Effect of Bushen Huoxuetang on Expression of Bax/Bcl-2 and Cleaved Caspase-3 in Nude Mice with Bone Metastasis of Breast Cancer / 中国实验方剂学杂志

ObjectiveTo study the effect of Bushen Huoxuetang on the apoptosis and the expression of B-cell lymphoma （Bcl-2）-associated X protein （Bax）/ Bcl-2 and cleaved cysteine-containing aspartate proteolytic enzyme-3 （cleaved Caspase-3） in the nude mouse model of bone metastasis of breast cancer， and explore the mechanism of Bushen Huoxuetang in inhibiting bone destruction. MethodThirty BALB/c female nude mice were randomly assigned into blank group （n=6） and model group （n=24）. The suspension of 4T1 breast cancer cells was injected into the tibia of mouse right lower limb to establish model of bone metastasis of breast cancer. The successfully modeled nude mice were randomly assigned into model group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group， with 6 mice in each group. Bushen Huoxuetang was administrated at a dose of 36.67 g·kg-1， once a day， and zoledronic acid was administrated by subcutaneous injection at a dose of 100 μg·kg-1， twice a week. The combined drug group was administrated with the same doses of Bushen Huoxuetang group by gavage and zoledronic acid by subcutaneous injection. The mice in the blank group and the model group were administrated with the same volume of distilled water by gavage for 14 days. On the next day at the end of drug administration， the mice were sacrificed by cervical dislocation. The general situation and weight changes of the mice were examined. The right lower limb was collected， and X-ray scanning and hematoxylin-eosin （HE） staining methods were used for observation of pathological changes in the bone. The terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） was employed to detect the apoptosis of bone tissue in nude mice， and Western blot to determine the expression of Bax/Bcl-2 and cleaved Caspase-3 in the bone tissue. ResultCompared with the blank group， the modeling reduced the body weight （P<0.01） and increased the right lower limb weight of the nude mice （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination increased the body weight （P<0.01） and decreased the right lower limb weight （P<0.01）. Compared with the blank group， the other groups showed obvious tumor cell atypia， deep nuclear staining， and clear bone metastasis， and the model group showed obvious osteolytic damage in right lower limb and loss of proximal tibia and knee joint. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination reduced the osteolytic lesions in the right lower limb and recovered part of the bone structure， demonstrating an inhibitory effect on bone destruction. The TUNEL assay showed that the model group had lower apoptosis rate of bone metastatic tumor cells than the blank group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group （P<0.01）. Compared with the blank group， the modeling down-regulated the expression of Bax and cleaved Caspase-3 （P<0.01） and up-regulated the expression of Bcl-2 （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination up-regulated the expression of Bax （P<0.01） and cleaved Caspase-3 （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 （P<0.05， P<0.01）. ConclusionBushen Huoxuetang may inhibit bone destruction in the nude mouse model of bone metastasis of breast cancer by up-regulating the expression of Bax， down-regulating the expression of Bcl-2， activating cleaved Caspase-3， and further inducing apoptosis.

Jinxiangdan Inhibits NLRP3/IL-1β/Caspase-1 Pathway to Alleviate Myocardial Ischemia-reperfusion Injury in Rats / 中国实验方剂学杂志

Objective:To observe the effect of Jinxiangdan （JXD） on NOD-like receptor pyrin domain-containing-3 （NLRP3）/cysteine-dependent aspartate-directed protease-1 （Caspase-1）/interleukin-1<italic>β</italic> （IL-1<italic>β</italic>） signaling pathway in myocardium of rats with myocardial ischemia-reperfusion injury （MIRI） and explore the protective effect and mechanism of JXD against MIRI. Method:Fifty male SD rats were randomly divided into the sham operation group， model group， high- and low-dose JXD groups， and positive drug （Di'ao Xinxuekang） group， with 10 rats in each group. Seven days before modeling， rats in the JXD groups were separately treated with intragastric administration of 0.72 and 0.18 g·kg^-1 JXD tablets， the ones in the sham operation group and model group with the same volume of normal saline， and those in the positive drug group with 1.29 g·kg^-1 Di'ao Xinxuekang. Twelve hours after the last intragastric administration， the anterior descending branch of the left coronary artery was ligated for 30 min and then re-perfused for 60 min for inducing MIRI. ST segment elevation was detected by electrocardiogram（ECG） for model evaluation. The contents of creatine kinase （CK） and lactate dehydrogenase （LDH） in cardiac tissue were measured by colorimetry. Hematoxylin-eosin （HE） staining was conducted for observing myocardial histopathological changes， followed by the detection of cardiomyocyte apoptosis by DNA in situ nick end-labeling （TUNEL） assay. The protein and mRNA expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> were detected by Western blot and real-time polymerase chain reaction （Real-time PCR）， respectively. Result:Compared with sham operation group， the model group exhibited obviously elevated ST segment （<italic>P</italic><0.01）， enhanced CK and LDH activities in the myocardium （<italic>P</italic><0.01）， increased apoptotic cardiomyocytes （<italic>P</italic><0.01）， and up-regulated NLRP3， Caspase-1， and IL-1<italic>β</italic> protein and mRNA expression （<italic>P</italic><0.01）. Compared with model group， JXD at both the high and low doses and Di'ao Xinxuekang significantly lowered the ST segment （<italic>P</italic><0.05，<italic>P</italic><0.01）， diminished the CK and LDH activities in myocardial tissue （<italic>P</italic><0.05，<italic>P</italic><0.01）， improved the apoptosis of cardiomyocytes （<italic>P</italic><0.05，<italic>P</italic><0.01）， and down-regulated the mRNA and protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> in myocardial tissue （<italic>P</italic><0.05，<italic>P</italic><0.01）. The ST segment of ECG in the low-dose JXD group was increased as compared with that in the Di'ao Xinxuekang group （<italic>P</italic><0.05）， while the ST segment in the high-dose JXD group was obviously elevated （<italic>P</italic><0.05）. Besides， the green fluorescence intensities in the low- and high-dose JXD groups and the Di'ao Xinxuekang group remarkably declined （<italic>P</italic><0.05，<italic>P</italic><0.01）. The mRNA and protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> in the high-dose JXD group were down-regulated （<italic>P</italic><0.05）. Conclusion:JXD alleviates MIRI possibly by lowering NLRP3 and IL-1<italic>β</italic> expression and inhibiting cardiomyocyte apoptosis.

Combating protein energy wasting in end stage kidney disease: role of exogenous proteolytic enzyme as an adjuvant to dietary protein

Background: Despite interventions, the prevalence of protein energy wasting in patients on dialysis continues to be unacceptably high. The objective of the study is to evaluate the efficacy of exogenous proteolytic enzyme Aminace 70000 (Hemoglobin Tyrosine Unit), as an adjuvant to dietary protein in improving the nutritional status.Methods: This is a retrospective, real world, single centre, observational study, aimed at assessing the changes in key nutritional indices, over 6 months in patients with chronic kidney disease (CKD) initiated on continuous ambulatory peritoneal dialysis (CAPD). The intervention included addition of egg protein and use of an exogenous proteolytic enzyme. Three cohorts were identified. Cohort 1, had access to a nephrologist and CAPD counsellor; Cohort 2, in addition had access to a dietician who emphasized the need for increase in dietary protein in form of 4-6 eggs a day; and cohort 3, were in addition given an exogenous proteolytic enzyme with the major protein meal.Results: The absolute fall in serum albumin at 6 months for the cohort 1, 2 and 3 is 0.48, 0.29 and 0.09 gm/dl respectively. Not only was the fall in serum albumin significantly less with the use of exogenous proteolytic enzyme, a higher proportion of patients were able to maintain or improve their serum albumin. The fall in midarm circumference was maximum in cohort 1 (2.08 cm) and least in cohort 3 (0.45 cm). This positive trend however, did not achieve statistical significance.Conclusions: Use of exogenous proteolytic enzyme, when combined with egg protein, improves key nutritional indices in patients of CKD on CAPD.

Optimization of cultural conditions for the production of an extra-cellular protease by Pseudomonas species.

The aim of the study was to identify new sources of protease and a protease producing bacteria was isolated from slaughter house soil and identified as Pseudomonas species. The protease production efficiency of the organism was measured with different environmental and nutritional parameters. Optimization of the fermentation medium for maximum protease production was carried out. The culture condition like temperature, pH, incubation time, carbon and nitrogen sources were optimized. The optimum condition found for protease production was 30°C at pH 7 in the medium for 48 hour. Carbon source casein and nitrogen source beef extract stimulated the production of protease. Protease production was higher with immobilized cells. Pseudomonas species can be used profitably for the large scale production of protease to meet the present day demand of the industrial sector.

Optimization of cultural conditions for the production of an extra-cellular protease by Pseudomonas species.

The aim of the study was to identify new sources of protease and a protease producing bacteria was isolated from slaughter house soil and identified as Pseudomonas species. The protease production efficiency of the organism was measured with different environmental and nutritional parameters. Optimization of the fermentation medium for maximum protease production was carried out. The culture condition like temperature, pH, incubation time, carbon and nitrogen sources were optimized. The optimum condition found for protease production was 30°C at pH 7 in the medium for 48 hour. Carbon source casein and nitrogen source beef extract stimulated the production of protease. Protease production was higher with immobilized cells. Pseudomonas species can be used profitably for the large scale production of protease to meet the present day demand of the industrial sector.

Optimization of cultural conditions for the production of an extra-cellular protease by Pseudomonas species.

The aim of the study was to identify new sources of protease and a protease producing bacteria was isolated from slaughter house soil and identified as Pseudomonas species. The protease production efficiency of the organism was measured with different environmental and nutritional parameters. Optimization of the fermentation medium for maximum protease production was carried out. The culture condition like temperature, pH, incubation time, carbon and nitrogen sources were optimized. The optimum condition found for protease production was 30°C at pH 7 in the medium for 48 hour. Carbon source casein and nitrogen source beef extract stimulated the production of protease. Protease production was higher with immobilized cells. Pseudomonas species can be used profitably for the large scale production of protease to meet the present day demand of the industrial sector.

Queijo prato com teor reduzido de gordura adicionado de enzima proteolítica: características físicas e sensoriais / Reduced fat prato cheese added of proteolytic enzyme: physical and sensorial

O queijo Prato, segundo queijo mais consumido no Brasil, obtido por coagulação enzimática do leite e maturado por pelo menos 25 dias, é classificado como gordo e de média umidade. Devido à preocupação com a saúde, os consumidores de queijos têm procurado produtos em suas versões com menor teor de gordura. Contudo, a gordura confere as características sensoriais desejáveis, como sabor, cremosidade, maciez e textura aos queijos. Alterações têm sido introduzidas no processo tecnológico de fabricação dos queijos com teor reduzido de gordura, com o intuito de efetuar melhoria nesses produtos; e o uso de enzimas proteolíticas é uma importante estratégia a ser considerada. A capacidade de derretimento, cor e avaliação sensorial são fundamentais indicadores da qualidade dos produtos obtidos. O presente trabalho analisou as características físicas e sensoriais de queijo Prato com teor reduzido de gordura adicionado da enzima proteolítica fastuosaína, extraída do fruto verde do gravatá. A adição da fastuosaína não interferiu na capacidade de derretimento, tampouco promoveu o desenvolvimento de amargor, que é característica comum não apreciada em queijos com teor reduzido de gordura.

The Prato cheese is the second most consumed cheese in Brazil. It is produced by milk enzymatic coagulation,and maturated for at least 25 days; it is classified as fatty cheese and of medium moisture. Due to the concern to health, the cheeses consumers have been seeking for products with low fat contents; however fat is essential for providing desirable sensory and physiologic characteristics, such as flavor, softness and texture to cheeses. Alterations on the technological processing of low fat cheeses have been made seeking for improved products, and the use of proteolytic enzymes has been a significant strategy. The meltability, color and sensory characteristics are fundamental quality indicators of the final products. This study reports the findings from the analyses on the physical and sensory characteristics of low fat Prato cheese with addition of proteolytic enzyme – fastuosain, that is extracted from unripe gravata fruit. The addition of fastuosainimproved the quality of the product, as this additive neither affected the meltability, nor produced bitterness,which is a common unpleasant taste in low fat cheeses.

THE COMPARATION OF EXPERIMENTAL CONDITIONS EXTRACTING THE PROTEOLYTIC ENZYME FROM THE ALIMENTARY CANAL OF PNEUMATOPHORUS JAPONICUS / 中国海洋药物

Various conditions were compared for extracting the proteolytic enxyme from the alimentary canal of Pneumatophorus juponicus (Houttuyn). The results showed that when using CaCl2 as activator, the suitable conditions were pH7. 5,extracting temperature 20 C,extracting time 2h,activiting time 16h,70% alcohol precipitaion.

Effect on cell proliferation and proteolytic enzyme of human gastric carcinoma by MUC2 antisense oligodeoxynucleotide in vitro / 第三军医大学学报

Objective To investigate the inhibition effect in vitro of mucin gene MUC2 antisense oligodeoxynucleotide (ASODN) on its gene expression and proteolytic enzyme on gastric cancer cell line. Methods Phosphorothioated MUC2 ASODN were synthesized and transfected to SGC7901 cells mediated by lipofectin. MUC2 mRNA and MUC2 protein expressed on SGC7901 cells was detected by RT-PCR and immunohistochemical method. The inhibitory effects of ASODN on proteolytic enzyme, such as cathepsin D, MMP-2 and MMP-9 protein were detected by immunohistochemical staining. The cell cycle and apoptosis were determined by flow cytometry (FCM). Results The inhibition effects peaked at 48th hour after transfection, and the inhibition rate reached 55% when the concentration of ASODN was 0.5 ?mol/L. The number of the cells treated with MUC2 ASODN was decreased as compared to the control cells. MUC2 ASODN transfection could inhibit the transition period of S phase to G2/M phase, and G2/M did not change much. The apoptosis rate was about 4.38%. As compared with blank control group, MUC2 ASODN could significantly inhibit MUC2 mRNA expression of SGC7901 cells. ASODN could downregulate the expression levels of MUC2 protein, MMP-2 and cathepsin D protein. Conclusion MUC2 ASODN transfection could specifically inhibit SGC7901 cells by downregulating the expression levels of proteolytic enzyme.

Effects of brain-derived neurotrophic factor on extracellular proteolytic enzymes / 中国病理生理杂志

AIM:To investigate the effects of brain-derived neurotrophic factor(BDNF)on extracellular proteolytic enzymes including matrix metalloproteinases(MMPs)and serine proteases,in particular,the urokinase-type plasminogen activator(uPA)-plasmin system in a human umbilical vein endothelial cell(HUVEC)model.METHODS:The HUVEC was activated with different doses of BDNF(25-200 ?g/L)for different time period(6-48 h).Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to assay MMP-2,MMP-9,TIMP-1,and TIMP-2 mRNA in HUVEC.The cultured conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography,respectively.uPA,PAI-1,TIMP-1,and TIMP-2 were quantified by Western blotting analysis.RESULTS:The stimulation of serum-starved HUVECs with BDNF caused marked increase in MMP-2 and MMP-9 mRNA expression and induced the pro-MMP-2 and pro-MMP-9 activation without significant differences in proliferation.However,BDNF had no effect on TIMP-1 and TIMP-2 production.BDNF increased uPA and PAI-1 production in a dose dependent manner up to 100 ?g/L,while effects of 200 ?g/L were approximately equal to those of 100 ?g/L.BDNF stimulated uPA and PAI-1 production beyond that in control cultures from 12 h until 48 h after BDNF addition.Protease activity for uPA was also increased by BDNF in a dose dependent manner.CONCLUSION:BDNF activates MMP and uPA/PAI-1 proteolytic network in HUVEC.