OBJECTIVETo design and synthesize ribozymes targeting 138 and 218 sites of the mRNA nucleotide of mouse caspase-12, a key intermedium of ER stress mediated apoptosis, and to identify their activities through in vitro transcription and cleavage.

METHODSThe mouse caspase-12 gene fragment was obtained by RT-PCR and cloned into the PGEM-T vector under the control of T7 RNA polymerase promoter. The transcription product of the target was labeled with a-32P UTP, while ribozymes were not labeled. Ribozyme and target RNA were incubated for 90 min at 37 degree C in a reaction buffer to perform the cleavage reaction.

RESULTSIt was found that under a condition of 37 degree C, pH 7.5 and with Mg2+ in a concentration of 10 mmol/L, Rz138 and Rz218 both cleaved targets at predicted sites, and the cleavage efficiency of Rz138 was 100%.

CONCLUSIONRz138 and Rz218 prepared in vitro possess the perfect specific catalytic cleavage activity. Rz138 has excellent cleavage efficiency. It may be a promising tool to prevent ER stress induced apoptosis through catalytic cleavage of caspase-12 mRNA in vivo. It also can be used to verify whether caspase-12 is necessary in ER stress induced apoptosis.

Animals ; Base Sequence ; Caspase 12 ; genetics ; Endoplasmic Reticulum ; metabolism ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Oxidative Stress ; genetics ; RNA, Catalytic ; chemistry ; genetics ; RNA, Messenger ; genetics

OBJECTIVESTo study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs) against caspase-12.

METHODSThe Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type IV, and apoptosis were induced with 4 micromol/L thapsigargin (TG). The three kingds of siRNAs targeting different gene sites (130, 214, 521) were synthetized chemically. The single-stranded RNAs were annealed to produce double-stranded siRNAs, then the mouse primary hepatocytes were transfected by oligofectamine package. The inhibition of caspase-12 was analyzed with RT-PCR and Western-blot. The viable hepatocytes following the induction of apoptosis were evaluated with MTT.

RESULTSAll the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA. The siRNA (214) were more effective than the other two when the concentration was 100 nmol/L. The caspase-12 mRNA expression was inhibited by 52.08%, while that of siRNA (521) was 30.73% (t=4.30, P <0.05). However when the concentration was 200 nmol/L, the inhibitions were similar (88.07%, 86.22% and 89.41% respectively). siRNA (214) could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43% ( t=4.30, P <0.01). Contrasted with apoptotic hepatocytes, the cell activity, which was analyzed with MTT, increased by 48.76% (t=2.23, P <0.01).

CONCLUSIONsiRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.

Animals ; Apoptosis ; physiology ; Caspase 12 ; biosynthesis ; genetics ; Hepatocytes ; cytology ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics

OBJECTIVEGRP78 is a sensitive marker of endoplasmic reticulum stress. Caspase-12 is involved in apoptosis induced by endoplasmic reticulum stress. This study was designed to explore the changes of GRP78 and caspase-12 mRNA in neonatal rats with experimental hypoxic-ischemic white matter damage (WMD) and investigate the roles of endoplasmic reticulum stress in the WMD.

METHODSTwo-day-old rats were randomized to WMD and control groups (n=49 each). The pups were sacrificed at 0, 2, 4, 6, 12, 24 and 72 hrs after hypoxia-ischemia (HI). The light microscope was used to observe the brain pathological changes. Real time PCR was used to detect the expression of GRP78 mRNA and caspase-12 mRNA in the white matter tissue.

RESULTSThe expression of GRP78 mRNA began increasing 2 hrs after HI and peaked at 6 hrs in the WMD group, demonstrating significant differences at 2, 4, 6, 12, 24 and 72 hrs compared with the control group (P<0.05). The caspase-12 mRNA expression in the WMD group began increasing 6 hrs after HI and demonstrated significantly increased levels 6, 12 and 24 hrs after HI compared with those in the control group (P<0.05).

CONCLUSIONSGRP78 and caspase-12 mRNA expression increased significantly in neonatal rats with WMD. This suggests that endoplasmic reticulum stress may be induced following HI. Endoplasmic reticulum stress seems to be involved in the apoptosis of oligodendrocytes induced by HI in neonatal rats with WMD.

Animals ; Animals, Newborn ; Brain ; pathology ; Caspase 12 ; genetics ; Disease Models, Animal ; Heat-Shock Proteins ; genetics ; Hypoxia-Ischemia, Brain ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To observe the effect of moxibustion at oppositely-located points "Mingmen" (GV 4) and "Shenque" (CV 8) on the motor function of the hind limbs and bladder function in rats with neurogenic bladder after suprasacral spinal cord injury (SCI), so as to explore the effect of this therapy on bladder tissue apoptosis mediated by endoplasmic reticulum stress pathway. METHODS: Twenty-eight female Wistar rats were randomly divided into a sham-operation group (8 rats) and a model establishment group (20 rats). Using the modified Allen's method, the spinal cord of T₁₀ segment was injured to establish a neurogenic bladder model in the model establishment group. Sixteen rats were modeled successfully and then divided into a model group (8 rats) and a moxibustion group (8 rats). In the moxibustion group, 2 h after consciousness regaining from modeling anesthesia, moxibustion was exerted at "Shenque" (CV 8) and "Mingmen" (GV 4), 2 cones at each acupoint in one intervention. The intervention was administered once every two days and 5-time intervention was required totally. After intervention, Basso, Beattie and Bresnahan locomotor rating scale (BBB) score for the motor function of the hind limbs, and the urodynamics indexes (maximum bladder capacity, urine leakage pressure and bladder compliance) were compared among groups. HE staining method was adopted to observe the morphological changes of bladder tissue. With Western blot method and real-time PCR assay, the protein and mRNA expressions of the endoplasmic reticulum stress-related genes (glucose- regulated protein 78 [GRP78], activating transcription factor 4 [ATF4] and cysteinyl aspartate specific proteinase-12 [Caspase-12]) were determined. RESULTS: The transitional epithelial cells were arranged irregularly, the bladder wall was getting thinner, and the cellular vacuolar degeneration and neutrophil infiltration were found in the model group. Whereas, compared with the model group, in the moxibustion group, the arrangement of transitional epithelial cells was clear and continuous in layers, the cellular vacuolar degeneration was mild and the infiltration presented in a small amount of neutrophil granulocytes. Compared with the sham-operation group, in the model group, the BBB score was reduced (P<0.01), the maximum bladder capacity and bladder compliance were increased (P<0.01), and the protein expression levels of GRP78, ATF4 and Caspase-12, as well as mRNA expressions were all increased (P<0.01). In comparison with the model group, in the moxibustion group, BBB score was increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), and the protein and mRNA expression levels of GRP78, ATF4 and Caspase-12 were all decreased (P<0.01). CONCLUSION Moxibustion at the "oppositely-located points" improves the urination function, alleviate urine retention in neurogenic bladder rats after spinal cord injury. The underlying mechanism may be related to the down-regulation of the expressions of GRP78, ATF4 and Caspase-12 in the endoplasmic reticulum stress pathway of the bladder tissues, and thus to alleviate the apoptosis of bladder tissue.
Animals ; Caspase 12/genetics* ; Electroacupuncture ; Endoplasmic Reticulum Stress ; Female ; Moxibustion ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Spinal Cord ; Spinal Cord Injuries/therapy* ; Urinary Bladder, Neurogenic/therapy*

OBJECTIVETo study the role of caspase-12 expression on hepatocyte apoptosis in an experimental model of acute hepatic failure (AHF).

METHODSA mouse experimental model of AHF was developed by intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-Gal). Hepatocyte apoptosis was examined by DNA agarose gel and liver pathology. Caspase-12 mRNA expression in liver was detected by reverse transcriptase PCR (RT-PCR) method. The expression of caspase-12, GRP78 proteins in livers was determined by Western blot.

RESULTSCaspase-12 mRNA expression in the livers increased significantly from 5 to 7 hours after administration of LPS and D-Gal. Typical manifestation of hepatocyte apoptosis appeared at 5 hours after the drug administration. After 5 hours the level of serum ALT and AST were remarkably increased, and they reached the peak at 7 hours. The expression of procaspase-12 protein decreased obviously at 7 hours. Seven hours after the drug administration, hepatocyte apoptosis and necrosis both started. The marker of endoplasmic reticulum (ER) stress, Bip/GRP78 was activated during the development of hepatocyte apoptosis. The level of Bip/GRP78 protein was gradually increased at 5 hours after the drug induction.

CONCLUSIONHepatocyte apoptosis plays an important role in the pathogenesis of AHF. Caspase-12 induced ER stress involves in hepatocyte apoptosis. It suggests that inhibition of caspase-12 activation might be a potential strategy in the treatment of AHF in the future.

Animals ; Apoptosis ; drug effects ; Caspase 12 ; biosynthesis ; genetics ; Galactosamine ; Hepatocytes ; pathology ; Lipopolysaccharides ; Liver Failure, Acute ; chemically induced ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation

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

BACKGROUNDAccumulated evidence shows that hypoxia can induce endothelial apoptosis, however the mechanism is still unknown. We hypothesized whether intermittent or persistent hypoxia could induce endoplasmic reticular stress, leading to endothelial apoptosis.

METHODSTwenty-four 8-week male Sprague Dawley (SD) rats were divided into three groups: normoxia (NC) group, intermittent hypoxia (IH) group and persistent hypoxia (PH) group. TUNEL staining was performed to detect aortic arch endotheliar apoptosis, and immunohistochemistry for BIP, CHOP and caspase12 to test protein expression; human umbilical vein endothelial cells (HUVECs) of the line ECV304 were cultured (with or without taurodeoxycholic acid (TUDCA) 10 mmol/L, 100 mmol/L) and divided into four groups: NC group (20.8% O2 for 4 hours), PH1 group (5% O2 for 4 hours), PH2 group (5% O2 for 12 hours) and IH group (20.8% O2 and 5% O2 alternatively for 8 hours). Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in each group. The expressions of GRP78, CHOP and caspase12 were detected by real-time quantitative reverse-transcription PCR. Result Intermittent and persistent hypoxia could increase the rate of endothelium apoptosis and the expressions of GRP78, CHOP and caspase12 compared with the control, induction by intermittent hypoxia was slightly higher than persistent hypoxia. In the HUVEC experiment, TUDCA significantly reduced apoptosis and the expressions of GRP78, CHOP and caspase12.

CONCLUSIONHypoxia, especially intermittent, can induce endothelial cell apoptosis possibly through endoplasmic reticulum stress pathway, which can be attenuated by taurodeoxycholic acid.

Animals ; Apoptosis ; drug effects ; genetics ; physiology ; Caspase 12 ; genetics ; Endoplasmic Reticulum Stress ; drug effects ; genetics ; physiology ; Heat-Shock Proteins ; genetics ; Human Umbilical Vein Endothelial Cells ; Humans ; Hypoxia ; genetics ; physiopathology ; Male ; Rats ; Rats, Sprague-Dawley ; Taurodeoxycholic Acid ; pharmacology ; Transcription Factor CHOP ; genetics

BACKGROUNDPrevious studies have indicated that endoplasmic reticulum stress participates in and mediates liver injury and apoptosis in brain-dead (BD) rats. In this study, we observed the effect of salubrinal (Sal, Sigma, USA) on liver cells in BD rats and explored its relevant mechanisms.

METHODSThirty Sprague-Dawley rats were equally randomized into three groups: BD group, Sal group, and DMSO group. The BD models were established by increasing intracranial pressure in a modified, slow, and intermittent way. In the drug groups, Sal was administered 1 h before the induction of BD. After modeling was completed, the blood and liver samples were harvested. CHOP and Caspase-12 mRNA expression was detected using quantitative polymerase chain reaction. PKR-like ER kinase (PERK), P-eukaryotic translation initiation factor 2α (eIF2α), eIF2α, CHOP and caspase-12 expression was detected using western blotting (WB). CHOP and caspase-12 distribution and expression in liver tissues were determined using immunohistochemistry (IHC). Alanine aminotransferase and aspartate aminotransferase level were detected using an automatic biochemical analyzer. Hepatic cell apoptosis was detected using TUNEL. The results were analyzed using Quantity-one v4.62 software (Bio-Rad, USA).

RESULTSCHOP and caspase-12 expression and PERK, eIF2α, and P-eIF2α protein expression showed no significant difference between BD group and DMSO group. Compared with BD group, Sal group had a significantly higher P-eIF2C level and a lower P-PERK level 2 h and 6 h after BD (P < 0.05). However, eIF2α expression showed no significant difference (P > 0.05). After the Sal treatment, CHOP and caspase-12 mRNA expression significantly decreased 4 h after BD (P < 0.05). WB and IHC indicated that CHOP and caspase-12 expression also significantly decreased after Sal treatment. Sal was associated with improved liver function and decreased hepatic cell apoptosis.

CONCLUSIONSSal can significantly reduce apoptosis in hepatic cells of BD rats. This protective effect may be achieved via the PERK-eIF2α signaling pathway.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Brain Death ; metabolism ; Caspase 12 ; genetics ; metabolism ; Cinnamates ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; drug effects ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Immunohistochemistry ; Liver ; drug effects ; injuries ; Male ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Thiourea ; analogs & derivatives ; Transcription Factor CHOP ; genetics ; metabolism