Effects of Moxibustion on NLRP3 Inflammasome and Caspase-12 in Synovium of Experimental RA Rabbits / 世界科学技术-中医药现代化

Objective To observe the effects of moxibustion on the expression of Nucleotide binding oligomeric domain like receptor thermoprotein domain associated protein 3(Nod-like receptor protein 3,NLRP3)inflammasome and Cysteine aspartate protease 12(Caspase-12)in synovial tissue of experimental Rheumatoid Arthritis(RA)rabbits,and explore the anti-inflammatory mechanism of moxibustion on RA rabbits.Methods Twenty-four Japanese big-eared white rabbits were randomly divided into 3 groups according to body weight and sex:the control group,the model group and the moxibustion group.The Freund's Complete Adjuvant(FCA)was injected into the joint cavities of two knee joints of rabbits at the dose of 0.5 mL·kg-1 in the model group and the moxibustion group,and the control group was injected with the same amount of normal saline as control.For the moxibustion group,scar free moxibustion was operated at bilateral"BL 23"and"ST 36"acupoints,moxibustion for 3 times per acupoint per day,continuous treatment for 6 days,rest for 1 day,as a course of treatment,a total of 3 courses of treatment.The histological changes of synovium samples of rabbit knee joints were observed under light microscope,real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of NLRP3,Cysteine aspartate protease 1(Caspase-1),Caspase-12 in synovial tissues and Enzyme-linked immuno sorbent assay(ELISA)was used to measure the change of Interleukin-1β(IL-1β)content in the synovial fluid of experimental animals.Results Compared with the control group,the degree of synovial lesions(inflammatory cell infiltration,synovial tissue hyperplasia,fibrous tissue hyperplasia,macrophage hyperplasia,etc.)and pathological score were increased(P<0.05),the expression of NLRP3,Caspase-1 mRNA were increased(P<0.05),the expression of Caspase-12 mRNA was significantly downregulated(P<0.01)and the expression content of IL-1β was significantly up-regulated(P<0.01)in the model group;Compared with the model group,the synovial lesion degree and pathological score were decreased(P<0.05),the expression of NLRP3,Caspase-1 mRNA were decreased(P<0.05),the expression of Caspase-12 mRNA was significantly increased(P<0.01)and the expression of IL-1β was decreased(P<0.05)in the moxibustion group.Conclusion Moxibustion can significantly inhibit the pathological process of knee synovitis in RA rabbits;Moxibustion at"Shenshu(BL 23)"and"Zusanli(ST 36)"acupoints can significantly up-regulate the expression of Caspase-12 and then inhibit the NLRP3 inflammasome,which may be one of mechanisms of moxibustion to reduce RA inflammatory response.

Tanshinone D A improves acute liver injury in mice by inhibiting Grp78/caspase-12 pathway / 中国药理学通报

Aim To investigate the protective effect of tanshinone II A on endoplasmic reticulum stress-induced acute liver injury in mice based on Grp78/ caspase-12 pathway. Methods Sixty male Kunming mice were randomly divided into normal group, model group, positive drug group and tanshinone II A low, medium and high dose groups (10, 20, 40 mg • kg"1 • d"1). Tunicamycin (1 mg • kg"1) was administered to replicate the acute liver injury model in mice. The liver pathological changes and hepatocyte apoptosis were observed 24 hours after the model was established. The serum levels of inflammatory factors and the expressions of Grp78, caspase-12, caspase-3, Bcl- 2 and Bax in liver tissues were detected. Results Compared with control group, serum AST, ALT, MDA levels increased and SOD level decreased in model group. The pathological damage and hepatocyte apoptosis were observed. The expressions of Grp78, caspase-12, caspase-3 and Bax in liver increased, the expression of Bcl-2 decreased, and the mRNA levels of Grp78 and caspase-12 were elevated. After bifendate and tanshinone II A treatment, the changes of the a- bove indicators in acute liver injury mice group were reversed. Conclusions Tanshinone IIA can improve the acute liver injury induced by tunicamycin in mice by inhibiting the activation of GRP78/caspase-12 pathway in liver tissues and reducing endoplasmic reticulum stress level.

Protective effect of glutamine on hyperoxic lung injury of neonatal rats through endoplasmic reticulum stress pathway / 吉林大学学报(医学版)

Objective: To explore the protective effect of glutamine (GLN) on the hyperoxia-induced lung injury of the neonatal rats through endoplasmic reticulum stress (ERS) pathway, and to elucidate its mechanisms. Methods: A total of 90 Wistar rats were randomly divided into control group (FiO2 =21%), hyperoxia group (FiO2 85%), and hyperoxia+GLN group (Fi2 85%, the concentration of intraperitoneal injection of GLN was 0. 75 g · kg-1 · d-1); there were 30 rats in each group The body weights and water contents in the lung tissue of the neonatal rats were measured on the 3rd, 7th and 14th days of the experiment. HE staining was used to determine the morphology of lung tissue of the rats. The superoxide dismutase (SOD) activity in lung tissue of the rats was detected by nitro blue tetrazolium chloride (NBT), and the malondialdehyde (MDA) level was determined by thiobarbital acid (TBA). The expression levels of Caspase-12, GADD153, GRP78, Bel-2, and Bax in lung tissue of the rats were detected by Western blotting method. Results: Compared with control group at the same time, the body weights of the neonatal rats in hyperoxia group on the 3rd, 7th and 14th days were significantly decreased (P<0. 05), the water contents in lung tissue of the neonatal rats were increased (P<0. 05), the SOD activities were significantly decreased (P<0. 05), the levels of MDA in the lung tissue of the neonatal rats were increased (P<0. 05), the expressions levels of Caspase-12, GADD153, GRP78 and Bax proteins were significantly increased (P<0. 05), and the expression levels of Bcl-2 protein and the Bcl-2/Bax ratios were significantly decreased (P<0. 05). Compared with hyperoxia group at the same time, the body weights of the neonatal rats in hyperoxia + GLN group on the 3rd, 7th and 14th days were significantly increased (P<0. 05), the water contents in lung tissue of the neonatal rats were decreased (P<0. 05), the SOD activities were significantly increased (P< 0. 05), the levels of MDA in lung tissue of the neonatal rats were decreased (P<0. 05), the expression levels of Caspase-12, GADD153, GRP78 and Bax proteins were significantly decreased (P<0. 05), the expression levels of Bcl-2 protein and the Bcl-2/Bax ratios were increased (P<0. 05). The pathological sections of lung tissue of the rats in control group showed that lung tissue structure was regular, no alveolar edema was found, the alveolar size and alveolar septum were approximately the same, and no inflammatory cell infiltration was found; the histopathological sections of lung tissue of the rats in hyperoxia group showed swelling of brochial and alveolar epithelial cells, enlargement of alveolar lumen, edema of interstitial cells, inflammatory cell infiltration and fibrous exudation; the degrees of alveolar damage, the inflammatory exudation and the proliferation of fibrons tissue in hyperoxia+GLN group were alleviated which was between hyperoxia group and control group. Conclusion: GLN can alleviate the hyperoxia-induced lung tissue edema and inflammatory response of the neonatal rats, and one of mechanisms is that GLN can down-regulate the expression levels of Caspase-12, GADD153, GRP78 and Bax proteins and up-regulate the expression level of Bcl-2 protein through ERS pathway to protect hypoxic lung injury.

Icariside II improves left ventricular function by improving endoplasmic reticulum stress and caspase 12 signaling in SHR / 中国药理学通报

Aim: To explore the mechanism of icariside II (ICS II) on improving left ventricular function based on endoplasmic reticulum stress and caspase-12 signaling in spontaneously hypertensive rats (SHRs). Methods: Thirty 14-week-old male SHRs were divided into model group, ICS II low, middle and high dose groups and positive drug group (n = 6). WKY was used as control group (n =6). ICS II groups were respectively given ICS 114, 8, 16 mg · kg-1(ig, qd), and positive drug group was given losartan (20 mg · kg-1). At the end of 26th weeks, anesthetized rats were measured by ultrasound for detection of the left ventricular function, RT-PCR was used to determine the level of GRP78 mR- NA in the left ventricle tissue, and Western blot was used to assess the levels of GRP78 and cleaved-caspase- 12/9/3 protein in the left ventricle tissues. Results: Compared with WKY group, the internal diameter and posterior wall thickness of the left ventricular end diastolic increased, while the ejection fraction and fractional shortening decreased in SHR group. GRP78 mRNA and protein levels were up-regulated, and the levels of cleaved-caspase-12/9/3 protein were raised in left ventricle (P < 0.05). Compared with SHR group, the internal diameter and posterior wall thickness of the left ventricular end diastolic increased in ICS II medium and high dose groups and positive drug group (P <0. 05), while the ejection fraction and fractional shortening decreased (P<0.05). GRP78 mRNA and protein levels were down-regulated, the levels of cleaved-caspase-12/ 9/3 protein declined in left ventricle (P <0.05). Conclusions: ICS II could improve left ventricular function in SHRs, and its mechanism may be related to improving left ventricular endoplasmic reticulum stress and down-regulating the elevated caspase-12 signaling.

Advances in the study of relationship between Caspases and innate immunity / 中国药科大学学报

@#Caspases are a group of structurally related cysteine proteases present in cytosol. One of their important common points is that the active sites contain cysteine and can specifically break the peptide bonds after the aspartic acid residues. Caspases are broadly divided into two groups based on their functions, including inflammatory Caspases and apoptotic Caspases. Inflammatory Caspases include Caspase-1, Caspase-4, Caspase-5, Caspase-11 and Caspase-12, which play important roles in the process of innate immune defense. Unlike inflammatory Caspases, apoptotic Caspases(2/3/6/7/8/910)initiate and execute an immunologically silent form of programmed cell death known as apoptosis. However, ongoing investigations have uncovered essential functions of Caspase-8 in the regulation of immunity in cells and organisms. Accumulated studies have shown that Caspases play important roles in the occurrence and development of various immunity-related diseases. In order to comprehensively elucidate the relationship between Caspases and innate immunity, and to provide some scientific basis and theoretical reference for the treatment of various diseases, this article reviews the regulation of activity and inflammation mechanism of innate immunity-related Caspase-1/4/5/11/8/12.

Effect of iridoid glycosides of Scrophulariae Radix on endoplasmic reticulum stress induced by oxygen glucose deprivation and reperfusion in primary cortical neurons / 中草药

Objective: To investigate the effects and mechanisms of iridoid glycosides of Scrophulariae Radix (IGRS) via endoplasmic reticulum stress-mediated apoptosis pathway on the primary cortical neurons induced by oxygen glucose deprivation/reperfusion (OGD/R). Methods: Newborn SD rats were performed primary cortical neurons culture. And the primary cortical neurons were pretreated with IGRS (50, 100, and 200 μg/mL) for 24 h, and the in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was applied. The neurons purity and morphology were observed under inverted microscope, the cell viability was detected by MTT assay; the intracellular lactate dehydrogenase (LDH) level and superoxide dismutase (SOD) activity were detected by commercial kit. The apoptotic rate was detected by flow cytometry. The expression of C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78) and Caspase-12 protein were detected by western blotting. Results: The cultured primary cortical neurons were plump with high purity in good condition. Compared with the control group, the primary cortical neurons were retracted and rounded after OGD/R treatment, and the surface of the neurons became rough; The cell viability and SOD activity were significantly decreased; The LDH level and apoptotic rate were evidently increased; The expression of CHOP, Caspase-12, and GRP78 were significantly increased. Compared with the model group, IGRS could relieve neurons damage, increase cell viability and SOD activity, decrease LDH level and apoptotic rate, and down-regulate the expression of CHOP, Caspase-12, and GRP78. Conclusion: IGRS can antagonize the neuronal damage induced by OGD/R in primary cortical neurons, and its mechanism is related to the inhibition of endoplasmic reticulum stress-mediated apoptosis.

Effects of metformin on the expressions of ATF6 and Caspase12 in hippocampus of type 2 diabetic rats / 中华内分泌代谢杂志

Objective@#To investigate the effect of metformin on the expressions of activating transcription factor-6(ATF6)and Caspase12 in hippocampus of type 2 diabetic rats.@*Methods@#GK male rats with random blood glucose≥11.1 mmol/L were orally administrated with normal saline(DM group)and metformin(MET group, 85 mg·kg-1·d-1)for 8 weeks(n=10 each group). Wistar male rats were selected as normal contorl group(NC, n=10). After 8 weeks of continuous medication, body weight and fasting plasma glucose(FPG)were measured, and the morphology of HE stained cells and the expressions of ATF6 and Caspase12 by immunohistochemistry staining in the CA1 area of hippocampus were detected.@*Results@#Compared with NC group, the body weight of DM and MET groups decreased(P<0.05), but there was no significant difference between DM and MET groups(P>0.05). In comparison with NC group, FPG levels in DM and MET groups were markedly increased(P<0.05)while FPG level in MET group was significantly lower than that in DM group(P<0.05). The normal nerve cells in the CA1 region of hippocampal were lower in DM and MET groups than those in NC group, especially in DM group. The protein expressions of ATF6 and Caspase12 in DM and MET groups were higher than that in NC group(all P<0.05), and the expressions of the two protein in MET group were significantly decreased as compared with DM group(P<0.05).@*Conclusion@#Metformin reduces the expressions of ATF6 and Caspase12 in hippocampus of type 2 diabetic rats, which may be related to its protective effect on brain. (Chin J Endocrinol Metab, 2018, 34: 594-597)

The expression and role of Caspase3, Caspase12 and CHOP in pituitary tissues of rats with acute necrotizing pancreatitis / 中华内分泌外科杂志

Objective To observe the pathological changes of pituitary tissue in rats with acute necrotizing pancreatitis and to explore the mechanism of pituitary tissue injury in rats.Methods 24 SD rats were randomly divided into normal group (N group,n=8),sham operation group (SO group,n=8),and acute necrotizing pancreatitis group (ANP group,n=8).ANP model was established by retrograde injection of 5％ sodium taurocholate into the biliopancretic duct.The serum levels of amylase(AMY) and lipase (LIP) were detected by automatic biochemical.The serum levels of growth hormone (GH),adrenocorticotropic hormone (ACTH),thyroid stimulating hormone (TSH) and follicle-stimulating hormone (FSH) were measured by radioimmunoassay.The pathological changes of pancreatic tissue and pituitary tissue were observed by the light microscope.The expression of Casepase3,Caspasel2 and CHOP in pituitary tissue were determined by immunohistochemical method.Results Compared to SO group,the serum levels of AMY(8679.16±307.60) U/L and LIP(9376.83±380.92) U/L were significantly higher in ANP group (P＜0.05).The serum levels of ACTH (0.92±0.41) pg/ml,TSH (0.14±0.06) pg/ml,and FSH (2.01±0.38) pg/ml were significantly lower in ANP group(P＜0.05).The expression of Caspse 3 (65.66±7.58),Caspase12(70.66±4.76) and CHOP(143.16±19.05) in pituitary tissue were significantly increased in ANP group (P＜0.05).The pancreatic injury was more severe in ANP group under light microscope (P＜0.05).The degree of hyperemia of pituitary tissue of ANP group was aggravated.Conclusion Pathological changes occur in rat pituitary tissues and endoplasmic reticulum stress injury plays a role in pituitary injury during ANP.

Anti-lung cancer mechanisms of diterpenoid tanshinone via endoplasmic reticulum stress-mediated apoptosis signal pathway / 中国中药杂志

At present, lung cancer ranks second and first respectively in the incidence and the mortality among malignant tumors. It is urgent to find new effective anti-lung cancer drugs with less side effects and relatively defined mechanisms. Endoplasmic reticulum stress (ERS)-mediated apoptosis pathway is an effective way to promote tumor cell apoptosis; diterpenoid tanshinone (DT), an effective part separated from Salviae Miltiorrhizae Radix et Rhizoma, was found to have an anti-lung cancer effect in previous studies via ERS-induced PERK-EIF2α pathway. In this paper, human lung adenocarcinoma PC9 cell line and nude mouse transplantation tumor model were applied to verify the anti-lung cancer effect of DT in vivo and in vitro, and illuminate the potential mechanism via ERS induced IRE1α/caspase 12 apoptosis pathway. The results showed that in vivo, DT could promote PC9 cell apoptosis in a concentration-dependent manner, up-regulate Bip, IRE1 and TRAF2 protein expressions in tumor tissue, reduce tumor weight and alleviate bodyweight loss. In vitro, DT inhibited the proliferation of PC9 cell line in a concentration-dependent manner, and destroyed the structure of mitochondria in PC9 cell, promoted Bax, IRE1α, Bip, TRAF2 and caspase 12 protein expressions, lower Bcl-2 protein expression in a time-dependent manner. DT shows a good effect on anti-lung cancer both in vivo and in vitro. The mechanism is related to the activation of ERS-induced IRE1α/caspase 12 apoptosis pathway and the promotion of cell apoptosis. ERS-mediated apoptosis pathway may be an important target of DT on anti-lung cancer.

Allicin attenuates macrophage-derived foam cell apoptosis by inhibiting caspase-12 / 中国病理生理杂志

AIM:To investigate the inhibitory effect of allicin on apoptosis and caspase-12 activation of macro-phage-derived foam cells,and to elucidate the underlying molecular mechanisms. METHODS:RAW264.7 macrophages were pretreated with allicin (12.5,25 and 50 mg/L) or 4-phenylbutyric acid(PBA,4 mmol/L) for 1 h and then treated with oxidized low-density lipoprotein(ox-LDL,100 mg/L) or tunicamycin(TM,4 mg/L) for 24 h. The cell viability and apoptosis were examined by MTT assay and flow cytometry with Annexin V-FITC/PI staining,respectively. The activities of caspase-3 in the cells and lactic dehydrogenase (LDH) in the medium were measured. The protein levels of caspase-12 were determined by Western blot. The intracellular lipid accumulation was measured with oil red O staining and the content of intracellular total cholesterol was determined by enzymatic colorimetry. RESULTS:Similar to the endoplasmic reticulum stress (ERS) inhibitor PBA, allicin inhibited ox-LDL-induced injury of RAW264.7 macrophages in a concentration-de-pendent manner,as determined by the increased cell viability and the decreased LDH leakage,apoptosis and caspase-3 ac-tivity. The decrease in cell viability and increases in LDH leakage and apoptosis induced by TM (an ERS inducer) were also suppressed by allicin. Moreover, similar to PBA, allicin remarkably inhibited ox-LDL- or TM-induced activation of caspase-12. Furthermore, allicin remarkably attenuated ox-LDL-induced lipid accumulation in the RAW264.7 cells and foam cells formation in a concentration-dependent manner. CONCLUSION:Allicin may inhibit macrophage-derived foam cell apoptosis induced by ox-LDL,and the mechanism is partially related to suppressing the activation of caspase-12.

Inhibitory effects of Huoxue Jiedu decoction on cellapoptosis via down-regulating 8-OhdG in obstructive nephropathy rats / 中国药理学通报

Aim To observe the effect of eplerenone(EPL) and Chinese decoction on cell apoptosis in obstructive nephropathy rats.Methods Sixty male Wistar rats were randomly divided into sham group, UUO group, EPL group and ZY group(n=15).Except sham group, the rats in the other groups were ligated with unilateral ureteral obstruction(UUO) for renal interstitial fibrosis model.The rats were treated with eplerenone at 100 mg·kg-1·d-1 added to diet in EPL group, and orally 13.7 g·kg-1·d-1 decoction of Chinese medicine in ZY group.The kidneys were harvested on 14th day, the number of renal cell apoptosis were detected by TUNEL, and serum aldosterone and 8-OhdG were detected with radioimmunoassay and ELISA.Caspase-12, caspase-9, Bax and Bcl-2 were examined by immunohistochemistry and Western blot.Results The levels of serum aldosterone, serum and urine 8-OhdG and the number of positive apoptotic cells increased significantly in UUO rats compared with Sham group.The overexpression of caspase-9, caspase-12 and Bax and down-regulated Bcl-2 were obvious in UUO group(P<0.01).The level of 8-OhdG, expression of caspase-9, caspase-12 and Bax were down-regulated, and Bcl-2 expression was up-regulated in eplerenone and Chinese decoction treated rats(P<0.01).Conclusion Eplerenone and Chinese decoction could inhibit cell apoptosis induced by oxidative damage after UUO via caspases and(or) Bax pathway.

Caspase-12 expression in hyperoxia-induced corpus callosum damage in newborn mice / 中国新生儿科杂志

Objective To study the relationship between Caspase-12 expression and the hyperoxia-induced corpus callosum damage. Methods A total of 12 groups of C57 / BL6 mice were randomly assigned into hyperoxia group (80% O2 ) and control group (21% O2 ) at day 6 after birth (P6). The pups were sacrificed after 24 h and 48 h of hyperoxia exposure and at P10, P12, P15 and P30. Immunohistochemical ( IHC) method was used to detect the expression of myelin basic protein (MBP) in corpus callosum. Real-time PCR, Western Blot and IHC were used to detect the expression of mRNA and protein of Caspase-12 in corpus callosum. The corpus callosum apoptosis was measured using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling ( TUNEL ) method. Results The expression of MBP in hyperoxia group were significantly lower than the control group at P10 and P12 (P = 0. 004 and 0. 016); however, no significant differences existed between the two groups at P15 and P30 (P > 0. 05). The expression of Caspase-12 mRNA after 24 h and 48 h hyperoxia exposure were significantly higher than the control group [24 h: (1. 549 ± 0. 098) vs. (1. 080 ± 0. 101); 48 h:(1. 333 ± 0. 076) vs. (1. 022 ± 0. 089); P < 0. 05]. The expression of cleaved Caspase-12 protein after 24 h and 48 h hyperoxia exposure were also significantly higher than the control group [24 h: (1. 582 ± 0. 010) vs. (0. 994 ± 0. 078); 48 h: (1. 370 ± 0. 095) vs. (0. 978 ± 0. 069); P < 0. 05] . The Caspase-12 positive cell were significantly increased after 24 h and 48 h hyperoxia exposure comparing with the control group. The apoptosis index in hyperoxia group was significantly higher than the control group at P10 and P12 [P10: (18. 742 ± 2. 503) vs. (4. 587 ± 2. 353); P12 (36. 184 ± 3. 655) vs. (5. 351 ± 2. 678); P < 0. 05]. Conclusions Hyperoxia exposure induces corpus callosum damage in newborn mice. Over-expressed Caspase-12 may induce corpus callosum cell apoptosis excessively.

Myocardial expression of Caspase-12 and GRP78 in cardiac arrest and beating heart mitral valve replacement / 实用医学杂志

Objective To observe the expression of Caspase-12 and GRP78 of endoplasmic reticulum stress (ERS) in cardiac arrest and beating heart mitral valve replacement Methods Thirty patients with rheumatic heart disease mitral stenosis were randomly divided into beating heart group (BH,n=15) and cardiac arrest group(CA, n = 15). Both groups accepted MVR by beating heart surgery and cardiac arrest surgery under cardiopulmonary bypass (CPB) respectively. Right atrial myocardial tissues were collected at prior the start of CPB (T0), after aortic cross-clamping 30 minutes (BH group 30 minutes after CPB, T1) and stitched right atrium (T2) respectively. The method of reverse transcriptase polymerase chain reaction (RT-PCR) was applied to detect the expression level of Caspase-12 and GRP78 in two groups and positive staining of Caspase-12 and GRP78 of myocardial tissue slices in both groups was observed by immunohistochemical method. Results The expression of Caspase-12 in CA group heightened at T1and significantly increased at T2 (P < 0.05) but the expression of Caspase-12 in BH group had increased in T2 only (P < 0.05). Caspase-12 in CA group expressed higher than that in BH group at T1 and T2. The expression of GRP78 had increased at T1 in two groups but it in CA group expressed higher than that inBH group at T2. The number of positive staining of Caspase-12 and GRP78 in CA group was higher than that in BH group at T2. Conclusion MVR of beating heart can reduce the reaction of ERS to enhance the myocardial protection under CPB.

The protective effect of hydroxytyrosol on contrast-induced nephropathy and endoplasmic reticulum stress / 中华肾脏病杂志

Objective To investigate the expression of glucose-regulated protein 78(GRP78)and cysteine aspartic acid protease 12(Caspase-12) and evaluate the endoplasmic reticulum stress (ERS) in rats with contrast-induced nephropathy (CIN),and observe the protective effects of hydroxytyrosol on CIN rats.Methods Eighty-four Wistar rats,(220±20) g,were randomly divided into control group,CIN group,hydroxytyrosol treated group (group C+H).At 12th,24th,48th,72th day after the rats model were established,BUN and Scr were detected.ELISA were used to detect the expression of methane dicarboxylic aldehyde (MDA),superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).HE staining were used to evaluate the pathological change of kidney.TUNEL were used to detect the apoptosis of tubular ceils.Real-time PCR were used to detect the expression of GRP78 mRNA and Caspase-12 mRNA in tubular cells.Immunohistochemistry and Western blotting were used to detect the expression of GRP78 and Caspase-12 protein in tubular cells.Results BUN,Scr,the mRNA and protein expression of GRP78,Caspase-12 in hydroxytyrosol treated group were higher than that in control group(P ＜ 0.05),but were significantly lower than that in CIN group (P ＜ 0.05).Pathological changes and the apoptosis of tubular cells in CIN group were more serious than that in hydroxytyrosol treated group (P ＜ 0.05).Conclusions Endoplasmic reticulum stress may be associated with contrast-induced nephropathy.Hydroxytyrosol can protect kidney from contrast medium via reducing the endoplasmic reticulum stress.

Apolipoprotein A-I mimetic peptide D4F protects macrophages from oxi-dized low-density lipoprotein-induced apoptosis by inhibiting caspase-12 / 中国病理生理杂志

[ABSTRACT]AIM:ToinvestigatetheeffectofD4F,anapolipoproteinA-Imimeticpeptide,onoxidizedlow-density lipoprotein ( ox-LDL)-induced macrophage apoptosis and activation of caspase-12, a key molecule in endoplasmic reticulum stress ( ERS )-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms. METHODS:RAW264.7 macrophages were pretreated with D4F (12.5, 25 and 50 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium ( DPI, 5 μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin ( TM, 4 mg/L) for 24 h.The cell viability and apoptosis were determined by MTT assay and TUNEL detection, respective-ly.The levels of malondialdehyde ( MDA) and reactive oxygen species ( ROS) in the cells and the activities of superoxide dismutase ( SOD) and nicotinamide adenine dinucleotide phosphate ( NADPH) oxidase were determined.The protein level of caspase-12 was examined by Western blot analysis.RESULTS: Similar to the ERS inhibitor PBA, D4F protected RAW264.7 macrophages from ox-LDL or TM ( an ERS inducer)-induced decrease in the viability and increase in apoptotic rate in a dose-dependent manner.Like DPI (an oxidative stress inhibitor), D4F significantly inhibited ox-LDL-induced ox-idative stress, as expressed by the decreased generation of ROS and MDA ( P<0.01) , the increased activity of SOD and the decreased activity of NADPH oxidase (P<0.05).Moreover, similar to PBA and DPI, D4F significantly suppressed ox-LDL-induced activation of caspase-12 in a concentration-dependent manner ( P<0.05) .Furthermore, D4F also inhibi-ted the caspase-12 activation induced by TM (P<0.05).CONCLUSION: D4F inhibits macrophage apoptosis induced by ox-LDL, and the mechanism is at least partially by reducing oxidative stress and inhibiting the activation of caspase-12.

Ethanol extract of propolis protects macrophages from oxidized low-den-sity lipoprotein-induced apoptosis by inhibiting caspase-12 / 中国病理生理杂志

AIM:To investigate the inhibitory effect of ethanol extract of propolis (EEP) on oxidized low-den-sity lipoprotein ( ox-LDL )-induced macrophage apoptosis and the underlying molecular mechanisms . METHODS:RAW264.7 macrophages were pretreated with EEP (7.5, 15 and 30 mg/L), 4-phenylbutyric acid (PBA, 5 mmol/L) or diphenyleneiodonium ( DPI, 5μmol/L) for 1 h and then treated with ox-LDL (100 mg/L) or tunicamycin ( TM, 4 mg/L) for 24 h.The cell viability and apoptosis were determined by MTT assay and Annexin V-FITC apoptosis detection kit , re-spectively.The activity of superoxide dismutase (SOD), and the levels of reactive oxygen species (ROS) and malondial-dehyde (MDA) in the cells were measured.The protein levels of caspase-12, a proapoptotic molecule under endoplasmic reticulum stress ( ERS) , were examined by Western blot analysis .RESULTS:Like PBA ( an ERS inhibitor ) , EEP pro-tected RAW264.7 macrophages from ox-LDL-induced injury in a dose-dependent manner , as assessed by the increased cell viability and the decreased apoptotic rate .The decrease in cell viability and increase in apoptotic rate induced by TM , an ERS inducer, were also attenuated by EEP .Moreover, EEP suppressed ox-LDL-induced oxidative stress as revealed by the decreased generation of ROS and MDA as well as elevated SOD activity , which were similar to DPI , an oxidative stress in-hibitor.Furthermore, EEP significantly suppressed ox-LDL-or TM-induced activation of caspase-12.Similar results were observed in the cells pretreated with PBA or DPI and then treated with ox-LDL.CONCLUSION: EEP may protect RAW264.7 macrophages from ox-LDL-induced apoptosis and the mechanism is at least partially involved in the ability of EEP to suppress oxidative stress and subsequent activation of caspase -12.

Diterpene ginkgolides meglumine injection attenuates oxygen-glucose deprivation-induced apoptosis of nerve cells via inhibition of calpain signaling pathway / 中国药科大学学报

@#To investigate the anti-apoptotic effect of diterpene ginkgolides meglumine injection(DGMI)on SH-SY5Y cells induced by oxygen-glucose deprivation/reoxygenation(OGD/R), and to explore its mechanisms. After 4 h of OGD, the SH-SY5Y cells were treated with 25 mg/L DGMI for 1 h. The release of lactic dehydrogenase(LDH)was measured by cytotoxicity detection kitplus. Cell apoptosis was detected by caspase-3/7 assays. Cell death was detected by ELISA. The concentration of ［Ca2+］i in cytoplasm was measured by Fluo-3 AM and the levels of calpain and cleaved capaease-12 were evaluated by western blot. As we expected, DGMI significantly decreased the release of LDH, the concentration of ［Ca2+］i, the protein levels of calpain and cleaved caspase-12. Furthermore, DGMI injection also attenuated the activities of caspase-3/7 and the contents of cytoplasmic histone-associated- DNA-fragments. These data demonstrated that the DGMI injection showed good anti-apoptotic effect in SH-SY5Y cells induced by OGD/R. The mechanisms may be associated with the inhibition of Ca2+/calpain/caspase-12/caspase-3 signaling pathway.

Effect of dexmedetomidine pretreatment on expression of caspase-12 in lung tissues undergoing one-lung ventilation in rats / 中华麻醉学杂志

Objective To investigate the effect of dexmedetomidine pretreatment on the expression of caspase-12 in lung tissues undergoing one-lung ventilation (OLV) in rats.Methods Thirty male Sprague-Dawley rats, aged 6-8 weeks, weighing 180-220 g, were randomly allocated into 3 groups (n=10 each) using a random number table: two-lung ventilation (TLV) group, OLV group and dexmedetomidine group (Dex group).Bilateral lungs were ventilated for 2 h in group TLV.In OLV and Dex groups, unilateral lung was ventilated for 1.5 h followed by 0.5 h TLV.In group Dex, dexmedetomidine was infused intravenously at a rate of 3.0 μ g · kg-1 · h-1 over 60 min starting from 60 min prior to OLV.The equal volume of normal saline was given instead of dexmedetomidine in OLV and TLV groups.Peak airway pressure (Ppeak) and mean airway pressure (Paw) were recorded at 45 min of OLV and 15 min of TLV in OLV and Dex groups, and at 15 min of TLV in group TLV.The rats were then sacrificed, and left lungs were removed for microscopic examination of the pathologic changes (using HE staining) and the ultrastructure of lung tissues (with transmission electron microscope) and for determination of wet to dry lung weight ratio (W/D ratio), cell apoptosis in lung tissues (by TUNEL), caspase-12 mRNA expression (using real-time reverse transcriptase-polymerase chain reaction), and caspase-12 expression (by Western blot).Results Ppeak and Paw were significantly lower at 15 min of TLV than at 45 min of OLV in OLV and Dex groups (P＜0.05).Compared to group TLV, W/D ratio and AI were significantly increased, and the expression of caspase-12 protein and mRNA was up-regulated in OLV and Dex groups (P＜0.01).Compared to group OLV, W/D ratio and AI were significantly decreased, and the expression of caspase-12 protein and mRNA was down-regulated in group Dex (P ＜ 0.01).The pathologic changes of lung tissues were significantly alleviated in group Dex as compared with group OLV.Conclusion The mechanism by which dexmedetomidine pretreatment alleviates acute lung injury caused by OLV is associated with down-regulated expression of caspase-12 and inhibited cell apoptosis in rats.

Change of endoplasmic reticulum stress-related molecules in the course of sciatic nerve injury of diabetic rats / 中国医师杂志

Objective To investigate the dynamic changes of endoplasmic reticulum stress-related molecules including glucose regulated protein (GRP78), C/EBP homologous protein (CHOP), and caspase-12 in sciatic nerve of diabetic rats and explore its mechanisms.Methods Rats were randomly divided into normal control group (NC) and diabetes mellitus group (DM) that were induced by intraperitoneal injection of Streptozocin after 4 weeks of high-fat chow feeding.Sciatic nerves were isolated for three times at 4 weeks, 8 weeks and 12 weeks after induction of diabetes.The expressions of GRP78, CHOP,and caspase-12 were detected with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses.The morphology of sciatic nerve was investigated with electron microscope.Results With the extension of the course, demyelinating and axonal injury appeared in sciatic nerve of diabetic rats.The expressions of GRP78 mRNA and protein in DM group were significantly higher than NC group at 4 weeks and 8 weeks after induction of diabetes(P ＜0.05, P ＜0.01).The expressions of CHOP mRNA and protein in DM group were significantly higher than NC group at 8 weeks and 12 weeks after induction of diabetes (P ＜ 0.05).The expressions of caspase-12 mRNA and protein in DM group were significantly higher than NC group at 8 weeks after induction of diabetes(P ＜ 0.05, P ＜ 0.01).Conclusions Endoplasmic reticulum stress-related molecules (GRP78, CHOP, and caspase-12) contributed to the peripheral nerve injury of diabetic rats, and displayed dynamic changes.

Effect of fluvastatin on the expressions of caspase-12, CHOP and JNK in ischemia-reperfusion brain injury in rats / 中国医师杂志

Objective To investigate the effect of fluvastatin on the expressions of caspase-12,CCAAT/enhancer-binding protein homologous protein(CHOP), and c-Jun N-terminal kinases (JNK) in ischemia-reperfusion brain injury in rats.Methods Forty two rats were randomly divided into sham operation group (6 rats), ischemia-reperfusion (I/R) group (18 rats), and fluvastatin (Flu) group (18 rats).The rats of I/R and Flu groups were molded by modified Longa intraluminal thread, then put to death at 2 h occlusion and 24 h reperfusion point.Expressions of caspase-12, CHOP, and JNK were detected with immunohistochemistry and Western blot.Results Immunohistochemistry and Western blot showed that the expressions of caspase-12, CHOP, and JNK were increased at 24 h reperfusion.Compared to I/R group, the expressions of caspase-12 and CHOP in Flu group were decreased significantly (all P ＜0.01);and the expression of JNK had no difference between I/R and Flu groups(P ＞ 0.05).Conclusions The increased expression of caspase-12, CHOP, and JNK showed that endoplasmic reticulum stress was involved in the pathological process of ischemia-reperfusion brain injury.Fluvastatin could inhibit the expression of caspase12 and CHOP, and could delete endoplasmic reticulum stress (ERS) in ischemia-reperfusion brain injury.