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1.
China Occupational Medicine ; (6): 148-152, 2022.
Article in Chinese | WPRIM | ID: wpr-940881

ABSTRACT

@#Objective To investigate the role of tumor necrosis factor alpha stimulated gene/inducible protein 6(TSG-6)on free silica(SiO2 )-induced secretion of pro-inflammatory cytokine interleukin(IL)-1β by bone-marrow-derived macrophages (BMDMs). Methods i)BMDMs isolated from bone marrow were divided into eight groups:the control group was untreated; lipopolysaccharide (LPS) group was stimulated by LPS with a concentration of 50 µg/L;The TSG-6 control group was stimulated by 100 µg/L of recombinant mouse TSG-6. SiO2 model group was pre-stimulated with LPS for four hours,and then stimulated with SiO2 suspension at a final concentration of 250 mg/L;Different dose of TSG-6 groups were firstly treated with above concentrations of LPS and SiO2 suspension,then 10,20,50 and 100 µg/L of recombinant mouse TSG-6 were added respectively;After 16 hours of culture,the cells were collected and the level of IL-1β in BMDMs supernatant was detected by enzyme-linked immunosorbent assay(ELISA)to screen optimal concentration of TSG-6. ii)The cells of the control group,LPS group,SiO2 model group,TSG-6 optimal concentration group and TSG-6 control group were collected. The expression of IL-1β and components of its related pathways in BMDMs was detected by Western blot,including IL-1β,pro-IL-1β,caspase-1,pro dcaspase-1,asc type amino acid transport and NOD-like receptor protein 3(NLRP3). Results i)Compared with the control group,the expression of IL-1β in SiO2 model group was increased significantly(P<0.01). Compared with SiO2 model group,the expression of IL-1β in 20,50,100 µg/L dose of TSG-6 groups were decreased significantly(all P<0.01),and the optimal concentration of TSG-6 was found to be 100 µg/L. ii)Compared with the control group and LPS group,the relative expression levels of IL-1β,caspase-1 and NLRP3 in SiO2 model group were increased significantly (all P<0.05). Compared with SiO2 model group,the expression levels of IL-1β、caspase-1 and NLRP3 were decreased in 100 µg/L TSG-6 group(all P<0.05). Conclusion TSG-6 could inhibit BMDMs to secret pro-inflammatory cytokine IL-1β by down-regulating the expression of NLRP3 and caspase-1.

2.
Article in Chinese | WPRIM | ID: wpr-940757

ABSTRACT

ObjectiveTo explore the effect of Banxia Xiexintang (BXT) on the NOD-like receptor protein 3 (NLRP3)/cysteinyl aspartate-specific protease-1 (Caspase-1) pyroptosis pathway and its downstream factors in rats with ulcerative colitis (UC), and to explain the mechanism of BXT in the treatment of UC. MethodSD rats were randomly divided into normal control group, model group, low- and high-dose BXT groups (6.3, 12.6 g·kg-1·d-1), and salazosulfapyridine (SASP) group (0.42 g·kg-1·d-1), with 7 rats in each group. The UC model was induced by intragastric administration of 2.5% dextran sodium sulfate (DSS) solution for 10 days, followed by drug intervention for 7 days. The general state of rats was observed during the experiment, and the disease activity index (DAI) score was calculated during the administration period. At the end of the experiment, colonic tissues were collected for hematoxylin-eosin (HE) staining to observe the pathological changes and the curative effect of BXT. Real-time fluorescence-based quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA transcriptional levels of NLRP3, Caspase-1, gasdermin D (GSDMD), and interleukin (IL)-1β in colonic tissues. Western blot was used to detect the protein expression of NLRP3, Caspase-1, GSDMD, and IL-1β in colonic tissues to explore the therapeutic mechanism of BXT. ResultCompared with the normal control group, the model group showed increased DAI score, pathological changes in colonic tissues, and up-regulated mRNA and protein expression of NLRP3, Caspase-1, GSDMD, and IL-1β (P<0.05, P<0.01). Compared with the model group, the groups with drug intervention showed reduced DAI scores and improved pathological changes in colonic tissues. The mRNA and protein expression levels of NLRP3, Caspase-1, GSDMD, and IL-1β in colonic tissues of the BXT groups were significantly down-regulated or tended to be down-regulated, especially in the low-dose BXT group (P<0.05, P<0.01). ConclusionBXT can inhibit pyroptosis and alleviate inflammation in rats with UC by regulating the NLRP3/Caspase-1 pathway.

3.
Article in Chinese | WPRIM | ID: wpr-940454

ABSTRACT

ObjectiveTo observe the effects of Hedysarum polysaccharides(HPS)on the signaling pathways of B-cell lymphoma 2 (Bcl-2), cysteinyl aspartate-specific protease 3 (Caspase-3), and Bcl-2-associated X protein (Bax) in Schwann cells(SCs)cultured in high glucose,and explore the possible mechanism of HPS against diabetic peripheral neuropathy(DPN). MethodFour SD suckling mice aged 5-7 days were randomly divided into a normal group,a high-glucose group,an HPS + high-glucose group,and an α-lipoic acid(α-LA)+ high-glucose group. SCs were extracted from the sciatic nerve and cultured in a 37 ℃,5% CO2 incubator. After the cells reached 80% confluence,Cell Counting Kit-8(CCK-8)was used to screen the experimental concentrations suitable for high glucose,HPS, and α-LA interventions. Western blot and Real-time polymerase chain reaction (Real-time PCR)were used to detect the protein and mRNA expression of Bcl-2,Bax,and Caspase-3. The apoptosis rate of SCs was detected by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). ResultAs revealed by Western blot and real-time PCR,compared with the normal group,the high-glucose group showed reduced protein and mRNA expression of Bcl-2 and increased protein and mRNA expression of Bax and Caspase-3(P<0.01). Compared with the high-glucose group,the HPS + high-glucose group and the α-LA + high-glucose group showed increased protein and mRNA expression of Bcl-2 and decreased protein and mRNA expression of Bax and Caspase-3(P<0.01). As displayed by the results of flow cytometry using Annexin V/PI, compared with the normal group,the high-glucose group showed increased apoptosis rate;compared with the high-glucose group,the HPS + high-glucose group and the α-LA + high-glucose group showed reduced apoptosis rate(P<0.01). ConclusionHPS can alleviate the apoptotic response of SCs,and its mechanism may be related to the inhibition of the activation of the Bcl-2/Caspase-3 signaling pathway.

4.
Article in Chinese | WPRIM | ID: wpr-940452

ABSTRACT

ObjectiveTo explore the mechanism of coking death and apoptosis of A549 cells induced by Tingli Dazao Xiefeitang. MethodA549 cells were randomized into blank group, traditional Chinese medicine(TCM) low, medium, and high concentration groups, which were treated with 20, 40, 60 mg·L-1 Tingli Dazao Xiefeitang, and TCM low, medium, and high concentration groups, respectively, and blank group was treated with equal volume culture medium. After 48 h of treatment, cell migration was detected by scratch assay and cell apoptosis was detected by flow cytometry. The relative expression levels of cysteine aspartate protease-1(Caspase-1), NOD-like receptor protein 3 (NLRP3), dermoderin D (GSDMD), Survivin protein and nuclear transcription factor -κB (NF-κB) pathway proteins were detected by Western blot. The levels of intracellular reactive oxygen species (ROS) were determined by DCFH-DA fluorescence probe, and the contents of tumor necrosis factor -β (TNF-β) and interleukin-1β (IL-1β) in supernatant were determined by enzyme-linked immunosorbent assay (ELISA). ResultCompared with blank group, the scratch healing rate, apoptosis rate, relative expression of Survivin protein, Caspase-1, GSDMD, NLRP3, ROS and NF-κB phosphorylation levels were significantly increased in low, medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased (P<0.05). Compared with the low concentration group, the scratch healing rate, apoptosis rate, Survivin protein relative expression, Caspase-1, GSDMD, NLRP3 relative expression, ROS and NF-κB phosphorylation levels were significantly increased in the medium and high concentration groups. The contents of TNF-β and IL-1β in supernatant were significantly increased (P<0.05). Compared with the TCM group, the scratch healing rate, apoptosis rate, Survivin protein relative expression, Caspase-1, GSDMD, NLRP3 relative expression, ROS and NF-κB phosphorylation levels were significantly increased in the high concentration group. The contents of TNF-β and IL-1β in supernatant were significantly increased (P<0.05). ConclusionTingli Dazao Xiefeitang can improve NLRP3 protein expression, inhibit Survivin protein expression and promote apoptosis of A549 cells. At the same time, it can activate NF-κB pathway and ROS system, up-regulate the expression of Caspase-1 and GSDMD, mediate scortosis of A549 cells.

5.
Article in Chinese | WPRIM | ID: wpr-940350

ABSTRACT

ObjectiveTo explore the possible mechanism of total flavonoids of peony flower (TFPF) in protecting rats from gouty nephropathy and provide data support for the pharmaceutical research on the treatment of gouty nephropathy. MethodGouty nephropathy rat model was established by adenine combined with ethambutol. Rats were randomly assigned into blank control group, model group, allopurinol (42 mg·kg-1) group, Tongfengshu tablets (600 mg·kg-1, positive control) group, and TFPF (260, 130, and 65 mg·kg-1) groups. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in rat serum and those of transforming growth factor-β1 (TGF-β1) and IL-1β in renal homogenate. Hematoxylin-eosin(HE) staining was carried out for observation of the morphological changes of renal cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was conducted for observation of the DNA damage in renal cells. The expression of NOD-like receptor protein 3 (NLRP3), cysteine aspartic acid protease(Caspase)-1 and IL-1β were observed by immunohistochemistry. The expression levels of NLRP3, Caspase-1 and nuclear transcription factor -κB (NF-κB) in renal tissues were detected by Western blot. ResultCompared with blank group, the contents of TNF-α, MCP-1, IL-1β, IL-18, and TGF-β1 in serum of model group were significantly increased (P<0.01), and the expressions of NLRP3, Caspase-1, NF-κB and IL-1β in kidney of model group were significantly increased (P<0.01). The renal tissue cells showed cytoplasmic swelling, cell membrane rupture, and the number of nuclear pyknotic fracture increased. The positive rate of TUNEL staining was significantly increased in model group (P<0.01), and the contents of IL-1β and TGF-β1 in renal tissue homogenate were significantly increased (P<0.01). Compared with model group, the contents of inflammatory factors TNF-α, IL-1β and IL-18 in serum of rats in TFPF high- and medium-dose groups could be decreased to different degrees (P<0.05, P<0.01), while the content of MCP-1 in TFPF high-dose group was significantly decreased (P<0.01). The content of TGF-β1 in renal tissue homogenate in TFPF high- and medium-dose groups was significantly decreased (P<0.05, P<0.01), and the content of IL-1β in renal tissue homogenate in TFPF medium-dose group was significantly decreased (P<0.01). HE staining showed that each dose group of TFPF could improve the status of renal tubular epithelial cells, reduce cytoplasmic swelling and the number of nuclear pyknosis to varying degrees. The positive rate of TUNEL staining was decreased (P<0.01) and DNA damage was decreased. The expression of NLRP3, Caspase-1, IL-1β and NF-κB protein in renal tissue cells was inhibited (P<0.05, P<0.01). ConclusionTFPF protects rats from gouty nephropathy by inhibiting the secretion of inflammatory cytokines. Specifically, it may inhibit the activation of NF-κB and NLRP3/Caspase-1 pathways to reduce the expression, maturation, and release of inflammatory cytokines such as IL-1β and IL-18 and further inhibit pyroptosis, thereby reversing the inflammatory injury of kidney in gouty nephropathy.

6.
Article in Chinese | WPRIM | ID: wpr-940195

ABSTRACT

Pyroptosis is a newly discovered programmed cell death. It is an important natural immune response and has obvious anti-infection function. Studies have shown that pyroptosis plays an important role in the occurrence and development of rheumatoid arthritis. Traditional Chinese medicine(TCM) has unique advantages in the prevention and treatment of rheumatoid arthritis. How to guide TCM to effectively prevent and treat rheumatoid arthritis using pyroptosis theory is a new research hotspot in this field. This paper discussed the overview of pyroptosis theory,its mechanism, signal pathway,and application in the treatment of rheumatoid arthritis as well as the research on the activity of TCM based on pyroptosis theory. It was found that the occurrence of pyroptosis was related to Caspase-1-dependent classical inflammatory body pathway and Caspase-1-independent non classical inflammatory body pathway, and pyroptosis produced distinct regulatory effect on the occurrence,development and treatment of rheumatoid arthritis,which would provide a new strategy for the treatment of rheumatoid arthritis. Additionally,TCM recipes such as Miao ethnomedicine prescription Sidaxue and Duhuo Jishengtang, and a variety of effective components such as punicalagin and paeoniflorin monomer derivatives exerted anti-rheumatic and other biological activities by regulating pyroptosis. This provided a theoretical basis and research ideas for the in-depth study of pyroptosis theory and guiding the prevention and treatment of rheumatoid arthritis with TCM.

7.
Article in Chinese | WPRIM | ID: wpr-940163

ABSTRACT

Pyroptosis is a newly discovered programmed cell death. It is an important natural immune response and has obvious anti-infection function. Studies have shown that pyroptosis plays an important role in the occurrence and development of rheumatoid arthritis. Traditional Chinese medicine(TCM) has unique advantages in the prevention and treatment of rheumatoid arthritis. How to guide TCM to effectively prevent and treat rheumatoid arthritis using pyroptosis theory is a new research hotspot in this field. This paper discussed the overview of pyroptosis theory,its mechanism, signal pathway,and application in the treatment of rheumatoid arthritis as well as the research on the activity of TCM based on pyroptosis theory. It was found that the occurrence of pyroptosis was related to Caspase-1-dependent classical inflammatory body pathway and Caspase-1-independent non classical inflammatory body pathway, and pyroptosis produced distinct regulatory effect on the occurrence,development and treatment of rheumatoid arthritis,which would provide a new strategy for the treatment of rheumatoid arthritis. Additionally,TCM recipes such as Miao ethnomedicine prescription Sidaxue and Duhuo Jishengtang, and a variety of effective components such as punicalagin and paeoniflorin monomer derivatives exerted anti-rheumatic and other biological activities by regulating pyroptosis. This provided a theoretical basis and research ideas for the in-depth study of pyroptosis theory and guiding the prevention and treatment of rheumatoid arthritis with TCM.

8.
International Eye Science ; (12): 1257-1261, 2022.
Article in Chinese | WPRIM | ID: wpr-934994

ABSTRACT

AIM:To investigate the effect of epigallocatechin gallate(EGCG)on the apoptosis of human retinal pigment epithelium(ARPE-19)cells and its mechanism. METHODS:The ARPE-19 cells were cultured in vitro and treated with 0,40,80 and 160 μg/mL EGCG, respectively. At the proposed time of treatment the morphological changes were detected by hoechst 33258 staining. The apoptosis rate was detected by flow cytometry. The expression of apoptosis-related factors B lymphocytoma-2 gene(bcl-2), BCL2-Associated X protein(Bax),caspase-3 and p53 were detected by quantitative RT-PCR and Western blotting.RESULTS: Hoechst 33258 staining showed that the ARPE-19 cells with the increase of EGCG drug concentration, the number of apoptotic cells gradually increased and the apoptotic bodies were observed. Flow cytometry showed that the apoptosis rate increased gradually with the increase of EGCG drug concentration. The apoptosis rates at 40, 80 and 160 μg/mL were 4.95%±0.071%, 11.75%±0.075% and 21.25%±0.919% respectively, which was significantly different compared with the control group(2.8%±1.556%)(P<0.01), presented with a drug concentration-dependent. The results of quantitative PCR and Western blotting showed that EGCG could significantly up-regulate the expression of apoptosis-promoting factors Bax, caspase-3 and the mRNA and protein expression of p53, and down-regulate the apoptosis-inhibiting factor bcl-2, all of these showed concentration-dependent effects.CONCLUSION:EGCG can obviously induce the apoptosis of ARPE-19 cells. The mechanism is related with the inhibition of bcl-2 and increase the expression of Bax, caspase-3 and p53.

9.
Clinical Medicine of China ; (12): 97-102, 2022.
Article in Chinese | WPRIM | ID: wpr-932152

ABSTRACT

Objective:To investigate the relationship between caspase recruitment domain protein 9 (CARD9) gene polymorphism and acute pancreatitis (AP) and the clinical efficacy of somatostatin.Methods:A total of 86 patients with AP treated in Shanghai Songjiang District Central Hospital from June 2019 to may 2020 were selected as the research object, and 81 healthy volunteers were selected as the control group for a prospective cohort study. The nucleotide database of National Center for Biotechnology Information (NCBI) was consulted to screen 10 common single nucleotide polymorphisms of CARD9.The single nucleotide polymorphism of CARD9 was detected by SNapShot micro sequencing. All patients with AP were treated with somatostatin. The relationship between CARD9 single nucleotide polymorphism and clinical symptoms and auxiliary examination indexes was observed.The measurement data of normal distribution were compared by independent sample t-test. The measurement data of non normal distribution are represented by M (Q1, Q3), and the rank sum test is used for comparison between groups. The comparison of counting data between groups was adopted χ 2 inspection. Results:The frequency of CARD9 rs10870077 C>G SNP in patients of AP group was significantly higher than that in healthy controls ( OR=1.934, 95% CI=1.011-3.700, P=0.046). Compared with CC genotype, the disappearance time of abdominal pain and abdominal distension in the somatostatin treatment group of CARD9 rs10870077 C>G moderate and severe AP patients was significantly longer ((5.64±2.06) d and (3.76±1.23) d, t=2.98, P=0.006), and the average hospital stay in the somatostatin treatment group of CARD9 rs10870077 C>G severe AP patients was increased by ((13.25±5.31) d and (9.00±3.68) d, t=1.51, P=0.170). Conclusion:CARD9 rs10870077 C>G is a predisposing factor for AP, which is related to the individual differences in the clinical efficacy of somatostatin in severe AP.

10.
Article in Chinese | WPRIM | ID: wpr-930315

ABSTRACT

Objective:To investigate the role of Caspase-1/gasdermin D (GSDMD) -mediated cell pyroptosis in anti-tumor effect of cisplatin (DDP) in triple-negative breast cancer (TNBC) .Methods:HE staining and immunohistochemical staining were performed to detect the morphological changes and the expression of pyroptosis/apoptosis pathway related proteins in TNBC tissues before and after DDP-based neoadjuvant chemotherapy (NACT) . The TNBC cell line MDA-MB-231 was treated with DDP and the morphological changes were observed. The type of cell death induced by DDP was analyzed by Annexin V-FITC/PI double staining and flow cytometry. Lactate dehydrogenase (LDH) release assay and ELISA were performed to detect the release of LDH and inflammatory factors (IL-18 and IL-1β) in cell culture supernatant after DDP treatment. Western blot (WB) was performed to detect the expression of pyroptosis/apoptosis pathway related proteins in cells after DDP treatment. MDA-MB-231 cells treated with DDP were co-treated with caspase-1 specific inhibitor to inhibit pyroptois or co-treated with caspase-3 specific inhibitor to inhibit apoptosis. The effect of caspase-1 inhibitor or caspase-3 inhibitor on the anti-tumor effect of DDP was detected by MTT assay, clone formation assay, transwell assay and would healing test.Results:Reactive changes in the breast surgical specimen after DDP-based NACT included cell swelling and inflammatory cell aggregation around the tumor bed, which were more similar to pyroptosis. The up-regulation of key molecules of pyroptosis pathway post-NACT was significantly higher than that of key molecules of apoptosis pathway. Further experiments in vitro showed that DDP could induce MDA-MB-231 cells to show pyroptosis-like changes characterized by large bubbles blowing from the cellular membrane. Flow-cytometry analyses showed that the death type of MDA-MB-231 cells caused by DDP was mainly Annexin V +PI + cells (mainly lytic cells, such as pyroptosis) . Additionally, DDP treatment induced significant activation of caspase-1 and GSDMD, increased the release of LDH, IL-18 and IL-1β, however, the activation level of caspase-3, which dominates the apoptosis pathway, was significantly lower than that of caspase-1/GSDMD. Moreover, caspase-1 inhibitors (blocking the classical pyroptosis pathway) had a significantly greater inhibitory effect on the anti-tumor effect of DDP than caspase-3 inhibitors (blocking the apoptosis pathway) . Conclusion:Caspase-1/GSDMD mediated pyroptosis may play a leading role in the anti-tumor effect of DDP in triple-negative breast cancer.

11.
China Pharmacy ; (12): 1573-1580, 2022.
Article in Chinese | WPRIM | ID: wpr-929694

ABSTRACT

OBJECTIVE To explore t he protective mechanism of Yangxin dingji capsules on the cardiomyocytes of diabetic cardiomyopathy(DCM)model golden hamsters. METHODS In this study ,golden hamsters were divided into control group (n= 10,no modeling ,no drug administration ),model group (n=9,modeling,no drug administration ),TCM high-dose group [ n=8, modeling,Yangxin dingji capsules 2 g/(kg·d)],TCM low-dose group [ n=8,modeling,Yangxin dingji capsules 1 g/(kg·d)] and empagliflozin group [ n=9,positive control ,modeling,10 mg/(kg·d)]. All the golden hamsters were gavaged continuously for 8 weeks. The general conditions of golden hamsters were observed during the experiment. Blood glucose ,total cholesterol (TC)and creatine kinase MB (CK-MB),ejection fraction (EF),fractional shortening (FS),interleukin 1β(IL-1β)and transforming growth factor β1(TGF-β1)were detected ;the histopathological changes of myocardium were observed. mRNA and protein expression of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),caspase-1,aspirin D (GSDMD),nuclear factor κB (NF-κB)and IL- 1β were detected and observed;DNA damage in myocardial was detected. RESULTS Compared with control group,the blood glucose ,TC,CK-MB,serum IL- 1β,TGF-β1 levels,the mRNA expressions and positive protein expression of NLRP 3,caspase-1,GSDMD,NF-κ B and IL-1 β and protein expression of GSDMD in golden hamsters were significantly increased in model group (P<0.05 or P<0.01) EF and FS were significantly decreased (P<0.01);the fibers of myocardial cells was disordered , and the blue-stained collagen fibers between the myocardium increased ; DNA damaged positive cells in myocardial tiss ue of gold hamsters increased significantly. Compared with model group,the above indexes of administration groups were reversed to varying degrees ;the gap of myocardial cells were clear ,and the fibers disorder was improved ;the DNA damaged positive cells in the myocardial tissue were reduced to varying degrees. CONCLUSIONS Yangxin dingji capsule can inhibit the cardiomyocyte pyroptosis and relieve the inflammatory injury of DCM in DCM model golden hamsters by regulating the NLRP 3/caspase-1/GSDMD signaling pathway ,so as to protect the cardiomyocytes.

12.
Chinese Journal of Biotechnology ; (12): 1025-1038, 2022.
Article in Chinese | WPRIM | ID: wpr-927760

ABSTRACT

In order to explore the antitumor effect and mechanism of different extracts of cultivated Phellinus vaninii fruit body on H22 tumor bearing mice, 150 ICR mice were randomly divided into blank group, model group, CTX group, P. vaninii water extract group, ethanol extract group, petroleum ether extract group and crude polysaccharide group. H22 liver cancer cells were used to establish a solid tumor model and the mice were sacrificed on the 10th day after administration. The spleen and thymus organ index and tumor inhibition rate were calculated, the serum levels of TNF-α, INF-γ, VEGF, and hematoxylin-eosin were detected, and the immunohistochemical staining method was used to observe the pathological changes of tumor tissues, while Western blotting was used to detect the expression of tumor-related proteins. The high-dose petroleum ether extract group showed the best tumor inhibition rate (73.21%), increased serum levels of TNF-α, IFN-γ, and VEGF, as well as significantly promoted tumor necrosis and ablation. The immunohistochemistry of the water extract group showed negative regulation, indicating an insignificant tumor suppression. Western blotting showed the apoptosis genes Caspase-3, Caspase-9 and pathway genes NF-κB and JAK were all highly expressed in each administration group compared with the model group, and their expression levels gradually decreased with increasing doses. In summary, the petroleum ether extract of P. vaninii fruit body showed a significant anti-tumor effect which is presumably mediated through the mitochondrial pathway. The metabolism of drug in the body induces activation of Caspase-3 and Caspase-9 apoptotic proteins by Bax, Bcl-2, and TNF, which further caused nuclear chromatin or DNA to condense or degrade, and subsequently destroy the normal proliferation of tumor cells, thereby inducing their apoptosis and inhibiting tumor growth.


Subject(s)
Animals , Apoptosis , Basidiomycota , Mice , Mice, Inbred ICR , Neoplasms/metabolism
13.
Biol. Res ; 552022.
Article in English | LILACS-Express | LILACS | ID: biblio-1383906

ABSTRACT

Abstract Background: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. Results: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. Conclusions: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

14.
Int. j. med. surg. sci. (Print) ; 8(2): 1-12, jun. 2021. graf, ilus
Article in English | LILACS | ID: biblio-1284445

ABSTRACT

Background/aim: Autophagic cell death and apoptosis of tumor cells has become one of the main objectives in cancer treatment, whereas tumor cell lines are mainly used in studies for providing important data for the evaluation of potential anti cancer substances. In this study, our objective was to evaluate morphological and biochemical changes including rate of apoptosis and Alpha Fetoprotein (AFP) levels at different concentrations of Carnosic Acid (CA) on Human Hepatocellular Carcinoma HepG2 Cells.Materials and methods: Human Hepatocellular Carcinoma (7th passage HepG2 cells) Cell lines were cultured on 11 µM D263M schott glass coverslips placed in 12-well plates and were treated with DMSO, 1, 2.5, 5 and 10 µM concentrations of CA for 24, 48 and 72 hours. Morphological and biochemical data were recorded daily including apoptosis rates demonstrated by Caspase 3, Annexin V expressions under inverted light and Immunofluorescence microscopy, then data were analyzed for statistical significance. AFP, albumin and total protein levels were analyzed spectrophotometricaly for biochemical evaluation.Results: Our results showed that CA significantly inhibited HepG2 cell proliferation in a dose and time dependant manner and significantly caused the formation of autophagic vacuoles starting from 5µM and reaching significance at 10 µM concentrations. Significant decrease was observed in AFP when 48 and 72 hours expressions were examined, with the lowest level reached at 72 hours in the 10 µM CA group. Additionally, increase in albumin levels reached significance only in the 48 h group whereas non-significant increases were also observed in 24 h and 72 h groups.Conclusion: Our current study demonstrates significant increase in apoptosis rates by Carnosic Acid mainly at 10µM concentrations, supporting its anticancer effect on HepG2 cells. These findings are also supported by changes in biochemical analyses of Albumin and AFP levels at 10 µM concentrations.


Antecedentes / objetivos: La muerte celular autofágica y la apoptosis de células tumorales se ha convertido en uno de los principales objetivos en el tratamiento del cáncer, mientras que las líneas celulares tumorales se utilizan principalmente en estudios para proporcionar datos importantes para la evaluación de posibles sustancias anticancerígenas. En este estudio, nuestro objetivo fue evaluar los cambios morfológicos y bioquímicos, incluida la tasa de apoptosis y los niveles de alfa fetoproteína (AFP) a diferentes concentraciones de ácido carnósico (CA) en células de carcinoma hepatocelular humano HepG2.Materiales y métodos: Carcinoma hepatocelular humano (HepG2).Las líneas celulares se cultivaron en cubreobjetos de vidrio Schott D263M de 11 µM colocados en placas de 12 pocillos y se trataron con DMSO, concentraciones de CA 1, 2,5, 5 y 10 µM durante 24, 48 y 72 horas. Los datos morfológicos y bioquímicos se registraron diariamente, incluidas las tasas de apoptosis demostradas por Caspasa 3, las expresiones de Anexina V bajo luz invertida y microscopía de inmunofluorescencia, luego se analizaron los datos para determinar la significación estadística. Los niveles de AFP, albúmina y proteínas totales se analizaron espectrofotométricamente para evaluación bioquímica.Resultados: Nuestros resultados mostraron que CA inhibió significativamente la proliferación de células HepG2 de una manera dependiente de la dosis y el tiempo y causó significativamente la formación de vacuolas autofágicas comenzando desde 5 µM y alcanzando significancia a concentraciones de 10 µM. Se observó una disminución significativa en la AFP cuando se examinaron las expresiones de 48 y 72 horas, alcanzando el nivel más bajo a las 72 horas en el grupo de CA 10 µM. Además, el aumento en los niveles de albúmina alcanzó significación solo en el grupo de 48 h, mientras que también se observaron aumentos no significativos en los grupos de 24 hy 72 h.Conclusión: Nuestro estudio demuestra un aumento significativo en las tasas de apoptosis por el ácido carnósico principalmente a concentraciones de 10 µM, lo que respalda su efecto anticancerígeno en las células HepG2. Estos hallazgos también están respaldados por cambios en los análisis bioquímicos de los niveles de albúmina y AFP a concentraciones de 10 µM.


Subject(s)
Humans , Carcinoma, Hepatocellular/drug therapy , Abietanes/administration & dosage , Hep G2 Cells/drug effects , Liver Neoplasms/drug therapy , Cell Survival , Cells, Cultured , Apoptosis/drug effects , Microscopy, Fluorescence
15.
Rev. bras. cir. cardiovasc ; 36(3): 331-337, May-June 2021. tab, graf
Article in English | LILACS | ID: biblio-1288248

ABSTRACT

Abstract Introduction: Blood cardioplegia (BC) and Custodiol cardioplegia (CC) have been used for a long time in open heart surgery and are highly effective solutions. The most controversial issue among these two is whether there is any difference between them regarding myocardial damage after ischemia surgery. In this study, autophagy, apoptosis, and hypoxia markers were investigated and that way we evaluated the differences between BC and CC patients. Methods: A total of 30 patients were included in this study, using two different cardioplegic solutions. Three different whole blood samples of the patients were taken from a central vein (preoperatively, immediately postoperatively, and one day after surgery). Total ribonucleic acid was extracted from these samples. Quantitative real-time polymerase chain reaction was performed, and changes in gene expression were determined by the 2-∆∆Ct method of relative quantification. Results: In the CC group, Beclin gene expression level was found to be higher and this difference was statistically significant (P=0.0024). Similarly, cysteine-aspartic acid protease (caspase) 9 and hypoxia-inducible factor 1α messenger ribonucleic acid (mRNA) gene expression level increased and were significantly different in the CC group. In the BC group, Beclin and microtubule-associated protein light chain 3 expressions were higher in the samples taken one day after surgery. Caspases 3 and 8 gene expressions were significantly different in the BC group. Conclusion: As a result of the analysis performed between the two cardioplegia groups, it has been shown that CC harms the myocardium more than BC at the level of mRNA expression of related markers.


Subject(s)
Humans , Cardioplegic Solutions/therapeutic use , Heart Arrest, Induced , Autophagy , RNA, Messenger , Apoptosis , Hypoxia/drug therapy
16.
Int. j. morphol ; 39(1)feb. 2021.
Article in English | LILACS-Express | LILACS | ID: biblio-1385291

ABSTRACT

SUMMARY: We aimed to investigate the possible protective effects of Potentilla fulgens on kidney tissue with ischemia- reperfusion using immunohistochemical methods. Wistar rats were grouped as sham, ischemia, ischemia-reperfusion (I/R) and I/R treated with Potentilla fulgens. Renal vessels of the left rat kidney were clamped for 60 minutes for ischemia, IR group had 6 h of reperfusion. 400 mg/kg Potentilla fulgens were given intraperitoneally 5 days before ischemia+reperfusion procedure. Biochemical analysis (MDA, GSH and MPO) of samples were performed. Kidney tissues were fixed with 10 % neutral formalin and routine paraffin tissue follow-up protocol was applied, stained with routine Hematoxylin and Eosin. ADAMTS-5 and Caspase-3 immunostaining was applied for immunohistochemistry and examined under a light microscope. In the ischemia group, inflammation and congestion in the vessels and increased ADAMTS-5 expression in glomerular cells and tubule cells were observed. In reperfusion, an increase in degenerative glomerular cells, tubule cells and intertubular connective tissue and inflammatory cells ADAMTS-5 expression was observed. In the P. fulgens group, degeneration and inflammation decreased and positive ADAMTS-5 expression was observed. In the ischemia and ischemia reperfusion group, increased apoptotic appearance and Caspase-3 positive expression in glomerular and tubular cells, and negative expression in most cells in the P. fulgens group. Potentilla fulgens are thought to stop apoptotic cell development at a certain stage, which affects the cytokine mechanism and plays an important role in the reduction of inflammatory cells and angiogenic regulation.


RESUMEN: El objetivo de este estudio fue investigar los posibles efectos protectores de Potentilla fulgens en el tejido renal con isquemia-reperfusión utilizando métodos inmunohistoquímicos. Se agruparon ratas Wistar como simulación, isquemia, isquemia-reperfusión (I / R) e I / R tratadas con Potentilla fulgens. Los vasos renales del riñón iz- quierdo de las ratas se fijaron durante 60 min por isquemia, el grupo de IR tuvo 6 h de reperfusión. Se administraron 400 mg / kg de Potentilla fulgens por vía intraperitoneal 5 días antes del procedimiento de isquemia + reperfusión. Se realizaron análisis bioquímicos (MDA, GSH y MPO) de muestras. Los tejidos renales se fijaron con formalina neutra al 10 % y se aplicó el protocolo de seguimiento de tejido de parafina de rutina y teñido con hematoxilina y eosina. Se aplicó inmunotinción de ADAMTS-5 y Caspasa-3 para inmunohistoquímica y se examinó con un microscopio óptico. En el grupo de isquemia, se observó inflamación y congestión en los vasos y el aumento de la expresión de ADAMTS-5 en células glomerulares y células tubulares. En la reperfusión, se observó un aumento en la expresión de ADAMTS-5 de células glomerulares degenerativas, células tubulares y tejido conjuntivo intertubular y células inflamatorias. En el grupo de Potentilla fulgens, la degeneración y la inflamación disminuyeron y se observó expresión positiva de ADAMTS-5. En el grupo de isquemia y reperfusión de isquemia, aumentó la apariencia apoptótica y expresión positiva de Caspasa-3 en células glomerulares y tubulares, y expresión negativa en la mayoría de las células del grupo de Potentilla fulgens. Se cree que Potentilla fulgens detiene el desarrollo de las células apoptóticas en una determinada etapa, lo que afecta el mecanismo de las citocinas y juega un papel importante en la reducción de las células inflamatorias y la regulación angiogénica.

17.
Braz. dent. sci ; 24(4, suppl 1): 1-13, 2021. tab, ilus
Article in English | LILACS, BBO | ID: biblio-1349352

ABSTRACT

Objective: Head and neck cancer is the sixth leading cancer by incidence worldwide and eighth by death. Recent reports revealed that, not only radiotherapy but also chemotherapy may induce xerostomia. The aim of this study was to compare the possible regenerative effect of BMSCs through systemic and local injections. Material and Methods: 52 male Albino rats were randomly divided into 4 groups: Group 1: 10 rats received 0.5 ml of PBS by injection. Group 2: 14 rats received an intraperitoneal injection of 5-FU drug. Group 3: 14 rats were injected the same dose of 5-FU then received an intraglandular transplantation of BMSCs suspended in 0.5 ml of PBS at day 1 after 5-FU administration. Group 4: 14 rats were injected the same dose of 5-FU then received an intravenous injection of BMSCs suspended in 0.5 ml of PBS via the tail vein at day 1 after 5-FU administration. Results: Histological examination showed that group 2 showed features of severe degenerative changes which increased over time. Group 3 showed increasing amelioration in the ductal structure overtime. Group 4 also showed regenerated ductal elements however concerning apoptotic changes, immunohistochemistry results revealed improvement in both group 3 and 4 over group 2 with no statistical difference between groups 3 and 4. Conclusion: Histological and immunohistochemical features in group 3 and group 4 revealed similar amelioration in regenerative potentials. On the other hand, regenerative features of both experimental groups were statistically significant as compared independently to group 1 (AU)


Objetivo: O carcinoma de cabeça e pescoço é o sexto câncer de maior incidência no mundo sendo a oitava causa de morte por cancer. Relatos recentes revelaram que não apenas a radioterapia, mas também a quimioterapia podem induzir xerostomia. O objetivo desse estudo foi comparar a possivel ação regenerative de BMSCs através de injeção local e sistêmica. Material e Métodos: 52 ratos Albino foram aleatoriamente alocados em 4 grupos: Grupo 1: 10 ratos que receberam 0.5 ml de injeção de PBS. Grupo 2: 14 ratos que receberam injeção intraperitoneal da droga 5-FU. Grupo 3: 14 ratos que foram injetados com a mesma dose de 5-FU e receberam transplante intraglandular de BMSCs ressuspendidas em 0.5mL de PBS no dia 1 após a administração do 5-FU. Grupo 4: 14 ratos que foram injetados com a mesma dose de 5-FU e receberam injeção intravenosa de BMSCs ressuspendidas em 0.5mL de PBS via veia caudal 1 dia após a administração de 5-FU. Resultados: O exame histológico demonstrou que o grupo 2 apresentou alterações degenerativas severas que se agravaram com o tempo. O Grupo 3 mostrou melhora da estrutura ductal ao longo do experimento. Group 4 também mostrou elementos ductais regenerados. Referente a alterações apoptóticas,análise imunohistoquimica mostrou melhora nos grupos 3 e 4 comparados ao grupo 2, sendo que os grupos 3 e 4 foram estatisticamente semelhantes. Conclusão: Análises histológicas e imunohistoquímicas mostram que os grupos 3 e 4 apresentam melhora no potencial regenerativo Por outro lado, os resultados observados para os dois grupos foi estatisticamente semlhante quando comparados independentemente ao grupo 1 (AU)


Subject(s)
Animals , Rats , Salivary Glands , Mesenchymal Stem Cells , Squamous Cell Carcinoma of Head and Neck
18.
J. vasc. bras ; 20: e20210040, 2021. tab, graf
Article in Portuguese | LILACS | ID: biblio-1279382

ABSTRACT

Resumo Contexto Estudos demonstraram, por análise histológica e Dopplerfluxométrica, a interferência da isquemia renal unilateral, realizada em algumas cirurgias, sobre o rim contralateral, identificando o fenômeno de kidney-kidney crosstalk. Objetivos Identificar o efeito da isquemia de duas estratégias de oclusão da vasculatura renal esquerda sobre o rim contralateral através do volume de células renais positivas para Caspase 3. Métodos Suínos foram divididos em 2 grupos: A (n = 8), artéria renal esquerda clampeada, e AV (n = 8), artéria e veia renais esquerdas clampeadas. Foi realizado o estudo imuno-histoquímico (anti-Caspase 3), com o material de biópsias coletadas do rim isquêmico e contralateral em 0, 30, 60 e 90 minutos de isquemia, e análise morfométrica, sendo que a média representou o volume de área de Caspase 3 positiva (%). Resultados A análise morfométrica do rim contralateral nos tempos 30, 60 e 90 minutos de isquemia mostrou que a média da área marcada por Caspase 3 foi estatisticamente superior à média do rim isquêmico nos dois grupos: artéria renal clampeada (A) e artéria e veia renais clampeadas (AV). Comparando o rim isquêmico e contralateral nos dois tipos de clampeamento, não houve diferença estatisticamente significante da área marcada por Caspase 3. Conclusões No modelo experimental de isquemia renal unilateral, o rim não isquêmico apresentou dano celular, demonstrado pela expressão da Caspase 3 de forma aguda em decorrência da isquemia contralateral. O tipo de clampeamento do hilo não parece ter influência sobre o volume de área marcada por Caspase 3.


Abstract Background Studies have demonstrated with histological analysis and Doppler flow measurement analysis that unilateral renal ischemia, which is performed in some surgeries, interfered with the contralateral kidney, identifying the phenomenon of kidney-kidney crosstalk. Objectives To identify the effects on the ischemic and contralateral kidney of renal ischemia induced by two types of clamping technique by analyzing the volume of kidney cells positive for Caspase 3. Methods Sixteen pigs were divided into 2 groups, as follows: A (n = 8) - clamping of left renal artery only and AV (n = 8) - clamping of left renal artery and vein. Immunohistochemical analyses (anti Caspase 3) were conducted with biopsy specimens collected from the ischemic and contralateral kidney at 0, 30, 60, and 90 minutes of ischemia and morphometric analysis was performed, taking the mean to represent the volume of the Caspase 3 positive area (%). Results Morphometric analysis of specimens collected at 30, 60, and 90 minutes of ischemia showed that the mean area marked for Caspase 3 was statistically larger in the contralateral kidney than the ischemic kidney in both groups: clamped renal artery (A) and clamped renal artery and vein (AV). Comparing the ischemic and contralateral kidney, there was no statistically significant difference in the area marked for Caspase 3 between the two types of clamping. Conclusions In the experimental model of unilateral renal ischemia, the non-ischemic kidney exhibited cell damage, demonstrated by Caspase 3 expression. The type of hilum clamping does not appear to influence the area marked for Caspase 3.


Subject(s)
Animals , Renal Circulation , Ischemia , Swine , Intervention Studies , Apoptosis , Constriction , Caspase 3
19.
Chinese Journal of Digestion ; (12): 457-465, 2021.
Article in Chinese | WPRIM | ID: wpr-912202

ABSTRACT

Objective:To explore the role of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in non-alcoholic steatohepatitis (NASH).Methods:The liver tissue samples of 24 patients admitted the Second Affiliated Hospital of Fujian Medical University were selected, including 12 NASH samples from liver biopsy and 12 normal liver tissues from the margin of hepatic hemangioma. The expression of NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, interleukin (IL)-1β and the content of triglyceride (TG) were detected. Wild-type and NLRP3 -/- C57BL/6 mice were fed with normal diet or methionine-choline deficient diet (MCD) for 8 weeks. The wild-type mice were divided into MCC950 NASH, 0.9% sodium chloride (NaCl) NASH, MCC950 and 0.9% NaCl group, 8 mice in each group, and were fed with MCD diet and treated with MCC950, fed with MCD diet and treated with 0.9% NaCl, fed with normal diet and treated with MCC950, and fed with normal diet and treated with 0.9% NaCl respectively for eight weeks. After eight weeks, the pathologic changes of liver tissues were observed with hematoxylin-eosin staining and immunohistochemistry. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), free fatty acid (FFA), IL-1β and TG in serum were determined by enzyme linked immunosorbent assay. The expression levels of NLRP3, ASC, caspase-1 and IL-1β in liver tissues were examined by Western blotting and real-time quantitative polymerase chain reaction. Primary Kupffer cells were isolated and cultured from the livers of wild-type and NLRP3 -/- mice and divided into control group and palmitic acid group. The expression levels of related proteins in the supernatant of cells culture were detected by Western blotting. Independent sample t test and one-way ANOVA were used for statistical analysis. Results:The expression levels of NLRP3, ASC, caspase-1, IL-1β and the content of TG of the liver tissues of the NASH patients were all higher than those of healthy control group (all P<0.05). The formation of steatohepatitis in hepatocyte of MCD-fed mice was more obvious than that of nomal diet-fed mice, with more hepatocyte ballooning and inflammatory cell infiltration. The expression of NLRP3, ASC, caspase-1, IL-1β, caspase-1 activity and the content of TG in liver tissue of NASH mice were all higher than those of normal diet-fed group (all P<0.05); and serum levels of ALT, AST, IL-1β, and the content of FAA were all higher than those of normal diet-fed group (all P<0.05). The serum levels of ALT, AST, IL-1β and IL-18 of NLRP3 -/- NASH mice were all lower than those of wild-type NASH mice (all P<0.05). The serum level of ALT, the expression of ASC, caspase-1 and IL-1β in liver tissues, and the degrees of liver fibrosis of wild-type MCC950 NASH group were all lower than those of 0.9% NaCl NASH group (all P<0.05). The expression of NLRP3, ASC, caspase-1, caspase-1 activity, and secretion of IL-1β and IL-18 in Kupffer cells from wild-type mouse treated with palmitic acid were all higher than those of the negative control group (all P<0.05). However, the changes of the above indicators in Kupffer cells from NLRP3 -/- mouse were not affected by palmitic acid treatment. Conclusion:NLRP3 blockade can significantly alleviate the liver injury and fibrosis in NASH mice and prevent the development of NASH.

20.
Article in Chinese | WPRIM | ID: wpr-912112

ABSTRACT

Objective:To detect the expression of cysteinyl aspartate specific proteinase 4 (caspase-4), caspase-5, gasdermin D (GSDMD), nucleotide-binding oligomerization domain-like receptor protein-3 (NLRP3), Pannexin-1 and P2X7 involved in non-canonical pyroptosis pathway in muscle tissues of patients with dermatomyositis (DM)/polymyositis (PM) and to investigate the roles and significance of them in the pathogenesis of DM and PM.Methods:Altogether 13 DM patients, nine PM patients and 20 volunteers (control group) treated in the Affiliated Hospital of Qinghai University from January 2019 to September 2020 were enrolled in the present study. The 20 volunteers with no additional concomitant diseases underwent debridement due to simple orthopedic trauma. Pathological changes in muscle tissues were detected by hematoxylin & eosin (HE) staining. Expression of caspase-4, caspase-5, GSDMD, NLRP3, Pannexin-1 and P2X7 in muscle tissues was measured using immunohistochemistry (IHC).Results:(1) HE staining results showed that the muscle fibers in the control group had basically normal morphology and structure with no obvious inflammatory cell infiltration, atrophy, degeneration or necrosis. However, the size and thickness of muscle fibers in DM and PM groups were different with excessive inflammatory cell infiltration, atrophy, degeneration and necrosis to varying degrees. Moreover, the pathological scores of HE staining in muscle tissues of DM and PM groups were significantly higher than that of the control group and the differences were of statistical significance ( P<0.05). (2) IHC staining results suggested that the expression of caspase-4, caspase-5, GSDMD, NLRP3, Pannexin-1 and P2X7 in muscle tissues was higher in DM and PM groups than in the control group ( P<0.05). (3) As indicated by Pearson correlation analysis, the pathological scores of HE staining in muscle tissues of DM and PM groups were positively correlated with the IHC scores of caspase-4, caspase-5, GSDMD, NLRP3, PAnnexin-1 and P2X7 ( P<0.05). Furthermore, the IHC scores of caspase-4 and caspase-5 were positively correlated with the IHC scores of GSDMD and Pannexin-1 ( P<0.05); the IHC score of GSDMD was positively correlated with the IHC score of NLRP3 ( P<0.05); the IHC score of Pannexin-1 was positively correlated with the IHC score of P2X7 in muscle tissues ( P<0.05). Conclusions:The non-canonical pyroptosis pathway might be involved in the pathogenesis of DM and PM, which was possibly achieved by promoting inflammatory response. These results suggested that the non-canonical pyroptosis pathway played crucial roles in the immune pathogenesis of DM and PM.

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