This study aims to explore the mechanism through which Yishen Jiangtang Decoction(YSJTD) regulates endoplasmic reticulum stress(ERS)-mediated NOD-like receptor thermal protein domain associated protein 3(NLRP3) inflammasome to improve diabetic nephropathy(DN) in db/db mice. Thirty db/db mice were randomly divided into the model group, YSJTD group, ERS inhibitor 4-phenylbutyric acid(4-PBA) group, with 10 mice in each group. Additionally, 10 db/m mice were selected as the control group. The YSJTD group was orally administered YSJTD at a dose of 0.01 mL·g~(-1), the 4-PBA group was orally administered 4-PBA at a dose of 0.5 mg·g~(-1), and the control and model groups were given an equal volume of carboxylmethyl cellulose sodium. The treatments were administered once daily for 8 weeks. Food intake, water consumption, and body weight were recorded every 2 weeks. After the intervention, fasting blood glucose(FBG), glycosylated hemoglobin(HbA1c), urine microalbumin(U-mALB), 24-hour urine volume, serum creatinine(Scr), and blood urea nitrogen(BUN) were measured. Inflammatory markers interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected using the enzyme-linked immunosorbent assay(ELISA). Renal pathology was assessed through hematoxylin-eosin(HE), periodic acid-Schiff(PAS), and Masson staining, and transmission electron microscopy(TEM). Western blot was used to detect the expression levels of glucose-regulated protein 78(GRP78), C/EBP homologous protein(CHOP), NLRP3, apoptosis-associated speck-like protein containing CARD(ASC), cysteinyl aspartate-specific proteinase(caspase-1), and gasdermin D(GSDMD) in kidney tissues. The results showed that compared to the control group, the model group exhibited poor general condition, increased weight and food and water intake, and significantly higher levels of FBG, HbA1c, U-mALB, kidney index, 24-hour urine volume, IL-1β, and IL-18. Compared to the model group, the YSJTD and 4-PBA groups showed improved general condition, increased body weight, decreased food intake, and lower levels of FBG, U-mALB, kidney index, 24-hour urine volume, and IL-1β. Specifically, the YSJTD group showed a significant reduction in IL-18 levels compared to the model group, while the 4-PBA group exhibited decreased water intake and HbA1c levels compared to the model group. Although there was a decreasing trend in water intake and HbA1c in the YSJTD group, the differences were not statistically significant. No significant differences were observed in BUN, Scr, and kidney weight among the groups. Renal pathology revealed that the model group exhibited more severe renal damage compared to the control group. Kidney sections from the model group showed diffuse mesangial proliferation in the glomeruli, tubular edema, tubular dilation, significant inflammatory cell infiltration in the interstitium, and increased glycogen staining and blue collagen deposition in the basement membrane. In contrast, the YSJTD and 4-PBA groups showed varying degrees of improvement in renal damage, glycogen staining, and collagen deposition, with the YSJTD group showing more significant improvements. TEM analysis indicated that the model group had extensive cytoplasmic edema, homogeneous thickening of the basement membrane, fewer foot processes, and widening of fused foot processes. In the YSJTD and 4-PBA groups, cytoplasmic swelling of renal tissues was reduced, the basement membrane remained intact and uniform, and foot process fusion improved.Western blot results indicated that compared to the control group, the model group showed upregulation of GRP78, CHOP, GSDMD, NLRP3, ASC, and caspase-1 expression. In contrast, both the YSJTD and 4-PBA groups showed downregulation of these markers compared to the model group. These findings suggest that YSJTD exerts a protective effect against DN by alleviating NLRP3 inflammasome activation through the inhibition of ERS, thereby improving the inflammatory response in db/db DN mice.
Animals ; Endoplasmic Reticulum Stress/drug effects* ; Diabetic Nephropathies/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Inflammasomes/drug effects* ; Male ; Kidney/pathology* ; Endoplasmic Reticulum Chaperone BiP ; Humans ; Interleukin-18/genetics* ; Mice, Inbred C57BL

Microglial functions are linked to Ca²⁺ signaling, with endoplasmic reticulum (ER) calcium stores playing a crucial role. Microglial abnormality is a hallmark of Alzheimer's disease (AD), but how ER Ca²⁺ receptors regulate microglial functions under physiological and AD conditions remains unclear. We found reduced ryanodine receptor 2 (Ryr2) expression in microglia from an AD mouse model. Modulation of RyR2 using S107, a RyR-Calstabin stabilizer, blunted spontaneous Ca²⁺ transients in controls and normalized Ca²⁺ transients in AD mice. S107 enhanced ATP-induced migration and phagocytosis while reducing ramification in control microglia; however, these effects were absent in AD microglia. Our findings indicate that RyR2 stabilization promotes an activation state shift in control microglia, a mechanism impaired in AD. These results highlight the role of ER Ca²⁺ receptors in both homeostatic and AD microglia, providing insights into microglial Ca²⁺ malfunctions in AD.
Animals ; Microglia/pathology* ; Alzheimer Disease/pathology* ; Phagocytosis/drug effects* ; Ryanodine Receptor Calcium Release Channel/metabolism* ; Disease Models, Animal ; Mice ; Cell Movement/drug effects* ; Mice, Transgenic ; Calcium Signaling/physiology* ; Calcium/metabolism* ; Mice, Inbred C57BL ; Male ; Endoplasmic Reticulum/metabolism*

BACKGROUND: Pilea umbrosa (Urticaceae) is used by local communities (district Abbotabad) for liver disorders, as anticancer, in rheumatism and in skin disorders. METHODS: Methanol extract of P. umbrosa (PUM) was investigated for the presence of polyphenolic constituents by HPLC-DAD analysis. PUM (150 mg/kg and 300 mg/kg) was administered on alternate days for eight weeks in rats exposed with carbon tetrachloride (CCl). Serum analysis was performed for liver function tests while in liver tissues level of antioxidant enzymes and biochemical markers were also studied. In addition, semi quantitative estimation of antioxidant genes, endoplasmic reticulum (ER) induced stress markers, pro-inflammatory cytokines and fibrosis related genes were carried out on liver tissues by RT-PCR analysis. Liver tissues were also studied for histopathological injuries. RESULTS: Level of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione (GSH) decreased (p < 0.05) whereas level of thiobarbituric acid reactive substance (TBARS), HO and nitrite increased in liver tissues of CCl treated rat. Likewise increase in the level of serum markers; alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and total bilirubin was observed. Moreover, CCl caused many fold increase in expression of ER stress markers; glucose regulated protein (GRP-78), x-box binding protein1-total (XBP-1 t), x-box binding protein1-unspliced (XBP-1 u) and x-box binding protein1-spliced (XBP-1 s). The level of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) was aggregated whereas suppressed the level of antioxidant enzymes; γ-glutamylcysteine ligase (GCLC), protein disulfide isomerase (PDI) and nuclear erythroid 2 p45-related factor 2 (Nrf-2). Additionally, level of fibrosis markers; transforming growth factor-β (TGF-β), Smad-3 and collagen type 1 (Col1-α) increased with CCl induced liver toxicity. Histopathological scrutiny depicted damaged liver cells, neutrophils infiltration and dilated sinusoids in CCl intoxicated rats. PUM was enriched with rutin, catechin, caffeic acid and apigenin as evidenced by HPLC analysis. Simultaneous administration of PUM and CCl in rats retrieved the normal expression of these markers and prevented hepatic injuries. CONCLUSION Collectively these results suggest that PUM constituted of strong antioxidant chemicals and could be a potential therapeutic agent for stress related liver disorders.
Animals ; Carbon Tetrachloride ; adverse effects ; Chemical and Drug Induced Liver Injury ; drug therapy ; etiology ; pathology ; Endoplasmic Reticulum Stress ; drug effects ; Fibrosis ; drug therapy ; genetics ; Inflammation ; drug therapy ; genetics ; Liver ; drug effects ; enzymology ; metabolism ; Male ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Urticaceae ; chemistry

PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.
Anti-Bacterial Agents/pharmacology ; Apoptosis ; Blotting, Western ; C-Reactive Protein/biosynthesis/*genetics ; Cells, Cultured ; Endoplasmic Reticulum Stress/*drug effects/genetics ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Humans ; Polymerase Chain Reaction ; RNA, Messenger/*genetics ; Retinal Pigment Epithelium/*metabolism/pathology ; Serum Amyloid P-Component/biosynthesis/*genetics ; Tunicamycin/*pharmacology

OBJECTIVE: To explore the effects of 3-methyladenine (3-MA, an autophagy inhibitor) on sensitivities of nasopharyngeal carcinoma cells to radiotherapy and chemotherapy and the underlying mechanisms.
 METHODS: Cell proliferation was examined by MTT and colony formation assay, while cell apoptosis was evaluated by annexin V/PI double staining and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining. Mitochondrial membrane potential was measured by commercial kit (JC-1). The expression of endoplasmic reticulum stress (ERS)-related protein, glucose-regulated protein 78 (GRP78) and autophagy-related protein beclin1, microtubule-associated protein 1 light chain 3 (LC3) were examined by Western blot.
 RESULTS: Cisplatin (DDP), ionizing radiation (IR) or tunicamycin (TM) treatment obviously inhibited the proliferation of HONE-1 cells in a concentration-dependent and time-dependent manner. Compared with control group, pretreatment with 1 mmol/L of 3-MA significantly 
reduced cell viability and enhanced the apoptosis in the DDP (6.00 μmol/L), 4.00 Gy IR or TM (1.00 μmol/L) groups. There was no significant difference in the apoptosis between the DDP (5.8%) and 4Gy IR (6.7%) groups. Compared with the control group, protein levels of GRP78, beclin1 and lipid-conjugated membrane-bound form (LC3-II) were significantly increased after the treatment of DDP, 4.00 Gy IR or TM, which were inhibited by pretreatment of 3-MA.
 CONCLUSION 3-MA can sensitize HONE-1 cells to chemotherapy and radiotherapy, which is related to prevention of endoplasmic reticulum stress-induced autophagy in nasopharyngeal carcinoma cells.
Adenine ; analogs & derivatives ; pharmacology ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Beclin-1 ; Carcinoma ; Cell Line, Tumor ; drug effects ; radiation effects ; Cell Proliferation ; Cell Survival ; Cisplatin ; pharmacology ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; pathology ; Radiation, Ionizing ; Radiation-Sensitizing Agents ; pharmacology ; Tunicamycin ; pharmacology

OBJECTIVETo explore the effect of emodin on endoplasmic reticulum (ER) stress of pancreatic acinar AR42J cells.

METHODRat pancreatic acinar AR42J cells were cultured in 6-well plates, and divided into the normal control group, the model group (with the final concentration at 1 x 10(-7) mol · L(-1) for cerulean and lipopolysaccharide at 10 mg · L(-1)) and the emodin group (10, 20, 40 μmol · L(-1)). Cells in each group were cultured in three multiple pores for 24 h, and their supernate was removed after cell attachment. The normal control group was added with haploids, the model group was added with the modeling liquid for haploids, and the treatment groups were added with different concentrations of emodin at 15-20 min before the modeling liquid. The cells were continuously cultured for 3 h under 37 °C and 5% CO2. Their intracellular protease and lipase expressions were detected with kits. The cellular morphology was observed under optical microscope. The level of calcium in endoplasmic reticulum was measured under laser confocal microscopy. Western blot assay were used to determine the protein expression of ER-related signaling molecules.

RESULTEmodin could significantly inhibit levels of amylase, lipase and intracellular calcium and ER.

CONCLUSIONEmodin could reduce pancreatic acinar cell injury induced by the combination of cerulean and lipopolysaccharide. Its action mechanism is correlated with the inhibition of intracellular calcium overload and ER stress.

Animals ; Calcium ; metabolism ; Cell Line, Tumor ; Emodin ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Pancreatic Neoplasms ; metabolism ; pathology ; Rats ; Unfolded Protein Response ; drug effects

OBJECTIVETo investigate the effect of lonidamine on apoptosis of human breast carcinoma cells MCF-7 and the possible mechanisms.

METHODSMTT assay and colony-forming assay were used to evaluate the growth inhibition induced by lonidamine in breast cancer MCF-7 cells. PI/Annexin-V staining was used to detect the apoptotic cells. The ATP levels in the cells were detected using an ATP assay kit. The expression of glucose regulated protein 78 (GRP78), inhibitor of apoptosis protein (cIAP1) and caspase-8 were analyzed with Western blotting.

RESULTSMTT assay and colony-forming assay showed that 50-250 mmol/L lonidamine caused a time- and concentration-dependent inhibition of MCF-7 cell proliferation. Exposure to increased concentrations of lonidamine resulted in significantly increased apoptosis rate in MCF-7 cells. In MCF-7 cells treated with 50, 150 and 250 mmol/L lonidamine for 5 h, the intracellular ATP levels were lowered to 80.67%, 62.78% and 30.73% of the control level, respectively. Western blotting showed that lonidamine up-regulated the expression of GRP78, down-regulated the expression of cIAP1 and promoted caspase-8 activation as the treatment time extended.

CONCLUSIONLonidamine can inhibit the proliferation and induce apoptosis in MCF-7 cells, and these effects are probably mediated by reducing ATP level, inducing endoplasmic reticulum stress response, down-regulating cIAP1, and promoting caspase-8 activation in the cells.

Apoptosis ; drug effects ; Breast Neoplasms ; metabolism ; pathology ; Caspase 8 ; metabolism ; Cell Proliferation ; Down-Regulation ; Endoplasmic Reticulum Stress ; Heat-Shock Proteins ; metabolism ; Humans ; Indazoles ; pharmacology ; Inhibitor of Apoptosis Proteins ; metabolism ; MCF-7 Cells ; drug effects ; Ubiquitin-Protein Ligases ; metabolism ; Up-Regulation

OBJECTIVETo investigate the role of oxidative stress in the endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust.

METHODSSeventy-two healthy adult Wistar rats were randomly divided into control group, quartz dust group, quartz dust plus N-acetyl cysteine (NAC) group, and NAC group, with 18 rats in each group. One milliliter of sterile saline (for the control and NAC groups) or 1 ml of saline with 5%ultrafine quartz dust (for dust group and dust plus NAC group) was given to each rat by non-exposed endotracheal infusion. From the second day after dust infusion, rats in dust plus NAC group and NAC group received intragastric administration of NAC (100 mg/kg). In each week, the treatment with NAC lasted for 5 consecutive days, followed by 2 days' interval. For each group, 6 rats were randomly selected on the 14th, 28th, or 56th day after dust exposure; they were sacrificed by bloodletting from the femoral artery, and the lungs were collected. Bronchoalveolar lavage fluid was collected to separate macrophages. The protein expression of caspase-12 in alveolar macrophages, the apoptosis rate and reactive oxygen species (ROS) content of alveolar macrophages, and the protein carbonyl content of alveolar macrophages were determined by Western blot, flow cytometry, and colorimetry, respectively.

RESULTSIncreased protein expression of caspase-12, apoptosis rate, and content of ROS and protein carbonyl were discovered on the 14th day in the dust group, in comparison with the control group (P < 0.05), and the increase lasted till the 28th and 56th days. (P < 0.05). Compared with the dust group, the dust plus NAC group showed significant decreases in the content of ROS on the 14th, 28th, and 56th days (P < 0.05), significant decreases in the content of protein carbonyl on the 28th and 56th days (P < 0.05), and significant decreases in the protein expression of caspase-12 and apoptosis rate (P < 0.05).

CONCLUSIONOxidative stress is potentially involved in the endoplasmic reticulum stress-induced apoptosis of alveolar macrophages triggered by quartz dust. Oxidative damage of protein in the endoplasmic reticulum may play an important role in the process.

Animals ; Caspase 12 ; metabolism ; Dust ; Endoplasmic Reticulum Stress ; drug effects ; Macrophages, Alveolar ; drug effects ; pathology ; Male ; Oxidative Stress ; drug effects ; Protein Carbonylation ; Quartz ; toxicity ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism

OBJECTIVEThe study intended to investigate the effect and mechanism of endoplasmic reticulum stress on cisplatin resistance in ovarian carcinoma.

METHODSRT-PCR and Western blot were used to test the expression of mTOR and Beclin1 mRNA and protein in ovarian cancer SKOV3 cells after saquinavir induction. MTT assay was used to analyze the influence of saquinavir on cisplatin sensitivity in SKOV3 cells.

RESULTSThe IC50 of SKOV3 cells was (5.490 ± 1.148) µg/ml. After induced by Saquinavair 10 µmol/L and 20 µmol/L, the IC50 of SKOV3 cells was increased to (11.199 ± 0.984) µg/ml and (14.906 ± 2.015) µg/ml, respectively. It suggested that the sensitivity of ovarian cancer cells to cisplatin was decreased significantly (P = 0.001). The expression of mTOR and Beclin1 mRNA and protein was significantly different among the five groups: the (Saquinavair+DDP) group of, Saquinavair group, LY294002 group, DDP group and control group (P < 0.001) . The expressions of mTOR and Beclin1 mRNA were highest in the (Saquinavair+DDP) group, 0.684 ± 0.072 and 0.647 ± 0.047, respectively; Secondly, the Saquinavair group, 0.577 ± 0.016 and 0.565 ± 0.037, respectively. The expressions of mTOR and Beclin1 proteins were also highest in the (Saquinavair+DDP) group, 0.624 ± 0.058 and 0.924 ± 0.033, respectively, followed by the Saquinavair group, 0.544 ± 0.019 and 0.712 ± 0.024. 3-MA inhibited the autophagy and restored cisplatin sensitivity in the SKOV3 cells after Saquinavir induced ER stress (P < 0.001).

CONCLUSIONSSaquinavir can effectively induce endoplasmic reticulum stress in SKOV3 cells. Endoplasmic reticulum stress can decrease the sensitivity to cisplatin in SKOV3 cells. The mechanism of the decrease of sensitivity to cisplatin in SKOV3 cells may be that ERS regulates cell autophagy through the mTOR and Beclin1 pathways. ERS of tumor cells and autophagy may become a new target to improve the therapeutic effect of chemotherapy and to reverse the drug resistance in tumor treatment.

Antineoplastic Agents ; pharmacology ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Autophagy ; drug effects ; Beclin-1 ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Cystadenocarcinoma, Serous ; metabolism ; pathology ; Drug Resistance, Neoplasm ; Endoplasmic Reticulum Stress ; drug effects ; Female ; HIV Protease Inhibitors ; pharmacology ; Humans ; Membrane Proteins ; genetics ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; Saquinavir ; pharmacology ; TOR Serine-Threonine Kinases ; genetics ; metabolism

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered the key determinants of insulin resistance. Impaired mitochondrial function in obese animals was shown to induce the ER stress response, resulting in reduced adiponectin synthesis in adipocytes. The expression of inducible nitric oxide synthase (iNOS) is increased in adipose tissues in genetic and dietary models of obesity. In this study, we examined whether activation of iNOS is responsible for palmitate-induced mitochondrial dysfunction, ER stress, and decreased adiponectin synthesis in 3T3L1 adipocytes. As expected, palmitate increased the expression levels of iNOS and ER stress response markers, and decreased mitochondrial contents. Treatment with iNOS inhibitor increased adiponectin synthesis and reversed the palmitate-induced ER stress response. However, the iNOS inhibitor did not affect the palmitate-induced decrease in mitochondrial contents. Chemicals that inhibit mitochondrial function increased iNOS expression and the ER stress response, whereas measures that increase mitochondrial biogenesis (rosiglitazone and adenoviral overexpression of nuclear respiratory factor-1) reversed them. Inhibition of mitochondrial biogenesis prevented the rosiglitazone-induced decrease in iNOS expression and increase in adiponectin synthesis. These results suggest that palmitate-induced mitochondrial dysfunction is the primary event that leads to iNOS induction, ER stress, and decreased adiponectin synthesis in cultured adipocytes.
3T3-L1 Cells ; *Adipocytes/drug effects/metabolism ; Adiponectin/biosynthesis ; Adipose Tissue/metabolism ; Animals ; Endoplasmic Reticulum Stress/drug effects ; Insulin Resistance/genetics ; Mice ; Mitochondria/drug effects/*metabolism/pathology ; Mitochondrial Turnover/drug effects/genetics ; *Nitric Oxide Synthase Type II/genetics/metabolism ; Nuclear Respiratory Factor 1 ; Obesity/genetics/metabolism ; Palmitic Acid/pharmacology ; Thiazolidinediones/pharmacology