Cornel iridoid glycoside alleviates microglia-mediated inflammatory response via NLRP3/calpain pathway / 中国药理学与毒理学杂志

OBJECTIVE Vascular dementia(VaD)is associated with cerebral hypoperfusion,which results in long-term cognitive impairment and memory loss.Neuroin-flammation is an important mechanism of vascular demen-tia.Cornel iridoid glycoside(CIG)is the major active con-stituent isolated from the ripe fruit of Cornus officinalis.Previous studies have shown that CIG enhances neuro-logical function in VaD rats.In the present research,we attempted to clarify the molecular processes underlying the role of CIG on neuroinflammation in VaD.METHODS In vivo,we created a chronic cerebral ischemia rat model by ligation of the bilateral common carotid arteries(2VO).The rats were divided into sham operation,2VO,2VO + CIG(60 and120 mg·kg-1·d-1),and 2VO+ butylphthalide(100 mg·kg-1·d-1)groups and then treated rats with differ-ent concentrations of CIG.In vitro,BV2 microglia cells were induced with bacterial lipopolysaccharide(LPS)and interferon-γ(IFN-γ)to construct the model of microglias with analog neuroinflammation.Histopathology and biel-schowsky silver staining were used to detect myelin integrity and neuronal loss.Immunofluorescence was used to observe changes in microglia.Magnetic Luminex Assay was used to detect changes in inflammatory fac-tors.Western blotting,ELISA or calpain activity assay was used to measure the expression and activity of cal-pain,as well as the expression of NLRP3 inflammasome protein.Furthermore,NLRP3 overexpressing cells were used to further elucidate the potential anti-inflammatory molecular mechanism of CIG.RESULTS ① CIG improved neuronal impairment in the brain of 2VO rats.②CIG increased white matter(WM)integrity in 2VO rats.③ CIG reduced microglia inflammatory response in the cortex and hippocampus of 2VO rats.④ CIG inhibited calpain activity in the cortex and hippocampus of 2VO rats.⑤ CIG exerted anti-inflammatory effects on BV2 cells stimulated by LPS and IFN-γ.⑥ CIG Inhibited the expression and activity of calpain in LPS/IFN-γ-activated BV2 cells.⑦ The main component of CIG had a weak binding force to calpain1.⑧ CIG inhibited the activation of the NLRP3 inflammasome.⑨CIG reduced the activity of calpain induced by NLRP3 overexpression.CONCLU-SION CIG inhibits microglial polarization into a proinflam-matory state by attenuating the assembly of the NLRP3 inflammasome and calpain activation,thus reducing brain inflammation,WM injury,and the loss of neurons.To sum up,the present study suggests that CIG inhibits neuroinflammation.The NLRP3/calpain pathway may be the main pathway by which CIG protects against neuroin-flammation.

Effect of extracellular signal-regulated kinase inhibitor on calpain in rat cerebral cortex after cardiopulmonary resuscitation / 中华危重病急救医学

Objective:To explore the effect of extracellular signal-regulated kinase (ERK) inhibitor PD98059 on calpain-related proteins in the brain, and to understand the pathophysiological changes of calpain in cerebral ischemia/reperfusion injury (CIRI).Methods:Forty-two rats were divided into sham operation (Sham) group ( n = 6), model group ( n = 12), dimethyl sulfoxide (DMSO) control group ( n = 12), and PD98059 group ( n = 12) by random number table. The rat model of CIRI induced by cardiac arrest-cardiopulmonary resuscitation (CA-CPR) was reproduced by transesophageal electrical stimulation to induce ventricular fibrillation. In the Sham group, only the basic operations such as anesthesia, tracheal intubation, and arteriovenous catheterization were performed without CA-CPR. The rats in the DMSO control group and PD98059 group were injected with DMSO or PD98059 0.30 mg/kg via femoral vein, respectively, 30 minutes after the restoration of spontaneous circulation (ROSC), and rats in the Sham group and model group were given the same amount of normal saline. The duration of CPR, 24-hour survival rate and neurological deficit score (NDS) after ROSC were recorded. Hematoxylin-eosin (HE) staining and Nissl staining were used to observe the pathological changes of the cerebral cortex. The expressions of phosphorylated ERK (p-ERK), ERK, calpastatin, calpain-1, and calpain-2 were detected by Western blotting. The co-expression of p-ERK and calpain-2 was detected by double immunofluorescence. Results:There were no significant differences in the duration of CPR and 24-hour survival rate among all groups. In the model group, the nuclei of the cerebral cortex were obviously deformed and pyknotic, cells vacuoles and tissues were arranged disorderly, Nissl corpuscles were significantly reduced, NDS scores were also significantly reduced, level of ERK phosphorylation was increased, and calpain-2 protein was significantly up-regulated compared with the Sham group. There was no significant difference in the above parameters between the DMSO control group and the model group. After intervention with PD98059, the pathological injury of brain tissue was significantly improved, Nissl corpuscles were significantly increased, the NDS score was significantly higher than that in the model group [75.0 (72.0, 78.0) vs. 70.0 (65.0, 72.0), P < 0.05], the level of ERK phosphorylation and calpain-2 protein expression were significantly lower than those in the model group [p-ERK (p-ERK/ERK): 0.65±0.12 vs. 0.92±0.05, calpain-2 protein (calpain-2/GAPDH): 0.73±0.10 vs. 1.07±0.14, both P < 0.05], while there was no significant difference in the expressions of calpastatin and calpain-1 in the cerebral cortex among all the groups. Double immunofluorescence staining showed that p-ERK and calpain-2 were co-expressed in cytosol and nucleus, and the co-expression rate of p-ERK and calpain-2 in the model group was significantly higher than that in the Sham group [(38.6±4.3)% vs. (9.2±3.5)%, P < 0.05], while it was significantly lowered in the PD98059 group compared with the model group [(18.2±7.0)% vs. (38.6±4.3)%, P < 0.05]. Conclusions:ERK together with calpain-2 participated in CIRI induced by CA-CPR. PD98059 inhibited the expression of calpain-2 and ERK phosphorylation. Therefore, ERK/calpain-2 may be a novel therapeutic target for CIRI.

Mechanism of Xinfeng Capsules improving rheumatoid arthritis based on CD19~+B cells regulating FAK/CAPN/PI3K pathway / 中国中药杂志

To observe the effect of Xinfeng Capsules on rheumatoid arthritis (RA) B lymphocytes,inflammatory mediators,FAK/CAPN/PI3K pathway,in order to explore the mechanism of Xinfeng Capsules in improving clinical symptoms of RA.Joint and systemic symptoms of RA patients were observed,and laboratory indicators[hemoglobin (HGB),platelet count (PLT),erythrocyte sedimentation (ESR),immunoglobulin (Ig) G,Ig A,Ig M,rheumatoid factor (RF),anti-cyclic citrulline antibody (CCP-AB),C-reactive protein (CRP)]were detected.ELISA was used to detect serum interleukin (IL)-1β，IL-10,IL-33,chemokine 5 (CCL5),and vascular endothelial growth factor (VEGF).CD3~-CD19~+B cells were measured by flow cytometry.Western blot was used to detect FAK,p-FAK,CAPN,PI3K protein.The results showed that Xinfeng Capsules could significantly alleviate RA joint and systemic symptoms and improve clinical efficacy.And Xinfeng Capsules could increase HGB,decrease PLT,CCP-AB,CRP,ESR index,upregulate IL-10 expression,and down-regulate IL-1β，IL-33,CCL5,VEGF,CD3~-CD19~+B cells,FAK,p-FAK,CAPN,PI3K expressions (P<0.01).Based on the above results,Xinfeng Capsules may reduce the expression of CD3~-CD19~+,regulate the balance of inflammatory cytokines and chemokines,inhibit abnormal activation of FAK/CAPN/PI3K pathway,and improve clinical symptoms of RA.

Advance in Mechanisms of Calpain Involved in Hyperphosphorylation and Truncation of Tau Protein (review) / 中国康复理论与实践

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, which is characterized by cognitive and memory dysfunction. Calpain is widely activated in cells. Disturbance of calpain is currently thought to a main cause of hyperphosphorylation and abnormal cleavage of tau protein in AD pathology. Calpain affects the activities of glycogen synthase kinase 3 and protein phosphatase 2A, which causes abnormal hyperphosphorylation of multiple sites of tau protein, and mediates truncation of tau protein monomers, and induces neurodegeneration. Calpain is expected to be a potential target for drug therapy of AD.

Calpain-2 as a Treatment Target in Prenatal Stress-induced Epileptic Spasms in Infant Rats

Stress can induce a serious epileptic encephalopathy that occurs during early infancy. Recent studies have revealed that prenatal stress exposure is a risk factor for the development of infantile spasms. Our previous work demonstrates that prenatal stress with betamethasone-induced alterations to the expression of the K⁺/Cl⁻ co-transporter (KCC2) in gamma-aminobutyric acid (GABA) interneurons lowers the seizure threshold in exposed animals. Here, we further investigated the mechanisms involved in this KCC2 dysfunction and explored possible treatment options. We stressed Sprague-Dawley rats prenatally and further treated dams with betamethasone on gestational day 15, which increases seizure susceptibility and NMDA (N-Methyl-D-aspartate)-triggered spasms on postnatal day 15. In this animal model, first, we evaluated baseline calpain activity. Second, we examined the cleavage and dephosphorylation of KCC2. Finally, we checked the effect of a calpain inhibitor on seizure occurrence. The phosphorylated-N-methyl-D-aspartate Receptor 2B (NR2B):non-phosphorylated NR2B ratio was found to be higher in the cortex of the prenatally stressed beta-methasone model. We further found that the betamethasone model exhibited increased phosphorylation of calpain-2 and decreased phosphorylation of KCC2 and Glutamic acid decarboxylase 67 (GAD67). After using a calpain inhibitor in prenatal-stress rats, the seizure frequency decreased, while latency increased. GABAergic depolarization was further normalized in prenatal-stress rats treated with the calpain inhibitor. Our study suggests that calpain-dependent cleavage and dephosphorylation of KCC2 decreased the seizure threshold of rats under prenatal stress. Calpain-2 functions might, thus, be targeted in the future for the development of treatments for epileptic spasms.

Hypoxia promotes migration and invasion of pancreatic cancer cells via inducing calpain 2 expression / 肿瘤

Objective: To investigate the expressions of Calpain 1 and Calpain 2 proteins, as well as the effects of regulating Calpain 2 expression on the migration and invasion of pancreatic cancer PANC-1 cells in hypoxia microenvironment. Methods: The expressions of Calpain 1 and Calpain 2 mRNAs in pancreatic cancer tissues and their survival prognosis value were analyzed using GEPIA (Gene Expression Profiling Interactive Analysis) database online tool. The pancreatic cancer PANC-1 cells were cultured in normoxia (21% O2) and hypoxic (1% O2) environment, then the expressions of Calpain 1 and Calpain 2 mRNAs and proteins were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The siRNA targeting CAPN2gene(encoding Calpain 2) was transfected into pancreatic cancer PANC-1 cells, then the silencing effect of CAPN2 gene was detected by real-time fluorescent quantitative PCR and Western blotting. The PANC-1 cells with Calpain 2 low expression were cultured under the normoxia and hypoxic conditions, then the migration and invasion abilities of these cells were detected by wound healing test and Transwell chamber assay, the expressions of E-cadherin and Vimentin were detected by Western blotting, and the cell morphology was observed by inverted microscopy. Results: The expressions of Calpain 1 and Calpain 2 mRNAs in human pancreatic cancer tissues were higher than those in normal pancreatic tissues (both P < 0.01), and the high expression of Calpain 2 was associated with the poor prognosis of pancreatic cancer patients (P = 0.013). Compared with the normoxia, the expression levels of Calpain 2 mRNA and protein were increased in hypoxia microenvironment (both P< 0.01), but the expressions of Calpain 1 mRNA and protein were not changed. The Calpain 2 low-expressed PANC-1 cells were established successfully. Under normoxia condition, the down-regulation of Calpain 2 expression inhibited the migration and invasion of PANC-1 cells (both P < 0.05); hypoxia promoted the migration and invasion of PANC-1 cells (both P < 0.05); but the down-regulation of Calpain 2 expression could reverse the promotion effects of hypoxia on the migration and invasion of PANC-1 cells (both P < 0.05). Furthermore, the expression of E-cadherin increased, the expression of Vimentin decreased in PANC-1 cells after the down-regualtion of Calpain 2 expression under normoxia condition (both P < 0.05), while the size of irregular cells increased, and the part of cells had apoptotic change; but the expression of E-cadherin decreased, the expression of Vimentin increased in PANC-1 cells cultured under hypoxia condition (both P < 0.05), while the most of cells had mesenchymal-like changes, showing long shuttle type; but the down-regulation of Calpain 2 expression could reverse the effect of hypoxia on the epithelial-mesenchymal transition of PANC-1 cells (both P< 0.05). Conclusion: Stimulated by hypoxia microenvironment, the expression of Calpain 2 was increased, so as to promote the migration and invasion of PANC-1 cells via regulating E-cadherin and Vimentin expressions.

Expression of calpain small subunit 1 in breast cancer and its clinical significance / 肿瘤研究与临床

Objective To investigate the expression of calpain small subunit 1 (Capn4) in breast cancer and its clinical significance. Methods The cancer tissues and the paraffin-embeded specimens of the corresponding adjacent normal tissues from 12 patients with breast cancer between May 2015 and July 2015 in Shanghai General Hospital were collected. The expression of Capn4 was detected by using immunohistochemistry. The paraffin-embeded specimens of cancer tissues from 70 breast cancer patients in Shanghai General Hospital between January 2009 and September 2011 were also collected. And the expression of Capn4 was detected. The correlation between Capn4 expression level and TNM stage, pathological grade, molecular markers as well as the prognosis of breast cancer was also analyzed. Results The expression of Capn4 in breast cancer tissues was higher than that in the corresponding adjacent normal tissues. The expression level of Capn4 was correlated with TNM stage (P = 0.002), T stage (P = 0.004), N stage (P = 0.004), M stage (P = 0.025), estrogen receptor (ER) status (χ 2 = 4.787, P = 0.029) and survival status (χ 2 = 5.826, P = 0.016). Kaplan-Meier survival analysis revealed that the median survival time in the higher expression of Capn4 group 90 months was shorter than that in the lower expression of Capn4 group (not reaching the median survival time), and there were statistical differences (χ 2 = 4.351, P = 0.037). Conclusions The expression of Capn4 in breast cancer tissues is upregulated. The expression difference is correlated with TNM stage, pathological grade, ER status and the prognosis of breast cancer, suggesting that it may become a new target and molecular marker for breast cancer treatment.

Limb-girdle muscular dystrophy / 中华神经科杂志

Limb girdle muscular dystrophy (LGMD) is characterized by progressive proximal muscle weakness with high genetic heterogeneity.LGMD is the fourth prevalent form of muscular dystrophies in the adult neurology department.Since most patients are juvenile-or adult-onset and present as limb muscle weakness,it would be easily misdiagnosed as myositis or metabolic myopathies.The final diagnosis depends on muscle immunohistochemical staining,Western blotting and genetic screening.In China,LGMD2B and LGMD2A are the most prevalent forms,accounting for 74.3％ in overall LGMD.Patients with LGMD2B usually have onset age between 19-27 years old.LGMD2B patients present as asymptomatic hyper creatine kinase emia (CK) at the early stage,and later develop to typical proximal muscle weakness with bilateral calf atrophy and extremely high serum CK.The onset age of LGMD2A patients is between 7-18 years old.LGMD2A patients presented as proximal muscle weakness with or without bilateral scapular winging and Achilles tendon contractures.Serum CK can be moderately or highly elevated.Current therapies are mainly supportive and the effective treatment is insufficient.The ongoing global elinical history study and gene therapy bring us new hope for treating LGMD in the coming future.

Effects of Pulchinenoside at Different Concentrations on the Proliferation and Migration of Squamous Cell Carcinoma Cells and Calpain 1, E-cadherin, N-cadherin Protein / 中国药师

Objective: To explore the influences of pulchinenoside on the proliferation and migration of oral squamous cell carcino-ma cells and the expression of calpain 1 ( calpain 1). Methods: The human oral squamous cell carcinoma CAL27 cells were treated with pulchinenoside at the concentrations of 0, 1. 0,2. 0, 4. 0, 8. 0, 12. 0 mg·L-1. MTT method was used to detect the effect of pul-chinenoside on the proliferation of CAL27 cells; the effects of pulchinenoside on the proliferation and migration of CAL27 cells were de-tected by cell scratch test and Transwell experiment; the expression changes of calpain 1, E-cadherin and N-cadherin protein in CAL27 cells were detected by Western blot. Results: MTT results showed that, compared with the control group, the inhibitory rate of pul-chinenoside for the proliferation of CAL27 cells increased significantly (P<0. 05) in a time- and dose-dependent manner. The light microscope results showed that the CAL27 cells morphology changed from the long spindle shape to the cobblestone like epithelioid after the 48-hour treatment with 12. 0 mg·L-1pulchinenoside. Cell scratch test results showed that with the increase of pulchinenoside con-centration, the CAL27 cell migration ability gradually weakened. Transwell experimental results show that with the increase of pul-chinenoside concentration, the CAL27 cell migration and invasion abilities decreased gradually (P <0.05). Western blot results showed that with the increase of the concentration of pulchinenoside, the expression levels of calpain 1 and N-cadherin protein in CAL27 cells decreased gradually (P<0. 05), the expression level of E-cadherin protein increased gradually (P>0. 05). Conclusion:Pulchinenoside can inhibit the proliferation, migration and invasion of oral squamous cell carcinoma CAL27 cells, and its mechanism may be related to the down-regulation of calpain 1 and N-cadherin expressions and up-regulation of E-cadherin expression.

Calpain inhibition improves erectile function in diabetic mice via upregulating endothelial nitric oxide synthase expression and reducing apoptosis / 亚洲男科学杂志(英文版)

Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction (ED) in mice. Thirty-eight-week-old male C57BL/6J mice were divided into three groups: (1) nondiabetic control group, (2) diabetic mice + vehicle group, and (3) diabetic mice + MDL28170 (an inhibitor of calpain) group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg-1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, calpain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.

Possible effect of N-acetyl-leu-leu-norleucinal, an inhibitor of Calpain, on H2O2-induced damage in 661W cells / 中国药理学通报

Aim To investigate the possible effect of N-acetyl-leu-leu-norleucinal (ALLN),an inhibitor of Calpain,on H2O2-induced damage in 661W cells.Methods The cellular survival of 661W treated with different doses of H2O2 without or with ALLN for 24 h was measured by MTT assay.The protein level of Calpain 1 and Calpain 2 was assessed by Western blot.Results Upon the H2O2 treatment at the concentrations of 50,100,500,1 000 μmol · L-1,the survival rate of 661W significantly decreased in a dose-dependent manner compared to that of the control group.Furthermore,the protein level of Calpain 1 and Calpain 2 showed an obviously time-dependent increase in 661W cells treated with 100 μmol · L-1 H2O2 for 12,18,24 h.Finally,the survival rate of 661W treated with ALLN and H2O2 was higher than that treated only with H2O2,and there was no difference in survival rate between ALLN groups (at the concentrations of 25,50,100,200 μmol · L-1) and control group.Conclusions Calpain is involved in the damage induced by H2O2.ALLN,the inhibitor of Calpain,attenuates the oxidative damage,which plays a promising protective role in photo-receptor cells under oxidative stress.

A meta-analysis of the association between Caipain-10 gene polymorphism and T2DM in Chinese population / 中国糖尿病杂志

Objective To evaluate the association between calpain-10 gene polymorphism and type 2 diabetes mellitus(T2DM) by system assessment method.Methods CNKI,CBM and WangFang Data were searched for studies assessing the genetic association between calpain-10 gene polymorphism and T2DM.The data from included studies were extracted to calculate for odds ratio (OR).The simplified version of STREGA list was used to evaluate the quality of researches.Review Manger 5.3.1 software was used to analyze the heterogeneity (I2 values) among the studies,α=0.05.Results A total of 21 randomized controlled studies were analyzed.3062 T2DM patients were involved in SNP43 loci polymorphism evaluation.The highest score of STREGA list was 6 points and the lowest score was 3 points.The proportions of 6 points,5 points,4 points and 3 points were 38.10％,33.33％,9.52％ and 19.05％,respectively.Both scores of "whether having description of genotyping method" item and "whether having sufficient data" item were 0 point.Meta analysis results showed that SNP 43 mutation type "A" of Calpain-10 gene was related to T2DM (OR 0.60,95％CI 0.52,0.68,P＜0.05).The ORs of SNP 19,44 and 63 for T2DM were 0.92,1.50 and 0.84,with P values 0.50,0.11 and 0.08,respectively.The sensitivity analysis showed the similar results,with a small publication bias.Conclusion SNP43 loci of Calpain-10 gene has a genetic association with T2DM.

Effect of matrine on Calpain/MAP-2 of experimental autoimmune encephalomyelitic rats / 中国基层医药

Objective To study the effect of matrine on Calpain and MAP-2 in rats with experimental autoimmune encephalomyelitis(EAE).Methods In accordance with the random number table ,60 Wistar rats were divided into 6 groups randomly: normal group,model group,dexamethasone(DEX)-treated group(1mg/kg),high-dose matrine(MAT)-treated group(250mg/kg),middle-dose MAT-treated group(200mg/kg) and low-dose MAT-treated group(150mg/kg).The EAE models were induced by immunized spinal cord extracts of guinea pig with complete Freunds'adjuvant.Rats of three MAT-treated groups and DEX-treated group were injected intraper-itoneally with MAT and DEX daily for 16 days respectively,whereas rats of normal group and model group were injected intraperitoneally with normal saline.Clinical signs of rats in six groups were observed daily .Hematoxylin-eosin (HE) was used to analyze histopathological evaluation of spinal cord .μ-Calpain,m-Calpain and MAP-2 in spinal cord were determined using RT-PCR and immunohistochemistry respectively .Results Compared with the model group[(2.85 ±0.78)points],the clinical scores were significantly decreased in high-dose-MAT group[(1.28 ± 0.59) points], middle-dose-MAT group [(1.45 ±0.64) points] and low-dose-MAT group [(2.09 ± 0.71)points](t =5.345,4.314,2.869,all P <0.05).The HE score of rats in model group[(2.49 ±0.29)points] was significantly higher than that in high-dose-MAT group[(1.04 ±0.26) points],middle-dose-MAT group [(1.29 ±0.20) points] and low-dose-MAT group[(1.77 ±0.24)points] (t =5.185,4.274,3.629,all P <0.01).The levels of μ-Calpain mRNA and m-Calpain mRNA in the three MAT-treated groups were significantly lower than those in model group(t =10.656,9.418,7.044,all P <0.01;t =6.332,5.416,3.978,all P <0.01).In addition,the expression of MAP-2 in the spinal cord of EAE rats showed a marked elevation after MAT treatment (t =12.841,9.924,7.038,all P <0.01).Conclusion Matrine may be an effective therapeutic approach for EAE by inhibiting Calpain and increase MAP-2 expression.

Calpain activation in retinal pigment epithelium (RPE) cells induced by oxidative stress / 眼科新进展

Objective To investigate the content of Calpain in H2O2-induced oxidative stress in cultured human retinal pigment epithelium (RPE) and the change of biologic activity of RPE cells.Methods Normal culture of human RPE cell line (ARPE19 cells) was assigned as the control group,while cells were cultured in the medium with 10 μmol · L-1 and 100 μmol · L-1 H2O2 were the experimental groups.After culture of 4 h,8 h,16 h,32 h,the proliferation rate of RPE cells induced with different concentrations of H2 O2 was measured by methyl thiazolyl tetrazolium (MTT) assay,and the Ca2 + concentration was measured with fluorescence dye Fluo-3/AM ester,laser scanning confocal microscopy (LSCM) and flow cytometry.Then irritation with 10 μmol ·L-1 H2 O2 for 8 hours,Calpain protein and mRNA in RPE cells were detected by WesternBlot and RT-PCR.Results The proliferation rates (r) of the RPE cells were increased significantly induced by 10 μmol · L-1 H2O2 for 4 h,8 h,16 h and 100 μmol ·L-1 H2 O2 for 4 h,8 h when compared with the control group,and the differences were statistically significant (all P ＜ 0.01),whereas the rates of the cells induced by 100 μ mol · L-1 H2O2 for 16 h and 32 h were less than those of the control group (all P ＜0.01).The Ca2+ concentration in RPE cells induced by 10 μmol · L-1 H2O2 for 8 h was significantly higher than that in the controls [(563.27 ± 77.10) U vs.(170.77 ± 20.11) U] (P ＜ 0.01),and the protein and mRNA levels of Calpain were significantly increased when compared with the control group (both P ＜ 0.05).However,these changes can be effectively suppressed using Calpain inhibitor SNJ-1945 (SNJ).Conclusion The Ca2+ inflow can make Calpain activation involve in oxidative stress stimulated by a certain concentration of H2 O2 in RPE cells.

The effect of metoprolol on Calpain I and CaN signaling pathway in pressure overload induced myocardial hypertrophy / 天津医药

Objective To study the mechanism of metoprolol preventing pressure overload induced myocardial hypertrophy through inhibiting calcineurin (CaN) and CalpainⅠsignaling pathway in rat model of coarctation of abdominal aorta. Methods Thirty SD rats were used for hypertension rat model induced by coarctation of suprarenal abdominal aorta. Model rats were divided into three groups, sham operation group (n=10), abdominal aortic coarctation group (n=9) and metoprolol group (n=9). The changes of blood pressure [systolic blood pressure (SBP) and diastolic blood pressure (DBP)] and myocardial hypertrophy [the ratio of left ventricular mass/ body mass (LVW/BW)] were measured. RT-PCR was used to detect the expression of CaN mRNA, and Western blot assay was used to detect expressions of CaN and CalpainⅠproteins. The activity of CaN enzyme was detected and compared between three groups. Results Compared with the sham-operated sham operation group, values of SBP, DBP, LVW/BW, protein expressions and activities of CaN mRNA, CaN and CalpainⅠwere significantly increased in operation group (P<0.05). There were no significant differences in SBP and DBP between metoprolol-treated group and the operation group (P>0.05). Furthermore, values of SBP and DBP were significantly higher in metoprolol-treated group than those of sham group (P<0.05). Compared with operation group, values of LVW/BW, the protein expression and activity of CaN mRNA and Calpain Ⅰwere significantly decreased in metoprolol group (P<0.05), which were no significant differences compared with sham group. Conclusion Metoprolol prevents myocardial hypertrophy in abdominal aorta coarctative rats, through inhibiting CalpainⅠand CaN signaling pathways.

Molecular mechanism of calpain in regulating macrophage polarization mediated by NF-κB/STAT3 signaling pathways / 复旦学报（医学版）

Objective To investigate the molecular mechanism of calpain in regulating macrophage polarization.Methods Macrophages (RAW264.7) were transfected with siRNA by Lipofectamine 2000 to konckdown calpain1 and calpain2,respectively.The mRNA levels of markers in M1 (IL-23,TNF-α and iNOS) and M2 (IL-10,TGF-β and Arg-1) were measured by qRT-PCR.The levels of proteins in signaling pathways (NF-κB/STAT3) were measured by Western blot.The ability of macrophage migration was detected by Transwell assay.Results The expression level of calpain1 was lower in M1 than that in M2 (P＜0.05),but the expression level of calpain2 was significantly higher in M 1 than that in M 2 (P＜0.05).In calpain1-siRNA group,the mRNA levels of M1-type macrophages markers and M2-type macrophages markers were decreased by lipopolysaccharide (LPS) stimulation;The mRNA levels of M1-type macrophages markers in calpain2-siRNA group were significantly reduced (P ＜ 0.05),while the mRNA levels of M2-type macrophages markers were significantly increased (P ＜ 0.05).In calpain2-siRNA intervention group,the total phosphorylation inhibitor of nuclear factor kappa-B kinase (p-IKK) protein level of LPS-induced macrophages was decreased;in IL-4-induced macrophages,the protein level of plasmic signal transducers and activators of transcription 3 (STAT3) was also decreased,but there was no significant difference in total level of phosphorylation-p65 (p-p65).It was also found that the ability of migration was reduced by the interventions of calpain1-siRNA and calpain2-siRNA as compared with control-siRNA intervention (P＜0.05).Conclusions Calpain2 may potentially promote M1 polarizations by NF-κB and STAT3 signaling pathways,and inhibit the ability of its migration by the interventions of calpain1-siRNA and calpain2-siRNA.

Study on the relationship of the expression level of Calpain-9 with occurrence and metastasis of gastric carcinoma / 中华老年医学杂志

Objectives To explore the correlation of the expression lcvel of Calpain-9 in gastric carcinoma with clinico pathological features and metastasis of gastric cancer.Methods The expression level of Calpain-9 in gastric carcinoma and matched para-cancerous normal mucosa tissues were detected by Western blotting and immunohistochemistry in 146 patients with gastric cancer respcctively.The relationship between the expression level of Calpain 9 and the various clinicopathological indexes was analyzed.Results The expression level of Calpain-9 was significantly lower in cancerous tissues than in matched para cancerous normal mucosa tissues (P ＜ 0.05),with significantly different expression levels of Calpain-9 in different gastric cancerous histological differentiation degrees,different clinical stages,and with or without a lymph node metastasis(all P＜ 0.05).But no statistical difference was found in different sexes,different ages,different tumor sizes,with or without a distant metastasis (all P＞0.05).The spearman's correlation analysis showed that thc expression level of Calpain-9 in gastric cancer was negatively correlated with lymph node metastasis (r=-0.3826,P＜0.05).Conclusions The expression level of Calpain-9 in gastric cancer tissues is decreased,is negatively correlated with clinical stages,pathological differentiation,and cancerous lymph node metastasis.

Calpain-10 and Adiponectin Gene Polymorphisms in Korean Type 2 Diabetes Patients

BACKGROUND: Genetic variations in calpain-10 and adiponectin gene are known to influence insulin secretion and resistance in type 2 diabetes mellitus. Recently, several single nucleotide polymorphisms (SNPs) in calpain-10 and adiponectin gene have been reported to be associated with type 2 diabetes and various metabolic derangements. We investigated the associations between specific calpain-10 and adiponectin gene polymorphisms and Korean type 2 diabetes patients. METHODS: Overall, 249 type 2 diabetes patients and 131 non-diabetic control subjects were enrolled in this study. All the subjects were genotyped for SNP-43 and -63 of calpain-10 gene and G276T and T45G frequencies of the adiponectin gene. The clinical characteristics and measure of glucose metabolism were compared within these genotypes. RESULTS: Among calpain-10 polymorphisms, SNP-63 T/T were more frequent in diabetes patients, and single SNP-63 increases the susceptibility to type 2 diabetes. However, SNP-43 in calpain-10 and T45G and intron G276T in adiponectin gene were not significantly associated with diabetes, insulin resistance, nor insulin secretion. CONCLUSION: Variations in calpain-10, SNP-63 seems to increase the susceptibility to type 2 diabetes in Koreans while SNP-43 and adiponectin SNP-45, -276 are not associated with impaired glucose metabolism.

Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host

A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites’ physiology.

The inhibitory effects of calpain inhibitors on epithelial-mesenchymal transition of MCF-10A mammary epithelial cells induced by fibronectin / 药学学报

The aim of this research is to investigate the inhibitory effects of calpain inhibitors (ALLN and calpain inhibitor IV) on mammary epithelial-mesenchymal transition (EMT) of MCF-10A cells induced by fibronectin (FN). After FN treatment of MCF-10A cells for 48 h, cell migration and invasion were determined by scratch repair assay and matrigel coated transwell assay, respectively. Vimentin, E-cadherin, snail and calpain-2 protein expression were measured by Western blot. The results suggest that FN induced morphological changes in MCF-10A cells, significantly increased the migration and invasion abilities of MCF-10A cells, upregulated the expression of calpain-2, vimentin and snail, and downregulated the expression of E-cadherin. All such changes by FN were reversed with ALLN and calpain inhibitor IV. In conclusion, the upregulation of calpain-2 was involved in FN-induced EMT of MCF-10A mammary epithelial cells, which may be inhibited by ALLN and calpain inhibitor IV.