Apoptosis ; Autophagy ; Endoplasmic Reticulum ; Endoplasmic Reticulum Chaperone BiP ; Endoplasmic Reticulum Stress ; Hedyotis ; Humans ; Laryngeal Neoplasms/drug therapy* ; Polysaccharides

OBJECTIVE: To investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced hepatocyte apoptosis. METHODS: Cells of the rat hepatocyte line BRL were cultured. The hepatocytes were treated with LPS, ERS inducer thapsigargin (TG), and ERS inhibitor 4-phenylbutyric acid (4-PBA), respectively or in their different combination. The cell viability was measured by MTT assay. The cyto-nuclear morphological changes of apoptosis cells were detected by the fluorescent dye Hoechst 33258. The apoptosis rate was assessed by flow cytometry with Annexin V-FITC/PI double-staining. Expressions of GRP78 as ERS marker protein, CHOP, caspase-12 and cleaved-caspase-3 as ERS related protein were detected by Western blotting. RESULTS: LPS could cause a decrease in cell viability and an increase in apoptosis rate in a dose- and time-dependent manner. The expression of GRP78, CHOP, caspase-12 and cleaved-caspase-3 proteins were significantly increased with LPS treatment. TG led to a marked decrease in cell viability and an increase in apoptosis rate, which aggravated the hepatocyte injury induced by LPS; whereas 4-PBA alleviated LPS-induced apoptosis. CONCLUSION ERS mediates LPS-induced hepatocyte injuries, indicating that ERS may play a vital role in the pathogenesis of LPS-induced hepatocyte injuries.
Animals ; Apoptosis ; Caspase 3 ; Cell Survival ; Endoplasmic Reticulum Chaperone BiP ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; Hepatocytes ; Lipopolysaccharides ; Phenylbutyrates ; Rats

OBJECTIVES: To explore the role of high mobility group B1 （HMGB1） protein in the post-traumatic endoplasmic reticulum stress （ERS） in rat lung tissues. METHODS: The rat model of acute lung injury was established by crushing the hind limbs of rats with standard weight. The first experiment was to divide rats into postural control group and crush groups （6 h, 18 h and 30 h after crushing）. The second experiment was to divide rats into postural control group, 18 h crush group, HMGB1 inhibitor sodium butyrate （SB） group and 18 h crush+SB group. The protein expression changes of HMGB1 and ERS- related proteins （GRP78, caspase-12, CHOP and IRE1α） in rat lung tissues were detected with Western blotting. Meanwhile, the pathological changes of rat lungs were observed by HE stain. RESULTS: Compared with the postural control group, the expression levels of ERS-related proteins （GRP78, caspase-12, CHOP and IRE1α） and HMGB1 protein in rat lung tissues by crushing the hind limbs of rats were obviously increased. The protein levels reduced at 30 h after crushing but were still higher than those of postural control group and obvious pathological changes of acute lung injury were observed simultaneously in rats. Compared with the 18 h crush group, the expression levels of the ERS-related proteins and HMGB1 protein in rat lung tissues were attenuated in 18 h crush+SB group, and the pathological changes of rat lung injury began to alleviate. CONCLUSIONS HMGB1-ERS pathway activated by traumatic stress can lead to acute lung injury in rats.
Animals ; Apoptosis ; Endoplasmic Reticulum Chaperone BiP ; Endoplasmic Reticulum Stress ; Endoribonucleases ; HMGB1 Protein/metabolism* ; Heat-Shock Proteins ; Lung/metabolism* ; Protein Serine-Threonine Kinases ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To observe the effects of Fei-Liu-Ping ointment and chemotherapy on mice with lung cancer, and to explore the inherent mechanism of action from the point of acidic microenvironment and apoptosis. METHODS: First of all, the Lewis lung cancer transplanted mouse model was established. Therefore, they were treated by Fei-Liu-Ping ointment, cyclophosphamide, Fei-Liu-Ping ointment + cyclophosphamide and the saline as control. All the groups' tumor size, tumor growth rate and food consumption were recorded. The mice were sacrificed and the tumors were took out after 15 days' interventions. Then lactate relative concentrations were detected with lactate kits and the protein expressions of glucose transporter 4 (GLUT4), hexokinase 1 (HK1), glucose-regulated protein 78 (GRP78), carbonic anhydrase-IX (CA-IX) were detected through immunohistochemical staining. Flow cytometry was adopted to detect the percentage of apoptotic tumor cells and regulatory T cells (Treg), and the expression of hypoxia-inducible factor-1α (HIF-1α), Bcl-2, Bax, Caspase-3, interleukin-2 (IL-2) were tested through western blot. RESULTS: The strongest inhibition effect and the lowest tumor growth rate was found in Fei-Liu-Ping ointment + cyclophosphamide group. There were significant differences between Fei-Liu-Ping ointment + cyclophosphamide group and saline group(P<0.05). And the highest food consumption was found in Fei-Liu-Ping ointment + cyclophosphamide group while there were no significant differences between Fei-Liu-Ping ointment + cyclophosphamide group and saline group (P>0.05). Further molecular biological detections found that the lowest lactate level and regulatory T cells ratio were found in Fei-Liu-Ping ointment + cyclophosphamide group and these expressions of GLUT4, HK1, GRP78, CA-IX were suppressed. There were significant differences between Fei-Liu-Ping ointment+cyclophosphamide group and saline group (P<0.05). In addition, the Fei-Liu-Ping ointment + cyclophosphamide group's cell apoptosis increased significantly compared with saline group and there were significant differences on expressions of HIF-1α, Bcl-2, Bax, Caspase-3, IL-2 for this group compared with saline group. CONCLUSION Chemotherapy and Fei-Liu-Ping ointment had the synergistic effect on inhibiting tumor growth and improving the general conditions of tumor-bearing mice. The effect was partly owed to the improvement on tissue hypoxia, the inhibition of HIF-1α expression and the regulations on its downstream proteins, such as GLUT4, HK1, GRP78, and CA-IX. And then all these alterations led to the modulation tumor acidic microenvironment, the induced tumor cells apoptosis and suppression of T cells to regulatory T cells differentiation.
Animals ; Apoptosis ; Cell Proliferation ; Cyclophosphamide ; Drugs, Chinese Herbal ; Endoplasmic Reticulum Chaperone BiP ; Hypoxia-Inducible Factor 1, alpha Subunit ; Lung Neoplasms ; Mice ; Tumor Microenvironment

OBJECTIVE: To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14) on the expressions of Beclin-1 and GRP78 in spinal dorsal horn in rats with cervical spondylotic radiculopathy (CSR), and to explore the possible analgesic mechanism of wheat-grain moxibustion for CSR. METHODS: A total of 48 SD rats were randomly divided into a sham operation group, a model group, a wheat-grain moxibustion group and a wheat-grain moxibustion+3-MA group, 12 rats in each group. The CSR model was prepared by spinal cord insertion method. Three days after modeling, the rats in the model group were intraperitoneally injected with 1 mL of 0.9% sodium chloride solution; the rats in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time) on the basis of the model group; the rats in the wheat-grain moxibustion+3-MA group were intraperitoneally injected with 3-MA solution and wheat-grain moxibustion at "Dazhui" (GV 14, 6 cones per time). The three groups were intervened for 7 days, once a day. The gait score and mechanical pain threshold were observed before treatment and 7 days into treatment; after the treatment, the expressions of mRNA and protein of Beclin-1 in spinal dorsal horn were detected by real-time fluorescence quantitative PCR and immunohistochemistry; the expression of GRP78 protein in spinal dorsal horn was detected by Western blot method; the autophagosomes and ultrastructure in spinal dorsal horn neurons were observed by electron microscope. RESULTS: After the treatment, compared with the sham operation group, in the model group, the gait score was increased and the mechanical pain threshold was decreased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was increased (P<0.01). Compared with the model group and the wheat-grain moxibustion+3-MA group, in the wheat-grain moxibustion group, the gait score was decreased and mechanical pain threshold was increased (P<0.01), and the expression of GRP78 protein in spinal dorsal horn was decreased, and the expressions of mRNA and protein of Beclin-1 were increased (P<0.01). Under electron microscope, the ultrastructure of spinal dorsal horn neurons in the wheat-grain moxibustion group was not significantly damaged, and its structure was basically close to normal, and the number of autophagosomes was more than the other three groups. CONCLUSION Wheat-grain moxibustion at "Dazhui" (GV 14) has analgesic effect on CSR rats. The mechanism may be related to moderately up-regulate the expression of Beclin-1, enhance autophagy and reduce endoplasmic reticulum stress.
Animals ; Beclin-1/genetics* ; Endoplasmic Reticulum Chaperone BiP ; Moxibustion ; RNA, Messenger ; Radiculopathy/therapy* ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; Spinal Cord Dorsal Horn ; Spondylosis ; Triticum/genetics*

OBJECTIVE: To evaluate the inhibitory effect of cordycepin on oral cancer xenograft in nude mice and explore the underlying mechanisms. METHODS: Sixteen BALB/c mice bearing subcutaneous human tongue squamous cell carcinoma (TSCC) TCA-8113 cell xenografts were randomized into model group and cordycepin treatment group for daily treatment with saline and cordycepin for 4 weeks. After the treatment, the tumor xenografts were dissected and weighed to assess the tumor inhibition rate. Histological changes in the heart, spleen, liver, kidney, and lung of the mice were evaluated with HE staining, and tumor cell apoptosis was examined using TUNEL staining; The expressions of Bax, Bcl-2, GRP78, CHOP, and caspase-12 in the xenografts were detected using RT-qPCR and Western blotting. RESULTS: Cordycepin treatment resulted in a tumor inhibition rate of 56.09% in the nude mouse models, induced obvious changes in tumor cell morphology and significantly enhanced apoptotic death of the tumor cells without causing pathological changes in the vital organs. Cordycepin treatment also significantly reduced Bcl-2 expression (P < 0.05) and increased Bax, GRP78, CHOP, and caspase-12 expressions at both the RNA and protein levels in the tumor tissues. CONCLUSION Cordycepin treatment can induce apoptotic death of TCA-8113 cell xenografts in nude mice via the endogenous mitochondrial pathway and endoplasmic reticulum stress pathways.
Humans ; Animals ; Mice ; Carcinoma, Squamous Cell/drug therapy* ; Heterografts ; Mice, Nude ; Tongue Neoplasms/drug therapy* ; Cordyceps ; Caspase 12 ; Endoplasmic Reticulum Chaperone BiP ; bcl-2-Associated X Protein ; Tongue

This study aims to investigate the effect of atractylenolide Ⅲ(ATL-Ⅲ) on hydrogen peroxide(H_2O_2)-induced endoplasmic reticulum stress and apoptosis of H9 c2 cells via the ROS/GRP78/caspase-12 signaling pathway.The binding activity of ATL-Ⅲ to GRP78 was determined by molecular docking.The result showed that ATL-Ⅲ had a good binding activity to GRP78, and the binding activity of ATL-Ⅲ was stronger than that of its specific inhibitor.The endoplasmic reticulum stress model of H9 c2 was established by H_2O_2(100 μmol·L~(-1)) treatment.Five groups were designed: blank control group, model group, and ATL-Ⅲ(15, 30, and 60 μmol·L~(-1)) groups.Apoptosis was detected by Hoechst/PI double staining and flow cytometry.The levels of superoxide dismutase(SOD), malondialdehyde(MDA), and lactate dehydrogenase(LDH) were measured by colorimetry.The levels of reactive oxygen species(ROS) and calcium(Ca~(2+)) in cytoplasm were determined by the fluorescence probe DCFH-DA and the calcium fluorescence probe Flou-4, respectively.The protein levels of GRP78, caspase-12, and caspase-3 were determined by Western blot, and the mRNA levels of GRP78 and caspase-12 by RT-qPCR.N-acetyl-L-cysteine(NAC) and 4-phenylbutyric acid(4-PBA) were respectively used to inhibit ROS and GRP78, and then the mechanism of ATL-Ⅲ in protecting the cells from endoplasmic reticulum stress induced by H_2O_2 were deduced.ATL-Ⅲ(15, 30, and 60 μmol·L~(-1)) decreased the apoptosis rate and ROS, MDA, and LDH levels(P<0.01), increased the SOD activity(P<0.01), and down-regulated the protein levels of GRP78, caspase-12, and caspase-3 and the mRNA levels of GRP78 and caspase-12(P<0.05).The addition of NAC decreased the apoptosis rate and ROS, MDA, GRP78, caspase-12, and caspase-3 levels(P<0.01), while it elevated the SOD level(P<0.01).The addition of 4-PBA also decreased the apoptosis rate and the levels of GRP78, caspase-12, caspase-3, and Ca~(2+)(P<0.01).The effect of inhibitors were consistent with that of ATL-Ⅲ.In conclusion, ATL-Ⅲ can protect H9 c2 cardiomyocytes by regulating ROS/GRP78/caspase-12 signaling pathway to inhibit H_2O_2-induced endoplasmic reticulum stress and apoptosis.
Apoptosis ; Calcium/pharmacology* ; Caspase 12/metabolism* ; Caspase 3/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; Endoplasmic Reticulum Stress ; Lactones ; Molecular Docking Simulation ; RNA, Messenger ; Reactive Oxygen Species/metabolism* ; Sesquiterpenes ; Signal Transduction ; Superoxide Dismutase/metabolism*

OBJECTIVE: To investigate the effects of asperuloside on cervical cancer based on endoplasmic reticulum (ER) stress and mitochondrial pathway. METHODS: Different doses (12.5-800 µg/mL) of asperuloside were used to treat cervical cancer cell lines Hela and CaSki to calculate the half maximal inhibitory concentration (IC₅₀) of asperuloside. The cell proliferation was analyzed by clone formation assay. Cell apoptosis, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by flow cytometry. The protein expressions of cleaved-caspase-3, Bcl-2, Bax, Cyt-c, cleaved-caspase-4 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot. And the inhibitor of ER stress, 4-phenyl butyric acid (4-PBA) was used to treat cervical cancer cells to further verify the role of ER stress in the apoptosis of cervical cancer cells induced by asperuloside. RESULTS: Asperuloside of 325, 650, and 1300 µg/mL significantly inhibited the proliferation and promoted apoptosis of Hela and CaSki cells (P<0.01). All doses of asperuloside significantly increased intracellular ROS levels, reduced mitochondrial membrane potential, significantly reduced Bcl-2 protein expression level, and increased Bax, Cyt-c, GRP78 and cleaved-caspase-4 expressions (P<0.01). In addition, 10 mmol/L 4-PBA treatment significantly promoted cell proliferation and reduced apoptosis (P<0.05), and 650 µg/mL asperuloside could reverse 4-PBA-induced increased cell proliferation, decreased apoptosis and cleaved-caspase-3, -4 and GRP78 protein expressions (P<0.05). CONCLUSION Our study revealed the role of asperuloside in cervical cancer, suggesting that asperuloside promotes apoptosis of cervical cancer cells through ER stress-mitochondrial pathway.
Female ; Humans ; Uterine Cervical Neoplasms/metabolism* ; Caspase 3/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Reactive Oxygen Species/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; HeLa Cells ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Endoplasmic Reticulum Stress ; Cell Line, Tumor

Traditional Chinese medicine believes that the occurrence and development of tumors is related to the body's Qi deficiency. " Invigorating Qi for consolidation of exterior" has became an effective way to treat tumors by traditional Chinese medicine. This study is based on the " invigorating Qi for consolidation of exterior" to explore the effect of flavonoid components in Qi-invigorating herbs Astragali Radix( AR) on the growth and immune function of mouse Lewis lung cancer xenografts,and further explore its mechanism of action. In the present study,high performance liquid chromatography was performed to analyze the flavonoid components in AR.The Lewis lung cancer model of C57 BL/6 mice was constructed,and the tumor volume of mice was determined by Visual Sonics Vevo2100 high frequency color ultrasound. The levels of IL~(-1)7 and RORγt in serum and tumor tissues were detected by ELISA and immunohistochemistry. The expression of IRE~(-1)/XBP~(-1) pathway-related proteins in tumor tissues was detected by Western blot. The results revealed that treatment of 5 and 10 g·kg~(-1)·d~(-1) of flavonoid components in AR significantly inhibited tumor growth of C57 BL/6 tumorbearing mice. The inhibition rates at the dose of 5 and 10 g·kg~(-1)·d~(-1) of flavonoid components in AR were( 29. 5±4. 4) % and( 43. 4±5. 2) %,respectively. The expression of IL~(-1)7 and RORγt in serum and tumor tissues of Lewis lung cancer mice were decreased,and the spleen index and thymus index were significantly enhanced by the flavonoid components in AR. Flavonoid components in AR could decrease the expression of X-box binding protein 1( XBP1),inositol-requiring enzyme( IRE1) and glucose regulated protein 78 k D( GRP78),and increase the expression of C/EBP homologous protein( CHOP),and the high-dose group is better,suggesting that the anti-lung cancer effect of flavonoid components in AR is related to the regulation of XBP1 mediated ERs. This study provides new evidence that the flavonoid components in AR could inhibit the tumor growth of C57 BL/6 tumor-bearing mice by regulating the body's immune function through " invigorating Qi for consolidation of exterior".
Animals ; Astragalus Plant/chemistry* ; Carcinoma, Lewis Lung/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Endoplasmic Reticulum Chaperone BiP ; Flavonoids/therapeutic use* ; Mice ; Mice, Inbred C57BL ; Qi ; Xenograft Model Antitumor Assays

This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.
Rats ; Animals ; Proto-Oncogene Proteins c-akt ; Endoplasmic Reticulum Chaperone BiP ; Caspase 3 ; Caspase 9 ; Diabetes Mellitus, Experimental ; Caspase 12 ; Calcium/pharmacology* ; Molecular Docking Simulation ; Endoplasmic Reticulum Stress ; Protein Serine-Threonine Kinases/genetics* ; Liver ; Apoptosis ; Insulin ; Glucose ; Glycogen/pharmacology* ; RNA, Messenger