Aim To investigate the expression of IncRNA AC079466. 1 in non-small cell lung cancer (NSCLC) tissues and cells, and the effect of its overexpression on the proliferation, apoptosis, migration and invasion of A549 and H1299 cells. Methods Cancer tissues and corresponding adjacent tissues from 20 NSCLC patients were collected, and the expression of IncRNA AC079466. 1 in tissue and cells was detected by qRT-PCR. AC079466. 1 group was transfected with overexpression plasmid, NC group was transfected with empty plasmid, and no transfection was used in the Blank group. MTT, flow cytometry and Transwell were used to detect the effects of IncRNA AC079466. 1 overexpression on the viability, apoptosis, migration and invasion of A549 and HI299 cells. Western blot was used to detect the effect of overexpression of IncRNA AC079466. 1 on the expression of endoplasmic reticulum stress-related factors GRP78, PERK, eIF2a, ATF4, CHOP, Bax and caspase-3. Results Compared with adjacent tissues, the expression of IncRNA AC079466. 1 in cancer tissues significantly decreased. Compared with HBE cells, the expression of IncRNA AC079466. 1 significantly decreased in A549 and H1299 cells. Compared with the Blank group and NC group, the viability, migration and invasion abilities of A549 and H1299 cells in AC079466. 1 group all markedly decreased, the apoptosis rate apparently increased, and the expressions of endoplasmic reticulum stress-related factors GRP78, p-PERK, eIF2a, ATF4, CHOP, Bax and caspase-3 were significantly up-regulated. Conclusion The overexpression of IncRNA AC079466. 1 significantly inhibits the viability, migration and invasion of A549 and HI299 cells, and promotes cell apoptosis. The mechanism may be related to the promotion of endoplasmic reticulum stress-mediated cell apoptosis.

Objective:To investigate the related factors that affect the timing and prognosis of early tracheostomy in patients with multiple rib fractures.Methods:A retrospective case series study was conducted on medical data of 222 patients with multiple rib fractures who underwent tracheostomy in Affiliated Hospital of Yangzhou University from February 2013 to October 2019，including 160 males and 66 females，with the age of 18 to 85 years ［（49.5 ± 16.3）years］. According to the practice management guidelines for tracheostomy timing and the use of propensity score matching technology，there were 118 patients with tracheostomy within 7 days of tracheal intubation （early group） and 104 patients with tracheostomy after 7 days of tracheal intubation （late group） before matching，and there were 87 patients in early group and 87 patients in late group after matching. Data were compared between groups including the gender，age，underlying disease，injury severity score （ISS），Glasgow coma score （GCS），number of fractured ribs，total number of rib fractures （NTRF），first rib fracture，flail chest，traumatic brain injury，combined injuries （spine，maxillofacial，sternum），acute respiratory distress syndrome （ARDS），volume fraction of pulmonary contusion（VPC），blood lactic acid （within 24 hours of admission），hemothorax，pneumothorax，mechanical ventilation time，duration of tracheostomy，time from tracheal intubation to incision，length of hospital stay，length of stay in ICU，closed thoracic drainage，number of fiberoptic bronchoscopy，multi-drug resistant bacteria infection，ventilator-associated pneumonia，antibiotic use time，duration of sedative and analgesic drugs used and 28-day mortality. The multivariate Logistic regression analysis was used to predict independent risk factors for early tracheostomy. The Pearson method was used to compare the relationship between multiple factors. The receiver operating characteristic （ROC） curve was used to predict indicators that affect the prognosis of patients with early tracheostomy，and calculate the best cut-off value. The Kaplan-Meier single factor and COX multivariate survival were used to analyze the relevant factors affecting the 28-day mortality of patients.Results:（1） In early group，the NTRF，ARDS and VPC were higher than those in late group，and the time from tracheal intubation to incision and 28-day mortality rate were lower than those in late group （ P < 0.05），while the two groups showed no significant differences in the gender，age，underlying diseases and ISS （ P > 0.05）. （2） The multivariate Logistic regression analysis showed that there was statistical significance in NTRF （ OR = 1.775，95% CI 1.439-2.188），ARDS（ OR = 3.740，95% CI 1.441-9.711），VPC （ OR = 1.087，95% CI 1.052-1.124） （ P < 0.05）； the Pearson method analysis showed a significant correlation between VPC and NTRF （ r = 0.369， P < 0.05） and a low degree of correlation between ARDS and VPC （ r = 0.179， P < 0.05），but there was no significant correlation between ARDS and NTRF （ r = 0.132， P > 0.05）. （3） The ROC curve analysis showed that the area under the curve （AUC） of the VPC and NTRF ［AUC = 0.832 （95% CI 0.770-0.893），AUC = 0.804 （95% CI 0.740-0.868）］ were significantly higher than those of the number of rib fractures ［AUC = 0.437（95% CI 0.352-0.523），GCS ［AUC = 0.519 （95% CI 0.432-0.605）］ and ISS ［AUC = 0.484 （95% CI 0.398-0.571）］ （ P < 0.05）. After calculating the Yorden index，the best cut-off value for VPC was 23.9，and the best cut-off value for NTRF was 8.5. （4） The Kaplan-Meier single factor and multivariate COX model survival analysis showed that the 28-day survival ratio of patients with early tracheostomy was significantly better than that of late tracheostomy （ P < 0.05）. Conclusions:The NTRF，ADRS and VPC are independent risk factors for the timing and prognosis of early tracheostomy. There is a significant correlation between VPC and NTRF. The VPC ≥ 23.9% and or NTRF ≥ 8.5 can be used to predict early tracheostomy in patients with multiple rib fractures. Early tracheostomy may benefit the 28-day survival of patients with multiple rib fractures.

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.
Acetates ; Apoptosis ; Bidens ; Endoplasmic Reticulum Stress ; Hepatocytes ; Transcription Factor CHOP/genetics* ; eIF-2 Kinase/genetics*

This study aimed to investigate the effect and possible mechanism of Bidens pilosa decoction on non-alcoholic fatty liver disease(NAFLD) induced by high fat and high glucose in mice. Bald/c mice were randomly divided into normal group, model group, metformin(200 mg·kg~(-1)) treatment group, Bidens pilosa decoction(10 g·kg~(-1)) treatment group, metformin and B. pilosa decoction(100 mg·kg~(-1)+5 g·kg~(-1)) treatment group. Except for the normal group, mice in the other four groups were fed with high-fat and high-glucose diet for 8 weeks to establish the non-alcoholic fatty liver model. After 4 weeks of treatment, blood was collected from the eyeballs, the mice were sacrificed, and relevant indicators were detected. The results showed that compared with the model group, blood lipid and blood glucose levels of each treatment group were significantly lower(P<0.05); HE staining results showed that liver pathological damage in each treatment group was significantly improved; oil red O staining results showed fat distribution in each treatment group significantly reduced(P<0.01); immunohistochemical staining showed that glucose regulated the protein expression of protein 78(GRP78) in liver tissues of each treatment group was also significantly reduced(P<0.01); Western blot results showed that endoplasmic reticulum stress signal pathway-related factors GRP78, phosphorylated-protein kinase R-like ER kinase(p-PERK), eukaryotic translation-initiation factor 2α(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(Chop), inositol requiring 1α(IRE1α), and cleaved-cysteinyl aspartate specific proteinase 12(cleaved-caspase-12) were significantly reduced(P<0.01). The results of the combined drug treatment group were better than those of the single drug treatment group. These results showed that B. pilosa decoction had the effect in improving non-alcoholic fatty liver, and its mechanism may be related to the down-regulation of the expression of endoplasmic reticulum stress(ERS)-related factors, and the reduction of the apoptosis of hepatocytes caused by ERS and the down-regulation of blood lipid and blood glucose levels.
Animals ; Apoptosis ; Bidens ; Endoplasmic Reticulum Stress ; Endoribonucleases ; Glucose ; Mice ; Non-alcoholic Fatty Liver Disease ; Protein-Serine-Threonine Kinases

The aim of this study was to investigate the preventive and therapeutic effects of Erzhi Pills on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine( MPTP)-induced Parkinson's disease( PD) in mice,and explore its possible mechanism of action. Mice were intraperitoneally injected with MPTP( 30 mg·kg^-1,0. 01 m L·g^-1) once daily to induce PD for 8 days. In the treatment group,Erzhi Pills were given by intragastric administration( 2. 5 g·kg^-1,once daily for 30 days). The normal group received an equal volume of normalsaline. In terms of behavior,the limb movement coordination ability of the mice was detected by climbing,hanging and swimming experiments. The spatial learning and memory ability of the mice was detected by Morris water maze test. The content of MDA,as well as the activity of GSH-PX and SOD were determined in mice serum. Western blot was used to detect the protein expression levels of TH,MAOB and apoptosis-related factors CHOP and caspase-12 in brain tissues. Immunohistochemistry was used to detect the expression of TH in section of brain tissues in mice. The results showed that in behavioral aspects,as compared with the model group,the scores of limb movement ability as well as scores of spatial learning and memory ability were significantly improved in the treatment groups( P<0. 05). In terms of serological indicators,as compared with the model group,the activities of SOD and GSH-PX were significantly increased in the serum of treatment groups,and the content of MDA was significantly decreased( P<0. 05). The results of Western blot showed that as compared with the model group,the protein levels of TH in the brain tissues of the mice in treatment group were significantly up-regulated,while the protein levels of MAOB and apoptosis-related factors CHOP and caspase-12 were significantly down-regulated( P<0. 05). The results of immunohistochemistry showed that the number of TH positive cells in the brain tissues of the mice in the treatment group was significantly increased as compared with the model group( P<0. 05). In summary,Erzhi Pills have a certain preventive and therapeutic effect on MPTP-induced PD mice,which can significantly improve the limb motor coordination ability and spatial learning and memory ability of PD mice. Its mechanism may be related to down-regulating the expression of apoptosis-related factors CHOP and caspase-12,reducing the dopaminergic neuron damage and inhibiting dopaminergic neuronal apoptosis.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Disease Models, Animal ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Mice, Inbred C57BL ; Neuroprotective Agents/pharmacology* ; Parkinson Disease ; Substantia Nigra

This study focused on the protective effect of earthworm active ingredients (EWAs) on hepatocyte apoptosis induced by endoplasmic reticulum stress (ERS) in L-02 cells. The L-02 cells were cultured in vitro. The cell viability was measured with CCK-8, the apoptosis of L-02 cells was detected by flow cytometry, and the relevant protein and mRNA expressions were detected by Western blot and qPCR. According to the findings, tunicamycin (TM) could obviously reduce the survival rate of L-02 cells in a time-dependent and dose-dependent manner. Compared with normal group, the apoptosis rate in model group was significantly increased (<0.05 or <0.01). The protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, were significantly up-regulated (<0.05 or <0.01), while Bcl-2 was significantly down-regulated (<0.05 or <0.01). After the administration with different concentrations of EWAs, compared with model group, EWAs could significantly increase the survival rate ofL-02 hepatocyte and decrease the cell apoptosis rates. It could also reduce the protein and mRNA expressions of ERS-related signal molecules, such as GRP78, PERK, eLF2α, ATF4, CHOP and Bax, in a dose-dependent manner (<0.05 or <0.01) and increased the protein and mRNA expressions of Bcl-2(<0.05 or <0.01). These results showed that EWAs had a significantly protective effect on hepatocyte apoptosis induced by ERS in L-02 cells. Its mechanism may be related to the down-regulation of mRNA and protein expressions of GRP78, PERK, ATF4, eLF2α, CHOP and Bax, and the up-regulation, the relief of ERS and the promotion of the proliferation of impaired L-02 cells.

OBEJECTIVE Gecko has been clinically used in China for many years. It has been proved that the gecko polypeptide mixture(GPM)extracted from gecko could inhibit the growth of multiple types of tumor cells.In order to investigate the possible anti-tumor molecular mechanisms of GPM,we used RNA-seq technology to identify the differentially expressed genes of human hepatocellular carci-noma(HCC)HepG2 cells treated with or without GPM.METHODS The HepG2 cells were treated with different concentration of GPM(0,0.1,0.2,0.3,0.4 mg·mL-1)for 6 h,12 h and 24 h,respectively.MTT assay was used to detect the viability of HepG2 cells. DAPI fluorescence staining was performed to observe nucleus morphological changes of HepG2 cells.Western blot analysis was applied to observe the expres-sion of apoptosis- related proteins and endoplasmic reticulum stress (ERs)-related proteins in HepG2 cells.Flow cytometry was also applied to detect reactive oxygen species(ROS)generation.In this report, we showed that GPM could induce HepG2 cells apoptosis and influence HepG2 cells proliferation in a dose-dependent manner.We applied many analysis methods,including differentially expressed genes analysis,Gene Ontology(GO)enrichment analysis,KEGG pathway enrichment analysis,protein-protein interaction network analysis to screen out possible molecular mechanisms.RESULTS ER-nucleus signaling pathway, cellular response to stress and apoptotic processes were identified the potential anti-cancer molecular biological process of GPM.GPM may also induce apoptosis in HepG2 cells via endoplasmic reticulum stress pathway. The GPM could induce ROS generation and up-regulate ERs-related proteins. CONCLUSION The present study revealed the potential anti-tumor mechanism of GPM.

Objective To investigate the effect of Gecko polypeptide mixture (GPM) on the proliferation and endoplasmic reticulum stress (ERS) pathway of human hepatocellular carcinoma HepG2 cells.Methods The HepG2 cells were treated with differentconcentration of GPM(0,0.15,0.20,0.25,0.30,0.35,0.40,0.45 mg · mL-1) for 24 h,and then corresponding indicators were detected with respective methods.The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTF) assay was used to detect the viability of HepG2 cells.The concentration of blank group,GPM low-dose,middle-dose and high-dose experimental groups was respectively 0,0.1,0.2,0.3 mg · mL-1,according to the results of MTT.5-Fluorouracil was chosen as the positive control drug,which concentration was 10 μg · mL-1.Western blot analysis was applied to observe the expression of ERS-related proteins and apoptosis-related proteins in HepG2 cells.Results The GPM could inhibit the proliferation of HepG2 cells in a dose-and time-dependent manners.After treatment of GPM for 24,48,72 h,the 50％ inhibitory dose (IC50) values were 0.27,0.23,0.20 mg · mL-1.Compared with the normal group,the proteins expression levels of double-strand RNA-activated protein kinase-like ER kinase (PERK),glucose-regulated protein78 (GRP78),activating transcription factor-4 (ATF4),C/EBP-homologous protein (CHOP) and apoptosis-related poly-ADP-ribos polymerase (PARP),cleaved cysteinyl aspartate specific proteinase-3 (Caspase-3) were significantly up-regulated after treatment of GPM in vitro (P ＜0.05 or P ＜0.01).PERK and GAPDH grayscale average ratio in normal group,control group,and three concentration experimantal groups were (4.31 ±0.81) ×10-2,(8.92±0.91) ×10-2,(20.73±0.97) ×10-2,(24.04±0.95) ×10-2,(11.65±1.67) × 10-2;GRP78 and GAPDH grayscale average ratio in the five groups were (27.99 ±2.36) × 10-2,(35.58 ± 1.02) × 10-2,(42.55 ± 1.19) × 10-2,(54.91 ± 1.20) × 10-2,(7.31 ± 1.01) × 10-2;ATF4 and GAPDH grayscale average ratio in the five groups were (20.82 ± 1.42) × 10-2,(39.60 ± 0.56) × 10-2,(52.02 ± 1.83) × 10-2,(73.39 ± 1.83) × 10-2,(18.13 ± 2.28) × 10-2;CHOP and GAPDH grayscale average ratio in the five groups were (8.71 ±0.76) × 10-2,(11.27 ± 1.07) × 10-2,(41.29 ± 1.36) × 10-2,(48.55 ± 1.37) × 10-2,(33.01 ±3.95) × 10-2;PARP and GAPDH grayscale average ratio in the five groups were (13.06 ± 2.88) × 10-2,(36.79 ± 2.10) × 10-2,(58.72 ± 1.53) × 10-2,(67.61 ± 1.68) × 10-2,(34.88 ± 2.02) × 10-2;C aspase-3 and GAPDH grayscale average ratio in the five groups were (5.92 ±0.33) × 10-2,(14.71 ±1.11) × 10-2,(17.58±1.33) × 10-2,(35.41 ±2.91) × 10-2,(5.94 ± 1.61) × 10-2.Compared with the normal group,the expression levels of GRP78,ATF4,CHOP in the four groups were significantly up-regulated (P ＜ 0.05 or P ＜ 0.01).Conclusion GPM can inhibit proliferation and induce apoptosis of HepG2 cells,which may be associated with inducing the HepG2 cells endoplasmic reticulum stress.

Objective The aim of this study was to investigate the effects of gecko active components (GACs) on the levels of reactive oxygen species (ROS),calcium and mitochondrial membrane potential (MTP) in HepG2 cells.Methods The experiment was divided into 4 groups:control group (GACs,0),low,middle and high dose group (0.1,0.2,0.3 mg · mL-1,GACs).The growth inhibitory effect of GACs on HepG2 cells were assessed by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Intracellular reactive oxygen species (ROS) level,calcium level,mitochondrial membrane potential (MMP) level was measured by flow cytometry.Results The results showed that GACs could inhibit the proliferation of HepG2 cells in a dose-dependent manners.After treatment of GACs for 24 h,the 50％ inhibitory dose (IC50) values were 0.21 mg · mL-1.The cell growth inhibition rates of the control group,low-,middle-and high-dose groups were (0.07 ±0.01)％,(0.17 ±0.03)％,(0.49 ±0.12)％ and (0.67 ± 0.18) ％,respectively;the ROS levels of control group,low-,middle-and high-dose groups were (16.61 ± 1.12),(85.81 ±2.56),(268.91 ±2.34),(1741.5 ±5.64);the calcium levels of control group,low,middle and high-dose groups were (9.67 ±O.98),(12.30 ± 1.07),(94.80 ± 2.75),(910.99 ± 5.31);the MMP levels of control group,low-,middle-and high-dose groups were (27.02±1.8) ×10-2,(23.78 ±1.2) ×10-2,(18.27 ±1.5) ×10-2,(16.49 ±2.1) × 10-2,and significant differences were found in the above indexes between low/middle/high dose group and the control group(P ＜0.05 or P ＜0.01).Conclusion GACs can inhibit the proliferation of HepG2 cells,which may cause the increase of ROS content and calcium level and the decrease of MMP,and disrupt the normal function of mitochondria,eventually leading to the apoptosis of HepG2 cells.

To study the protective effect of earthworm active ingredients(EWAs) against endoplasmic reticulum stress(ERS)-induced acute liver injury in mice. The model of liver injury was induced through intraperitoneal injection of 10%CCl4. Serum glutamic-pyruvic transaminase(ALT), glutamic-oxaloacetic transaminase(AST), superoxide dismutase(SOD) and glutathione peroxidase(GSH-PX) activity and malondialdehyde(MDA) concentration were detected by colorimetric method. Histological examination was performed through hematoxylin-eosin staining and light microscopy, and apoptosis was detected using terminal transferase dUTP nick end labeling. The expressions of ERS related proteins, including glucose regulated protein 78(GRP78), protein kinase R-like ER kinase(PERK), eukaryotic transcription initiation factor 2α(eIF2α), active transcription factor-4(ATF4) and CCAAT/enhancer binding homologous protein(CHOP), were measured by immunohistochemistry and Western blot. According to the results, compared with the model group,serological indexes in the high, middle and low doses of EWAs were significantly improved (P<0.05 or P<0.01), the extent of liver lesion was decreased and the degree of injury was significantly reduced, and that the liver index and the spleen index of mice were significantly changed(P<0.05 or P<0.01). In liver tissue, the expressions of GRP78 and CHOP were significantly decreased(P<0.05 or P<0.01). The protein expressions of GRP78, CHOP and its upstream signaling pathway PERK-eIF2-ATF4 were significantly decreased in each dose group(P<0.05 or P<0.01). In summary, EWAs has a significant protective effect on ERS-induced acute liver injury, and its mechanism may be correlated with the inhibition of oxidative stress and ERS, and down-regulation of ERS marker protein CHOP expression, andinhibition of apoptosis.