Cysteinyl Aspartate-specific Protease Research Articles

Hyperglycemia-induced myocardial fibrosis may be associated with pyroptosis and apoptosis of cardiomyoctes in diabetic mice

Myocardial fibrosis is an important manifestation of diabetic cardiomyopathy. This study investigated the potential mechanism of diabetic myocardial fibrosis. Male C57BL/6J and db/db mice aged 8 weeks were randomly divided into the diabetic (DB) and control groups. At 20 weeks, the mouse heart was harvested and subjected to hematoxylin-eosin staining (HE) and Masson staining to investigate the degree of fibrosis. The expressions of transforming growth factor-beta 1 (TGF-β1), collagen-III, B-cell lymphoma-2 (Bcl2), Bcl2-associated X protein (Bax), cleaved gasdermin D (GSDMD), cysteinyl aspartate specific proteinase-1 (caspase-1), apoptosis-associated speck-like protein containing a CARD (ASC), and nucleotide-binding oligomerization domain (NOD)-like receptor 3 (NLRP3) were measured by western blotting. Immunohistochemistry and TdT-mediated dUTP nick end labeling (TUNEL) staining were performed to analyze the development of apoptosis and pyroptosis. A significant increase in body weight and blood glucose in the DB group was observed. Myocardial pathological injury, fibrosis, apoptosis, and pyroptosis were more obvious and serious in the DB group. The expression of anti-apoptotic Bcl2 significantly decreased, while the expression levels of pro-apoptotic Bax, caspase-3, and pyroptosis-related proteins, such as cleaved GSDMD, and caspase-1 in the DB group were significantly increased. Pyroptosis and apoptosis were probably the main mechanisms that caused myocardial fibrosis in mice with diabetes.

Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury

This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 μg·mL~(-1)) group, and TFR(30 μg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.

Mechanism underlying Müller cell pyroptosis and its role in the development of proliferative vitreoretinopathy

ObjectivesTo explore the mechanism underlying Müller Cell Pyroptosis (MCP) and its role in the development of Proliferative Vitreoretinopathy (PVR). MethodThe expression of pyroptosis-related factors, namely, cysteinyl aspartate-specific proteinase (caspase-1), interleukin (IL)-1β, IL-18, and Gasdermin D (GSDMD), was detected by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and western blotting at the mRNA and protein levels, respectively, in retinal tissues. Müller and spontaneously Arising Retinal Pigment Epithelia (ARPE)-19 primary cells with GSDMD overexpression or knockdown were cultivated. Western blotting was used to detect the levels of the following pyroptosis-related factors in retinal tissues: caspase-1, IL-1β, IL-18, and GSDMD. Through Cell Adhesion (CA) experiments, the changes in ARPE-19 CA in each group were observed. The migration and invasion of ARPE-19 cells were measured using the Transwell assay. The proliferation of ARPE-19 cells was measured with a Cell Counting Kit 8 (CCK-8) assay. Finally, the expression of the cytokines IL-1β and IL-18 in the ARPE-19 cell culture medium was detected using the Enzyme-Linked Immunosorbent Assay (ELISA). ResultsCompared with the surrounding normal tissues, the expression of caspase-1, IL-1β, IL-18, and GSDMD at the protein and mRNA levels in the retinal proliferative membrane samples of the patients decreased significantly (p < 0.05). MCP significantly enhanced ARPE-19 CA, migration and invasion, proliferation, and cytokine expression (p < 0.05). ConclusionsMCP can promote the development of PVR lesions.

Effect of Schizochytrium limacinum supplementation to a low fish-meal diet on growth performance, lipid metabolism, apoptosis, autophagy and intestinal histology of Litopenaeus vannamei

In this experiment, we aimed to evaluate the relationship between the addition of Schizochytrium limacinum to low fish meal diets on growth performance, apoptosis, autophagy, lipid metabolism, and intestinal health of Lipenaeus vanamei. The diet containing 25% fish meal was used as a positive control (FM) and the other three diets contained 15% fish meal and were supplemented with 0, 0.3, and 0.6% S. Limacinum (LF, LFLD, LFHD). The shrimp (0.22 ± 0.00 g) were divided into four replicates of 40 shrimp per tank and fed four times daily to apparent satiation for 8 weeks. Results showed that the final weight (FBW) and weight gain rate (WGR) of shrimp fed FM and LFHD diets were significantly increased compared to those fed the LFLD diet (P&amp;lt;0.05), and there was no significant difference in survival rate (SR) and feed conversion rate (FCR) among the groups (P&amp;gt;0.05). Supplementation of S. Limacinum in low fish meal diets had no effects on shrimp body composition (P&amp;lt;0.05). There were significant differences (P&amp;lt;0.05) in low-density lipoprotein (LDL-C) glucose (GLU), triglycerides (TG), and total cholesterol (TC) in the hemolymph of shrimp fed the LF diet compared to those fed the LFLD and LFHD diets. HE staining and transmission electron microscopy (TEM) results showed that the microvilli height, mucosal folds height, mucosal folds width and muscle layer thickness in the intestine of shrimp fed the LF diet were significantly reduced compared to those fed the other three diets (P&amp;lt;0.05). Swelling of the endoplasmic reticulum and irregular mitochondria in the gut of shrimp fed the LF diet was also observed by TEM, and the endoplasmic reticulum and mitochondria of shrimp fed the LFHD diet returned to a healthy state. Hepatopancreas genes expression results were showed that the gene expression of 5′ -AMP-activated protein kinase (ampk), stearoyl-CoA desaturase (scd1), acetyl-CoA carboxylase 1 (acc1), and malonyl-CoA decarboxylase (mcd) of shrimp fed the LF diet was significantly increased compared to those fed the FM diet (P&amp;lt;0.05). The gene expression of sterol regulatory element-binding protein (srbep) and carnitine palmitoyl transferase 1 (cpt-1) of shrimp fed the LFLD diet was significantly increased compared to those fed the LF diet (P&amp;lt;0.05). The gene expression of acc1, mcd and scd1 of shrimp fed the LFHD diet was significantly reduced compared to those fed the LF diet (P&amp;lt;0.05). Results of genes expression associated with apoptosis in the hepatopancreas showed that the gene expression of B lymphocytoma-2 (bcl-2), BCL2 associated X apoptosis regulator (bax) and cysteinyl aspartate specific proteinase 8 (caspase 8) of shrimp fed the LF diet was significantly reduced compared to those fed the FM diet (P&amp;lt;0.05). The gene expression of bcl-2 of shrimp fed the LFHD diet was significantly reduced compared to those fed the LF diet (P&amp;lt;0.05). Genes related to autophagy in the hepatopancreas showed that the expression of autophagy-related protein 12 (atg 12), autophagy-related protein 13 (atg 13) and beclin1 of shrimp fed LF the diet was significantly reduced compared to those fed the FM diet (P&amp;lt;0.05). The gene expression of atg 12 and atg 13 of shrimp fed the LFHD diet was significantly increased compared to those fed the LF diet (P&amp;lt;0.05). In summary, reducing fish meal is detrimental to the growth performance and intestinal health of shrimp, and 0.6% S. Limacinum supplementation can improve the growth performance, promotes hepatopancreas lipid metabolism, reduces apoptosis, promotes autophagy and improve intestinal health of Litopenaeus vannamei.

Network-based pharmacology and molecular docking exploring the "Bupleuri Radix-Scutellariae Radix" mechanism of action in the viral hepatitis B treatment.

Viral hepatitis B is caused by the hepatitis B virus, which is characterized by liver lesions. Bupleuri Radix and Scutellariae Radix are the main traditional medicine pairs with remarkable efficacy in hepatitis B. However, their molecular mechanisms are incompletely understood. The main active components of Bupleuri Radix and Scutellariae Radix, as well as therapeutic targets for the treatment of hepatitis B, were identified through network pharmacology techniques. We identified viral hepatitis B targets using the GeneCards, online mendelian inheritance in man, and therapeutic target databases. We discovered the active components of Bupleuri Radix and Scutellariae Radix as well as therapeutic targets using the encyclopedia of traditional Chinese medicine, HERB, traditional Chinese medicine systems pharmacology database, and a bioinformatics analysis tool for molecular mechanism of traditional Chinese medicine databases. VENNY obtained the intersections. Cytoscape and STRING were used to create the "active ingredient-potential target" network and protein interaction network. The DAVID database was used to enrich GO and KEGG pathways. The results were confirmed using the molecular docking method. There were 1827 viral hepatitis B targets, and 37 active ingredients for Bupleuri and Scutellariae Radix, with the main components being quercetin, wogonin, baicalein, and kaempferol. Tumor necrosis factor (TNF), mitogen-activated protein kinase 3 (MAPK3), interleukin-6 (IL-6), vascular endothelial growth factor A, cysteinyl aspartate specific proteinase 3, transcription factor AP-1 (JUN), RAC-alpha serine/threonine-protein kinase, and cellular tumor antigen p53 are among the 78 common targets of Bupleuri Radix and Scutellariae Radix intervention in viral hepatitis B. KEGG enrichment resulted in 107 pathways, including cancer, hepatitis B, and TNF signaling pathways. According to the molecular docking technique, quercetin, wogonin, baicalein, and kaempferol had strong binding activities with TNF, MAPK3, and IL-6. In this study, we initially identified various molecular targets and multiple pathways involved in hepatitis B treatment with Bupleuri Radix and Scutellariae Radix.

Anthocyanin from Lycium ruthenicum Murr. in the Qaidam Basin Alleviates Ultraviolet-Induced Apoptosis of Human Skin Fibroblasts by Regulating the Death Receptor Pathway.

The study aimed to investigate the potential protective role of anthocyanin from Lycium ruthenicum Murr. in the Qaidam Basin against ultraviolet B (UVB)-induced apoptosis of human skin fibroblasts (HSFs). HSFs cultured in vitro were randomly divided into a control group, UVB group, and anthocyanin groups (0.1, 0.5, and 1.0 mg/mL). HSFs in the UVB and anthocyanin groups were exposed to 30 mJ/cm2 UVB to establish a photoaging model. Then, apoptosis rate, tumor necrosis factor-α (TNF-α), cysteinyl aspartate specific proteinase-3 (caspase-3), cysteinyl aspartate specific proteinase-7 (caspase-7), and survivin expression were evaluated. UVB irradiation can increase the apoptosis rate of HSFs and expression of TNF-α, caspase-7, and survivin. Anthocyanin pretreatment (0.1, 0.5, and 1.0 mg/mL) decreased UVB-induced apoptosis rate and TNF-α and caspase-7 expression and increased survivin expression. Compared with the control group, the apoptosis rate and expression of TNF-α, caspase-7, and survivin of anthocyanin groups in UVB-irradiated HSFs were high. Among the three doses of anthocyanin (0.1, 0.5, and 1.0 mg/mL) groups, the apoptosis rate and TNF-α expression of anthocyanin at 1.0 mg/mL were the lowest. There was no significant change in caspase-3 expression in each group. Anthocyanin from Lycium ruthenicum Murr. in the Qaidam Basin could alleviate UVB-induced apoptosis by regulating the death receptor pathway.

Mechanism of total flavonoid extract from Dracocephalum moldavica on bleomycin-induced pulmonary fibrosis in mice based on pyroptosis pathway

This study investigated the mechanism of total flavonoid extract from Dracocephalum moldavica(TFDM) in mice with bleomycin(BLM)-induced pulmonary fibrosis(PF) and explored its mechanism against the pyroptosis pathway. A mouse model of PF was established by intratracheal infusion of bleomycin(4 mg·kg~(-1)), and the normal group was treated with the same dose of saline under the same conditions. After the second day of modeling, the distilled water was given to the normal and model groups by gavage, and the corresponding drug were given to the TFDM and the dexamethasone groups for 28 consecutive days. After 28 days, lung tissues of mice with PF were taken to determine the content of hydroxyproline(HYP). The degree of lung inflammation and fibrosis was observed by hematoxylin-eosin(HE) and Masson stainings, and the content of interleukin-18(IL-18) and interleukin-1β(IL-1β) in the serum of mice with PF were measured by enzyme-linked immunosorbent assay(ELISA). Western blot was used to determine the expression levels of proteins in the lung tissues of mice with PF. HE staining showed that the BLM group had abnormal lung tissue structures and showed more inflammatory cell infiltration. Masson staining showed plenty of collagenous fibrotic tissues that were stained blue in the lung tissues. As compared with the normal group, the content of HYP and levels of IL-18 and IL-1β in the serum of rats in the BLM group were up-regulated(P&lt;0.01). The protein expressions of type Ⅰ collagen(Col-1), fibronectin 1(FN1), α-smooth muscle actin(α-SMA), cysteinyl aspartate specific proteinase-1(caspase-1), gasdermin D(GSDMD), NOD-like receptor thermal protein domain associated protein 3(NLRP3), p62, and apoptosis-associated speck-like protein containing a CARD(ASC) in the lung tissues of mice with PF in the BLM group were increased(P&lt;0.01), whereas the protein expressions of autophagy-related 5(ATG5) and Beclin1 were decreased(P&lt;0.01). Compared with the BLM group, the TFDM groups and dexamethasone group showed normal lung tissue structures and reduced inflammatory cell infiltration. Less collagenous fibrous tissues in blue color were seen and the fibrosis in the lung tissue was alleviated in the TFDM groups and dexamethasone group, with the down-regulation of the content of HYP and the levels of IL-18 and IL-1β(P&lt;0.05, P&lt;0.01). In the TFDM groups and dexamethasone group, the protein expression levels of Col-1, FN1, α-SMA, caspase-1, GSDMD, NLRP3, p62, and ASC were decreased(P&lt;0.01), and the protein expressions of ATG5 and Beclin1 were increased(P&lt;0.01) in the lung tissues of mice with PF. From the above results, it is known that TFDM down-regulates the levels of inflammatory factors and related proteins, and effectively mitigates the process of BLM-induced PF by regulating the pyroptosis pathways and potentially affecting the autophagy.

Visual acupotomy intervention mitigates pain reaction by improving intervertebral disc degeneration and inhibiting apoptosis of nucleus pulposus cells in rabbits with cervical spondylosis

To investigate the effect of visual acupotomy intervention on intervertebral disc degeneration, nucleus pulposus cell apoptosis and expression of apoptosis related proteins in rabbits with cervical spondylosis (CS), so as to explore its mechanism underlying improvement of CS. A total of 48 male New Zealand rabbits were randomly divided into blank control, model, acupotomy and medication (meloxicam) groups, with 12 rabbits in each group. The neck type CS model was established by forcing the rabbit to make a neck flexion for 5 hours in a restrained chamber, once daily for 12 weeks. Rabbits of the medication group received an intramuscular injection of meloxicam (0.35 mg/kg), once daily for 4 consecutive weeks, and those of the acupotomy group received ultrasound-guided acupotomy intervention, once a week for 4 weeks. The pain threshold (PT) was measured by using a VonFrey electronic pain detector. The levels of prostaglandin E2 (PGE2), 5-hydroxytryptamine (5-HT) and substance P (SP) in serum were detected by ELISA. The severity of intervertebral disc degeneration was observed by using magnetic resonance imaging (MRI) and given scores in accordance with Suzuki's and colleague's "new classification system of cervical disk degeneration". The apoptosis of nucleus pulposus cells was analyzed by TUNEL staining. The protein expression levels of apoptosis-related protein Fas, cysteinyl aspartate-specific protease-3 (Caspase-3), B-cell lymphoma-2 asso-ciated X protein (Bax) and B-cell lymphoma-2 protein (Bcl-2) were measured by Western blot. Compared with the blank control group, the PT and Bcl-2 expression and MRI score were significantly down-regulated (P<0.01, P<0.001), whereas the contents of serum PGE2, 5-HT and SP, ratios of TUNEL-positive cells, and expression of Fas, Caspase-3 and Bax were considerably up-regulated (P<0.001, P<0.05, P<0.01) in the model group. In contrast to the model group, both the medication and acupotomy groups had an obvious increase in the levels of PT and Bcl-2 expression and MRI score (P<0.05, P<0.01), and a significant decrease in the contents of serum PGE2, 5-HT, SP, ratios of TUNEL-positive cells, and expression of Fas, Caspase-3 and Bax proteins (P<0.05). No significant differences were found between the medication and acupotomy groups in all the indexes mentioned above (P>0.05). Visual acupotomy intervention can mitigate the pain state of CS rabbits, which may be related to its functions in improving the intervertebral disc degeneration, reducing inflammatory reactions and apoptosis of nucleus pulposus cells.

Reduced graphene oxide quenched peptide probe for caspase-8 activity detection and cellular imaging.

Cysteinyl aspartate-specific protease 8 (caspase-8) plays a key role in various biological processes by regulating apoptosis. Therefore, this makes accurate detection and intracellular imaging of caspase-8 of great importance for drug screening, disease diagnosis, and prognostication. Here, by designing a reduced graphene oxide (rGO) quenched peptide probe, we constructed a new biosensing system for monitoring in vitro and intracellular caspase-8 activity. In this system, a fluorophore-labeled peptide and rGO were used as the substrate of caspase-8 and the fluorophore quencher, respectively. The hydrolysis of caspase-8 on the polypeptide probe substrate can generate two fragments with different lengths. The release of the short fragment labeled with the fluorophore causes recovery of the fluorescence signal (Ex/Em = 520/576nm). Under the optimized conditions, the proposed fluorescence method exhibited a linear response range of 0.2 to 5 U·mL-1 for caspase-8 with a limit of detection (LOD) of 0.2 U·mL-1 in vitro. Furthermore, this method has been successfully applied to monitoring the upregulation of intracellular caspase-8 activity caused by tert-butyl hydroperoxide (TBHP) and fluorouracil. Flow cytometry assay indicated the positive relation between the upregulation of intracellular caspase-8 activity and cell apoptosis rate. In summary, the above results demonstrated the practical application of this method for apoptosis-related cell imaging.

Dietary sodium butyrate supplementation improves fish growth, intestinal microbiota composition, and liver health in largemouth bass (Micropterus salmoides) fed high-fat diets

The present study evaluated the effect of dietary sodium butyrate (NaBT) supplementation on the growth performance, intestinal microbiota composition, and liver health of largemouth bass Micropterus salmoides fed high-fat diets. Four isonitrogenous and isolipid diets were formulated: a high-fat diet and the high-fat diet supplemented by 0.05% NaBT, 0.1% NaBT, or 0.2% NaBT. Each diet was randomly distributed to triplicate tanks (200L) containing 30 fish (initial weight 4.0 ± 0.1 g) per tank. The experimental fish were fed to apparent satiation twice daily for 60 days. The orthogonal polynomial contrasts showed that the weight gain rate (WGR) and specific growth rate (SGR) increased linearly (P < 0.05) with increasing dietary NaBT levels (P < 0.05). No significant differences were found for feed intake (FI), feed conversion ratio (FCR), condition factor (CF), viscerosomatic index (VSI), and hepatosomatic index (HSI). There were no differences in the alpha diversity indices (including Operational Taxonomic Units, Chao index, and Simpson index) of intestinal microbiota between the various dietary groups (P > 0.05). For the intestinal microbiota composition, the proportion of Proteobacteria were linearly and cubically increased, whereas the proportion of Tenericutes and Mesomycoplasma were linearly and cubically decreased with the increase of dietary NaBT levels (P < 0.05). The increasing dietary NaBT levels led to significantly quadratic decrease of alanine aminotransferase (ALT) (P < 0.05), linear and quadratic decrease of aspartate aminotransferase (AST) (P < 0.05), and linear, quadratic, and cubic decrease of diamine oxidase (DAO) (P < 0.05). Hepatic triglyceride (TG) and total cholesterol (TCHO) contents decreased linearly and quadratically with the increase of dietary NaBT levels (P < 0.05). Increasing dietary NaBT levels linearly, quadratically, and cubically decreased the malondialdehyde (MDA) contents (P < 0.05). Total superoxide dismutase (T-SOD) and glutathione peroxidase (GPx) activity levels increased linearly with the increasing dietary NaBT levels (P < 0.05). As the dietary NaBT levels gradually increased, the relative expression of carnitine palmitoyl transferase (CPT1) and peroxisome proliferator-activated receptor α (PPARα) increased linearly, quadratically, and cubically (P < 0.05), while the relative expression of sterol regulatory element binding protein 1 (SREBP 1) decreased linearly, quadratically, and cubically (P < 0.05). The relative expression of tumor necrosis factor α (TNF-α) and cysteinyl aspartate specific protease 3 (Caspase 3) decreased linearly and quadratically with the increasing NaBT levels (P < 0.05). Pearson correlation analysis showed that fish growth was closely correlated with microbiota composition, hepatic lipid accumulation, hepatic antioxidant-related parameters, and hepatic gene expression (P < 0.05). These results indicated that NaBT could be used to alleviate the negative influence of high-fat diets on fish growth, intestinal microbiota homeostasis, and liver health.

Acrylamide Induces Neurotoxicity in SH-SY5Y Cells via NLRP3-mediated Pyroptosis.

Acrylamide (ACR), a soft electrophile, is a typical environmental and food contaminant that presents potential health hazards and, consequently, is attracting increasing attention in the quest for its control. ACR neurotoxicity has been widely reported in experimental animals and attributed to neuroinflammation; however, the mechanisms involved therein require clarification. In this study, we used a neuron cell model to investigate the mechanisms of ACR-induced neuroinflammation and pyroptosis. The results showed that ACR treatment induced lytic cell death morphologically under both the canonical pyroptotic pathway (NOD-like receptor protein 3 (NLRP3)-apoptosis-associated speck-like protein containing CARD (ASC)-cysteinyl aspartate specific proteinase 1 (caspase-1)-gasdermin D (GSDMD)-interleukin-1β (IL-1β)/interleukin-18 (IL-18)) and an alternative pyroptotic pathway (cysteinyl aspartate specific proteinase 3 (caspase-3)-gasdermin E (GSDME)-IL-1β/IL-18) in SH-SY5Y cells. Moreover, the lactate dehydrogenase (LDH) production, cytokines release, and lytic cell death induced by ACR were diminished by caspase-1 and -3 inhibitors. Furthermore, the knockdown of caspase-1 by small interfering RNA attenuated ACR-induced lytic cell death, suggesting that canonical pyroptosis (the NLRP3-caspase 1-GSDMD-IL-1β signaling axis) played a primary role in the ACR-induced pyroptosis. Of the two pyroptotic-related pathways, the NLRP3 inflammasome cascade was activated first within the 6-h period of ACR exposure, while the activation of the alternative pyroptotic pathway was delayed. Collectively, these results indicate that ACR mainly induces NLRP3-related neuroinflammation and pyroptosis in SH-SY5Y cells, which is, thus, suggestive of an alternative mechanism for ACR-induced neurotoxicity.

Sini Decoction inhibits TLR4/NF-κB signaling pathway to improve airway remodeling of allergic asthmatic mice

This study aims to explore the effect of Sini Decoction on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) signaling pathway in the mice with allergic asthma(AA). Forty-eight SPF-grade BALB/c mice were randomly assigned into a blank control group, a model group, a dexamethasone group, and high-, medium-, and low-dose Sini Decoction groups, with 8 mice in each group. The sensitization solution made of ovalbumin and aluminum hydroxide powder was injected intraperitoneally in other groups except the blank control group which was injected with an equal volume of normal saline. The solution(or normal saline) was injected three times in total with an interval of 7 days. At the same time of sensitization, external cold stimulation and ice water were administered in a 4 ℃ climate box for 20 min every day. After modeling, the mice in each group were administrated with corresponding drugs by gavage for 3 weeks. At the end of administration, pentobarbital sodium(30 mg·kg~(-1)) was used for anesthesia, and then the samples were collected for the determination of various indexes. The phenol red test was conducted to evaluate tracheal excretion function. The histopathological changes of lung tissue were observed via hematoxylin-eosin(HE) staining. Masson staining was employed to reveal the deposition of blue collagen fibers around bronchi in lung tissue and the area occupied by blue collagen fibers was calculated. Immunofluorescence method was used to measure the expression of bronchial type Ⅰ collagen(Col-Ⅰ) and α-smooth muscle actin(α-SMA). The protein and mRNA levels of TLR4, NF-κB, cysteinyl aspartate specific proteinase-1(caspase-1), and interleukin-13(IL-13) were determined by Western blot and real-time fluorescence quantitative polymerase chain reaction(real-time PCR), respectively. Compared with the model group, Sini Decoction significantly increased the phenol red excretion from trachea, lowered the lung inflammation score, reduced subepithelial collagen deposition, and decreased Col-Ⅰ and α-SMA levels. Furthermore, the decoction down-regulated the protein and mRNA levels of TLR4, NF-κB, caspase-1, and IL-13 in mouse lung tissue. In conclusion, Sini Decoction can improve air remodeling by inhibiting the TLR4/NF-κB signaling pathway.

Acacetin improves cognitive function of APP/PS1 Alzheimer's disease model mice via the NLRP3 inflammasome signaling pathway.

Acacetin (5,7-dihydroxy-4'-methoxyflavone), one of the main extractions from Saussurea involucrata, has anti-inflammatory effects. Our previous study found that acacetin inhibited the Nod-like receptor pyrin domain containing 3 (NLRP3) signaling pathway after cerebral ischemia-reperfusion injury. NLRP3 inflammasome plays a role in Alzheimer's disease (AD) process. However, few studies have examined the effects of acacetin in AD. We randomly divided APP swe/PS1dE9 double transgenic mice into acacetin group (intraperitoneal injection of 25 mg/kg acacetin) and AD model group (intraperitoneal injection of same volume of saline). C57BL/6 mice were selected as control group (same treatment with AD model group). After treating for 30 days, a Morris water maze test was conducted to evaluate spatial learning and memory of the mice. Senile plaque (SP) formation was evaluated by immunohistochemistry. NLRP3 inflammasome-related inflammatory factors and amyloid-β-42 were detected by Western blot or enzyme-linked immunosorbent assay. Acacetin improved spatial learning and memory of AD mice and reduced APP/β expression, thereby decreasing SP formation in the brain. Acacetin also reduced the expression of NLRP3, cysteinyl aspartate-specific proteinase 1 (caspase-1), and interleukin-1β (IL-1β) and the release of inflammatory factors, tumor necrosis factor-α (TNF-α) and IL-1β. Acacetin improved the learning and memory abilities of AD mice and exerted a protective effect on AD by inhibiting the NLRP3 signaling pathway and reducing SP formation.

The Role of the GRP78/PERK/ATF4 Pathway in the Ability of Gua Lou Gui Zhi Decoction to Attenuate Apoptosis by Inhibiting Endoplasmic Reticulum Stress after Ischemia-Reperfusion Injury.

Endoplasmic reticulum stress (ERS) is a key part of the apoptotic cascade that is initiated after cerebral ischemia-reperfusion injury and is very important for research on poststroke rehabilitation. In addition, the unfolded protein response (UPR) plays an important role in ERS because it activates downstream apoptotic signal transduction and induces apoptosis through the glucose-regulated protein 78 (GRP78)/protein kinase R (PKR)-like ER kinase (PERK)/activating transcription factor 4 (ATF4) pathway. The Gua Lou Gui Zhi Decoction (GLGZD) ameliorated neuronal apoptosis of ischemia-reperfusion injury caused by middle cerebral artery occlusion (MCAO) had been proved in our previous study. The present study aims to underly the regulatory ability of GLGZD in ERS-induced apoptosis mediated by the GRP78/PERK/ATF4 pathway. GLGZD was analyzed by HPLC. The effects of GLGZD were obversed on MCAO-induced ischemic rats. The cerebral infarct volume was detected by 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) Staining. Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling (TUNEL) were used to detect apoptosis. Transmission Electron Microscopy (TEM), Ca2+ levels and reactive oxygen species (ROS) detection were used to determine the function of endoplasmic reticulum. The GRP78/PERK/ATF4 signaling pathway was assessed by western blotting and immunohistochemistry. Our results showed that GLGZD exerted its effects on ischemia-reperfusion injury by significantly promoting the restoration of the quantity and morphology of the rough ER and reducing the neuronal apoptosis rate in the ischemic cortex. Moreover, both of the intracellular ROS and Ca2+ levels in ischemic cortical cells were found significantly reduced by GLGZD. The GLGZD-treated group showed increased levels of phosphorylation in both of PERK and eukaryotic translation initiation factor 2α (eIF2α), activation of cysteinyl aspartate-specific proteinase-3 (Caspase-3), upregulation of the total protein levels of sarcoplasmic/endoplasmic Ca2+ ATPase 2α (SERCA 2α) and B-cell leukemia/lymphoma gene 2 (Bcl-2). These findings suggest that GLGZD reduces oxidative stress-induced injury and promotes a dynamic calcium balance, thereby inhibiting ERS and exerting an antiapoptotic effect on neuronal ischemic injury, which are closely related to the activation of GRP78/PERK/ATF4 signaling pathway.

CgCaspase-3 activates the translocation of CgGSDME in haemocytes of Pacific oyster Crassostrea gigas

Cysteinyl aspartate specific proteinase-3 (Caspase-3) is an important protein involved in the apoptosis and gasdermin E (GSDME)-mediated cell pyroptosis pathways in vertebrates. A Caspase-3 homologue (designated as CgCaspase-3) was previously identified as an immune receptor specific for lipopolysaccharide (LPS) to regulate apoptosis in the Pacific oyster Crassostrea gigas. In the present study, the binding activity of CgCaspase-3 to different pathogen associated molecular patterns (PAMPs) and its effects on CgGSDME translocation in haemocytes were further investigated in C. gigas. The mRNA expression of CgCaspase-3 could be detected in all the tested tissues, including hepatopancreas, labial palp, adductor muscle, gonad, gill, mantle and haemocytes, and it was highly expressed in labial palp, gonad, haemocytes, and adductor muscle. The mRNA expression of CgCaspase-3 in haemocytes increased significantly at 3, 24, 48 and 72 h after LPS stimulation, and it increased significantly at 6, 12, 24 and 48 h after Vibrio splendidus stimulation. The recombinant CgCaspase-3 displayed binding activity towards LPS, mannose (MAN), peptidoglycan (PGN), and polyinosinic-polycytidylic acid potassium salt (Poly (I:C)). The positive signals of CgGSDME on haemocyte membrane became stronger at 3 h after V. splendidus stimulation, compared with that of Seawater group, and the co-localization of CgCaspase-3 and CgGSDME was observed in the haemocyte membrane. After the injection of dsCgCaspase-3, the positive signals of CgGSDME on haemocyte membrane became weaker compared with that of EGFP-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgCaspase-3 was able to bind diverse PAMPs and activate the translocation of CgGSDME in haemocytes of oyster response against pathogen invasion.

Dietary Supplementation with Epicatechin Improves Intestinal Barrier Integrity in Mice

Epicatechin (EPI) is a dietary flavonoid that is present in many foods and possesses various bioactivities. We assessed the effects of EPI supplementation on intestinal barrier integrity in mice. Thirty-six mice were assigned to three groups and fed a standard diet or a standard diet supplemented with 50 or 100 mg EPI/kg (n = 12 per group). After 21 days of rearing, blood and intestinal samples were collected from eight randomly selected mice. Supplementation with 50 and 100 mg/kg EPI decreased (p < 0.05) the serum diamine oxidase activity and D-lactic acid concentration and increased (p < 0.05) the duodenal, jejunal, and ileal abundance of tight junction proteins, such as occludin. Moreover, it lowered (p < 0.05) the duodenal, jejunal, and ileal tumor necrosis factor-α contents and enhanced (p < 0.05) the duodenal and jejunal catalase activities and ileal superoxide dismutase activity. Supplementation with a lower dose (50 mg/kg) decreased (p < 0.05) the ileal interleukin-1β content, whereas supplementation with a higher dose (100 mg/kg) increased (p < 0.05) the duodenal and jejunal glutathione peroxidase activities. Furthermore, supplementation with 50 and 100 mg/kg EPI decreased (p < 0.05) cell apoptosis, cleaved cysteinyl aspartate-specific proteinase-3 (caspase-3), and cleaved caspase-9 contents in the duodenum, jejunum, and ileum. In conclusion, EPI could improve intestinal barrier integrity in mice, thereby suppressing intestinal inflammation and oxidative stress and reducing cell apoptosis.

Protective effects of glutamine on lipopolysaccharide/D-galactosamine-induced fulminant hepatitis in mice.

Fulminant hepatitis remains a critical health problem owing to its high mortality rate and the lack of effective therapies. An increasing number of studies have shown that glutamine supplementation provides protective benefits in inflammation-related disorders, but the pharmacological significance of glutamine in lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced fulminant hepatitis remains unclear. In the present study, the potential effects of glutamine on LPS/D-Gal-induced fulminant hepatitis were investigated. Pretreatment with glutamine decreased plasma activities of alanine and aspartate aminotransferases, and ameliorated hepatic morphological abnormalities in LPS/D-Gal-exposed mice. Glutamine pretreatment also inhibited LPS/D-Gal-induced tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) production. In addition, glutamine pretreatment decreased the level of cleaved cysteinyl aspartate-specific proteinase 3 (caspase-3), suppressed the activities of caspase-3, caspase-8, and caspase-9, and reduced the number of cells positive for TdT-mediated dUTP nick-end labeling in LPS/D-Gal-challenged mice. Interestingly, post-treatment with glutamine also provided protective benefits against LPS/D-Gal-induced acute liver injury, although these effects were less robust than those of glutamine pre-treatment. Thus, glutamine may have potential value as a pharmacological intervention in fulminant hepatitis.

Influence of Ginsenoside Rh2 on Cardiomyocyte Pyroptosis in Rats with Acute Myocardial Infarction.

Objective This paper intends to verify through in vivo experiments whether ginsenoside Rh2 (G-Rh2) can play an anti-inflammatory role by modulating cardiomyocyte (CM) pyroptosis in rats with acute myocardial infarction (AMI), thereby alleviating myocardial injury. Methods Twenty SD rats were randomized into control, L-Rh2, M-Rh2, and H-Rh2 groups, among which the latter three groups were modeled for AMI and given an intraperitoneal injection of G-Rh2 (L-Rh2: 2 mg/kg; M-Rh2: 4 mg/kg; H-Rh2: 8 mg/kg), while the control group was only treated with thoracotomy and sodium chloride injection. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), and left ventricular end-diastolic pressure (LVEDP) were recorded by ultrasonic diagnosis. Rats were killed under anesthesia, and the morphological characteristics of ventricular tissue were observed by electron microscope. Additionally, cardiac blood and ventricular tissues were collected to quantify the contents of myocardial injury markers (creatine phosphate kinase (CK), creatine phosphokinase-MB isoenzyme (CK-MB), and lactate dehydrogenase (LDH) by ELISA, as well as the expression of pyroptosis-related genes cysteinyl aspartate specific proteinase 1 (Caspase-1), gasdermin D (GSDMD), interleukin (IL)-1β, and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) by qRT-PCR and Western blot). Results Ultrasonic examination identified lower HR, SBP, DBP, MAP, and LVSP in the three Rh2 injection groups compared with the control group (P < 0.05); and in comparison with M- and H-Rh2 groups, HR, SBP, DBP, MAP, and LVSP were all lower in L-Rh2 group, while LVEDP was higher (P < 0.05). Microscopically, CMs and organelles in the L-RH2, M-RH2, and H-RH2 groups were damaged to varying degrees compared with the control group, with those in the L-RH2 group being the most serious. CK, CK-MB, and LDH were also the highest in the L-Rh2 group and the lowest in the control group, while their levels were obviously reduced in M- and H-Rh2 groups (P < 0.05). Finally, GSDMD, IL-1β, NLRP3, and Caspase-1 were found to be reduced in the control group, while pyroptosis-related gene expression in the M-Rh2 group was improved markedly (P < 0.05). Conclusion G-Rh2 can inhibit the pathological development of AMI by relieving the focal death of CM and inhibiting the release of proinflammatory factors in the body, and the effect is significantly related to the dosage, which is expected to become a new treatment option for AMI in the future.

Intervention effect of Jingfang Mixture on urticaria mice based on NF-κB/NLRP3/IL-1β signaling pathway

This study explored the curative effect of Jingfang Mixture on urticaria mice induced by aluminum hydroxide/ovalbumin, and discussed its mechanism. Sixty male Kunming mice were randomly divided into a normal group, a model group, three Jingfang Mixture(low-dose, medium-dose, and high-dose) groups, and a positive drug(cetirizine hydrochloride) group. The urticarial model in mice was induced by the intraperitoneal injection of the mixed solution of ovalbumin and aluminum hydroxide. The degrees of pruritus were observed after the second immunization. Pathological changes were detected by hematoxylin and eosin(HE) staining. Levels of interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) in the serum were detected by enzyme linked immunosorbent assay(ELISA). Expressions of NOD-like receptor protein 3(NLRP3) and IL-1β were detected by immunohistochemistry(IHC). Expressions of nuclear factor kappa-B(NF-κB p65), NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate-specific proteases 1(caspase-1), and IL-1β proteins were detected by Western blot. The results showed that, except for the normal group, the mice in all groups had different degrees of pruritus. Compared with the model group, the Jingfang Mixture groups and the positive drug group prolonged the scratching latency of mice(P&lt;0.05), and significantly reduced the number of scratching(P&lt;0.05). In addition, the Jingfang Mixture groups and the positive drug group improved the pathological morphology of skin tissue. The expression levels of IL-1β and TNF-α in serum were significantly reduced(P&lt;0.05), and the number of NLRP3 and IL-1β positive cells was decreased(P&lt;0.01). The expressions of p-NF-κB p65, NLRP3, ASC, cleaved caspase-1, and IL-1β protein were significantly down-regulated(P&lt;0.05). The results of the above study indicate that Jingfang Mixture inhibit the inflammatory response in urticaria mice, and the mechanism may be related to the inhibition of activating NF-κB/NLRP3/IL-1β signaling pathway.

ASIC1a induces mitochondrial apoptotic responses in acute lung injury

AimThis study aimed to investigate the promoting effect of acid-sensing ion channel 1a (ASIC1a) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and its mechanisms. Methods: In this experiment, the ALI rat model was induced by intratracheal injection of LPS, and the ASIC1a specific blocker psalmotoxin-1 (PcTx-1) was injected into the tail vein before LPS administration once. Western blot, immunofluorescence, immunohistochemistry and real-time PCR methods were used to detect ASIC1a and apoptosis-related proteins expressions in lung tissue and RLE-6TN rat type II alveolar epithelial cells. Confocal Laser Scanning Microscopy was used to detect Ca2+ fluorescence intensity in RLE-6TN cells. Results: PcTx-1 pretreatment not only inhibited the pathological changes of LPS-induced ALI in lung tissue, but also inhibited lung dysfunction. PcTx-1 also reduced the increased levels of the apoptosis-related proteins B-cell lymphoma-2-associated X (Bax) and cleaved cysteinyl aspartate specific proteinase 3 (Cleaved caspase-3) and increased the decreased level of B-cell lymphoma-2 (Bcl-2) in the lung tissue of the model group. LPS-induced changes in mitochondrial membrane potential and calcium influx in alveolar epithelial cells were also reversed by PcTx-1. Conclusion: ASIC1a induces an apoptotic response in ALI through mitochondrial apoptosis.