining and Masson staining were used to determine the degree of alveolitis and pulmonary fibrosis. ResultsHE staining and Masson staining of lung tissues proved that pulmonary fibrosis model was successfully constructed.The degree of alveolitis and pulmonary fibrosis in the model group was significantly more serious than that in the control group(P<0.01).RT-PCR and Immunohistochemistry results showed that the expression of Caspase-12 were remarkably increased in the pulmonary fibrosis model group(14 d group)(P<0.01),even more elevated in 28 d group compared with the 14 d group. ConclusionThe results demonstrate that the expression of Caspase-12 in paraquat poisoned rats is up-regulated,suggesting endoplasmic reticulum stress plays an important role in paraquat induced-pulmonary fibrosis.
Metabolic memory means if the hyperglycemia can't be controlled at early stage of diabetes, chronic complications such as diabetic retinopathy (DR) will continue to develop even if the blood glucose level maintains normal level at later stage. Oxidative stress plays an important role in the "metabolic memory" of DR, which interacts with the nitrative stress, advanced glycation end products, genetic modification and endoplasmic reticulum stress in the pathogenesis of DR. Further elucidation of the relationship between oxidative stress and "metabolic memory" of DR can open the way for the discovery of novel therapeutic targets to prevent DR progression.
ObjectiveTo investigate the influence of endoplasmic reticulum stress (ERS) on smoking-induced nucleus pulposus cells apoptosis and inflammatory response.MethodsBetween October 2016 and October 2018, 25 patients with cervical disc herniation receiving discectomy were collected and divided into smoking group (14 cases) and non-smoking group (11 cases). The baseline data of age, gender, herniated segment, and Pfirrmann grading showed no significant difference between the two groups (P>0.05). The obtained nucelus pulposus tissues were harvested to observe the cell apoptosis via detecting the apoptosis-related proteins (Caspase-3 and PRAP) by TUNEL staining and Western blot test. The nucleus pulposus cells were isolated and cultured with enzyme digestion, of which the third generation cells were used in follow-up experiments. Then, the expressions of inflammatory factors [interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α)] were detected by ELISA; the nuclear translocation of P65 was monitored by cell immunofluorescence staining. Furthermore, ERS-related proteins (GRP78 and CHOP) were detected by Western blot; and endoplasmic reticulum ultrastructure was observed under transmission electron microscope. To verify the regulatory effect of ERS, cells were pretreated by ERS specific inhibitor (4-PBA), then cell apoptosis and inflammatory response were tested.ResultsThe nucleus pulposus tissue observation showed that the cell apoptotic rate and the expressions of apoptosis-related proteins (Caspase-3 and PARP) were obviously higher in smoking group than in non-smoking group (P<0.05). The nucleus pulposus cells observation indicated that the expressions of the inflammatory factors (IL-1β and TNF-α) and the ERS-related proteins (GRP78 and CHOP) were also higher in smoking group than in non-smoking group (P<0.05). The results of cell immunofluorescence staining further confirmed that smoking stimulated nuclear translocation of P65 in nucleus pulposus cells. The ERS injury was much more serious in smoking group than in non-smoking group. Furthermore, after 4-PBA inhibiting ERS, the expressions of GRP78, CHOP, IL-1β, TNF-α, and P65 were significantly decreased (P<0.05), and flow cytometry results showed that cell apoptotic rate in smoking group was decreased, showing significant difference compared with the non-smoking group (P<0.05).ConclusionSomking can stimulate cell apoptosis and inflammatory response in nucleus pulposus cells via ESR pathway. Suppressing ESR may be a novel target to suspend smoking-induced intervertebral disc degeneration.
Objective Endoplasmic reticulum stress (ERS) mediated apoptosis is one of the eukaryotic cellularapoptotic pathways, to investigate the potential role of ERS during myocardium apoptosis in rats with severe burninjury. Methods Sixty-four 7-week-old male Wistar rats, weighing 200-220 g, were randomly divided into 2 groups. Thirtypercentage of total body surface area full-thickness thermal injury was produced in 32 rats of burn group, while sham burn wasproduced in 32 rats of control group. The heart tissues were harvested from 8 rats in each group at 1, 4, 7, and 14 days after burnto observe the changes of myocardium ultrastructure with transmission electron microscope (TEM). Myocardium apoptosis wasdetected with TUNEL assay. The expressions of glucose regulated protein 78 (GRP 78), C/EBP-homologous protein (CHOP),and cleaved Caspase 12 in different pathways of ERS were analysed with Western blot. Results All rats survived during theexperiment. Apoptotic changes of cardiomyocytes were observed in burn group, and the apoptosis index in burn group wassignificantly higher than that in control group at each time point (P lt; 0.05), and it reached peak at 7 days after burn injury(P lt; 0.05). The expressions of myocardial GRP 78, CHOP, and cleaved Caspase 12 showed persistent elevation in burn group.The expressions of GRP 78 and cleaved Caspase 12 in burn group were significantly higher than those in control group at eachtime point (P lt; 0.05), while the expression of CHOP was higher than that in control group at the other time points (P lt; 0.05)except 1st day after burn injury. Conclusion ERS and CHOP, Caspase 12 mediated apoptotic pathway are activated inmyocardium after severe burn injury, and this may be one pathway of myocardium apoptosis.