Cyclooxygenase-2 Protein Levels Research Articles

Phenolics and terpenoids with good anti-inflammatory activity from the fruits of Amomum villosum and the anti-inflammatory mechanism of active diterpene

The fruits of Amomum villosum are often considered a medicinal and food homologous material and have been found to have therapeutic effects in chronic enteritis, gastroenteritis, and duodenal ulcer. The aim of this study is to discover the anti-inflammatory active ingredients from dried ripe fruits of A. villosum and to elucidate the molecular mechanisms. We verified that the inhibitory activity of the ethyl acetate extract was superior to Dexamethasone (Dex), so we ultimately chose to study the ethyl acetate extract from the fruits of A. villosum. A total of 33 compounds were isolated from its ethyl acetate extract, including nine known diterpenoids (compounds 1–9), twelve known sesquiterpenoids (compounds 10–21), ten known phenolics (compounds 22, 23, 25–29, 31–33) and two new phenolics (24 and 30). On the basis of chemical evidences and spectral data analysis (UV, ECD, Optical rotation data, 1D and 2D-NMR, HR-ESI-MS, NMR chemical shift calculations), the structures of new compounds were elucidated. Among these compounds, isocoronarin D (5) was found to have good anti-inflammatory activity. Further research has found that isocoronarin D can down-regulate the protein levels of COX2 and NOS2, activate Nrf2/Keap1 and suppress NF-κB signaling pathway in LPS-induced RAW264.7 cells. In addition, isocoronarin D inhibited inflammasome assembly during inflammasome activation by hampering the binding of NLRP3 and ASC. Further evidence revealed that isocoronarin D suppressed the assembly of the NLRP3 inflammasome via blocking the formation of ASC specks. From these results, isocoronarin D may be the important bioactive compound of A. villosum and exhibits anti-inflammatory effects by regulating the NF-κB/Nrf2/NLRP3 axis in macrophages.

Xianlian Jiedu Decoction alleviates colorectal cancer by regulating metabolic profiles, intestinal microbiota and metabolites

BackgroundXianlian Jiedu Decoction (XLJDD) has been used for the treatment of colorectal cancer (CRC) for several decades because of the prominent efficacy of the prescription. Despite the clear clinical efficacy of XLJDD, the anti-CRC mechanism of action is still unclear. PurposeThe inhibitory effect and mechanism of XLJDD on CRC were investigated in the azoxymethane/dextran sulfate sodium (AOM/DSS)-induced mice. MethodsThe AOM/DSS-induced mice model was adopted to evaluate the efficacy after administering the different doses of XLJDD. The therapeutic effects of XLJDD in treating AOM/DSS-induced CRC were investigated through histopathology, immunofluorescence and ELISA analysis methods. In addition, metabolomics profile and 16S rRNA analysis were used to explore the effective mechanisms of XLJDD on CRC. ResultsThe results stated that the XLJDD reduced the number of tumor growth on the inner wall of the colon and the colorectal weight/length ratio, and suppressed the disease activity index (DAI) score, meanwhile XLJDD also increased body weight, colorectal length, and overall survival rate. The treatment of XLJDD also exhibited the ability to lower the level of inflammatory cytokines in serum and reduce the expression levels of β-catenin, COX-2, and iNOS protein in colorectal tissue. The findings suggested that XLJDD has anti-inflammatory properties and may provide relief for those suffering from inflammation-related conditions. Mechanistically, XLJDD improved gut microbiota dysbiosis and associated metabolic levels of short chain fatty acids (SCFAs), sphingolipid, and glycerophospholipid. This was achieved by reducing the abundance of Turicibacter, Clostridium_sensu_stricto_1, and the levels of sphinganine, LPCs, and PCs. Additionally, XLJDD increased the abundance of Enterorhabdus and Alistipes probiotics, as well as the content of butyric acid and isovaleric acid. ConclusionThe data presented in this article demonstrated that XLJDD can effectively inhibit the occurrence of colon inner wall tumors by reducing the level of inflammation and alleviating intestinal microbial flora imbalance and metabolic disorders. It provides a scientific basis for clinical prevention and treatment of CRC.

Isolation, structure modification, and anti-rheumatoid arthritis activity of isopimarane-type diterpenoids from Orthosiphon aristatus

Orthosiphon aristatus is a well-known folkloric medicine and herb for Guangdong soup for the treatment of rheumatism in China. Eight isopimarane-type and migrated pimarane-type diterpenoids (1-8), including a new one with a rarely occurring α,β-unsaturated diketone C-ring, were isolated from O. aristatus. Their structures were determined by spectroscopic methods and quantum chemical calculations. Furthermore, the most abundant compound, orthosiphol K, was structurally modified by modern synthetic techniques to give seven new derivatives (9-15). The anti-rheumatoid arthritis activity of these diterpenoids were evaluated on a TNF-α induced MH7A human rheumatoid fibroblast-like synoviocyte model. Compound 10 showed the most potent activity among these compounds. Based on their inhibitory effects on the release levels of IL-1β, the preliminary structure-activity relationships were concluded. Furthermore, western blot analysis revealed that 10 could increase the expression of IκBα and decrease the expression of NF-κB p65, and the expression levels of COX-2 and NLRP3 proteins were consequently down-regulated.

Modulation of autophagy and apoptosis can contribute to the anticancer effect of Abemaciclib/Celecoxib combination in colon cancer cells

Drug resistance and recurrence represent a great challenge in colorectal cancer management, highlighting the urgent need for novel therapeutics. Our objective is to evaluate the influence of Abemaciclib, Celecoxib, and their combination on both the autophagic and apoptotic machinery in an attempt to unravel the interplay between them in HCT-116 and Caco-2 cell lines. The MTT assay was used to assess the GI50 of the drugs. ELIZA was used to determine the protein levels of Beclin-1, LC3, Cox-2, and Bcl-2. Active Caspase-3 was determined by a colorimetric assay. Gene expression levels of ATG5, LC3, Beclin-1, and p62 were assessed by quantitative real-time PCR. In HCT-116 cells, the GI50s for Abemaciclib and Celecoxib were 15.86 and 92.67 μM, respectively, while for Caco-2 cells, the GI50s were 7.85 and 49.02 μM for Abemaciclib and Celecoxib, respectively. Upon treatment of HCT-116 and Caco-2 cells with Abemaciclib, Celecoxib, and their combinations, ATG5, p62, LC3, and Beclin-1 gene expression levels were up-regulated. The protein levels of Beclin-1, LC3, and Caspase-3 were significantly increased, while Bcl-2 was decreased in both cell lines due to single and combined treatments. Both drugs, either alone or in combination, decreased the migration ability of the cells in both cell lines. To conclude, the treatment protocol has the potential to induce cell cycle arrest, diminish the potentiality of cells for migration, and initiate apoptotic and autophagic cell death. Further research is recommended to unravel the potential antitumor effects of Abemaciclib/Celecoxib combination in different cancer types.

Anti-inflammatory effects of Mentha pulegium L. extract on human peripheral blood mononuclear cells are mediated by TLR-4 and NF-κB suppression

There is great interest in evaluating the anti-inflammatory properties of new herbal products. Thus, the effects of Mentha pulegium L. extract on gene and protein expressions of pro-inflammatory mediators and transcription factors were determined.The hydro-ethanolic extract of Mentha pulegium L. was obtained and optimal non-cytotoxic concentrations of the extract were determined by MTT assay. Then, three different concentrations of Mentha pulegium L. (10, 30, and 90 μg/mL) were used to pre-treat the lipopolysaccharide (LPS)-stimulated and non-stimulated peripheral blood mononuclear cells (PBMCs) of 10 healthy individuals. Finally, the tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, Toll-like receptor-4 (TLR-4), nuclear factor-kappa B (NF-κB) p65, activator protein-1 (AP-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) gene expressions and TNF-α, IL-1β, IL-6, TLR-4, prostaglandin E2 (PGE2), and COX-2 protein levels were measured.MTT results showed that there is no significant difference in cell viability among 10, 20, 40, and 80 μg/mL concentrations of Mentha pulegium L. extract at 24, 48, and 72 h (P > 0.05). The IC50 values were 236.1, 147.0, and 118.0 μg/mL after 24, 48, and 72 h respectively. TNF-α, IL-1β, IL-6, TLR-4, iNOS, and NF-κB p65 mRNA levels in the pre-treated LPS-stimulated PBMCs were concentration-dependently reduced (P < 0.01 for TNF-α, TLR-4, and NF-κB p65; P < 0.05 for IL-1β, IL-6, and iNOS). Also, the protein levels of pro-inflammatory mediators decreased and these differences were significant for TNF-α, IL-1β, and TLR-4 (P < 0.001, P < 0.01, and P < 0.001, respectively).Mentha pulegium L. extract decreased the expression and biosynthesis of pro-inflammatory mediators. These effects are mainly mediated by TLR-4 and NF-κB suppression. Thus, Mentha pulegium L. could be useful in treating or ameliorating chronic inflammatory diseases.

Protective Effect of Monoterpene Isoespintanol in a Rat Model of Prediabetes Induced by Fructose.

A high-fructose diet (HFD) induces murine alterations like those recorded in human prediabetes. Protective effects of isoespintanol (monoterpene isolated from Oxandra cf. xylopioides) on changes induced by HFD were evaluated. Animals were maintained for 21 days with a standard diet (C), 10% fructose (F), and F plus isoespintanol (FI, 10 mg/kg, i.p.). Glycemia, triglyceridemia, total and HDL-cholesterol, and insulin resistance index (IRX) were determined. Intraperitoneal glucose tolerance test (IGTT) was performed. In the liver, we measured glycogen, lipogenic gene expression (SREBP-1c, GPAT, FAS, and CPT1), oxidative stress (GSH and 3'-nitrotyrosine content), inflammation markers (iNOS, TNF-α, and PAI-1 gene expression; iNOS and COX-2 protein levels), p-eNOS, p-Akt, and p-GSK3β protein levels. Isoespintanol corrected enhanced triglycerides, lipogenic genes, and IRX, and reduced HDL-cholesterol induced by HFD. Increased liver glycogen and inflammatory markers and decreased GSH, p-Akt, and p-GSK3β measured in F rats were reversed by isoespintanol, and p-eNOS/e-NOS and iNOS/GADPH ratios were normalized. Isoespintanol restored glucose tolerance (IGTT) compared to F rats. These results demonstrate for the first time that isoespintanol prevents endocrine-metabolic alterations induced by HFD in prediabetic rats. These effects could be mediated by Akt/eNOS and Akt/GSK3β pathways, suggesting its possible use as a therapeutic tool for the prevention of diabetes at early stages of its development (prediabetes).

Danlou tablet attenuates ischemic stroke injury and blood‒brain barrier damage by inhibiting ferroptosis

Ethnopharmacological relevanceDanlou tablet (DLT) is a traditional Chinese medicinal formulation known for replenishing Qi, promoting blood circulation, and resolving stasis. Its pharmacological actions primarily involve anti-inflammatory, antioxidant stress reduction, antiapoptotic, proangiogenic, and improved energy metabolism. DLT has been confirmed to have favorable therapeutic effects on ischemic stroke (IS). However, the underlying mechanism through which DLT affects IS-induced brain injury remains unknown. Aim of the studyThis study aims to investigate the effects and underlying mechanisms of danlou tablet on ischemic stroke based on network pharmacology and experimental verification. Materials and methodsUsing a transient middle cerebral artery occlusion (tMCAO) mouse model, the impact of DLT on the blood‒brain barrier (BBB) and brain injury in mice was assessed. Network pharmacology and bioinformatics analyses were utilized to explore the potential mechanisms of DLT in treating IS. Endothelial cells were cultured to observe the effects of DLT on vascular endothelial cells after oxygen-glucose deprivation/reperfusion, and these findings were validated in the brains of tMCAO mice. ResultsDLT alleviated oxidative stress and brain damage in tMCAO mice, mitigating BBB damage. A total of 185 potential targets through which DLT regulates IS were identified, including COX2, a known critical marker of ferroptosis, which identified as a key target. In vitro and in vivo experiments demonstrated that DLT significantly (p < 0.05) improved cell death and vascular barrier damage in IS, reducing intracellular oxidative stress and COX2 protein levels while increasing SLC7A11 and GPX4 protein levels. ConclusionsThis study demonstrated that DLT maintained BBB integrity and alleviated brain injury of tMCAO mice by inhibiting ferroptosis. The study partially unraveled the mechanism through which DLT functioned in treating IS and further clarified the pivotal active components of DLT, thereby providing a theoretical scientific basis for treating IS with DLT.

Gypenoside XLIX attenuates sepsis-induced splenic injury through inhibiting inflammation and oxidative stress

BackgroundTo investigate the effect of Gypenoside XLIX (Gyp-XLIX) on acute splenic injury (ASI) induced by cecal ligation and puncture (CLP) in septic mice, a study was conducted. MethodsSixty healthy mice were randomly divided into six groups: the NC group, the Sham group, the Sham + Gyp-XLIX group, the CLP group, the CLP + Gyp-XLIX group, and the CLP + Dexamethasone (DEX) group. The NC group did not undergo any operation, while the rest of the groups underwent CLP to establish the sepsis model. The Sham group only underwent open-abdominal suture surgery without cecum puncture. After the operation, the groups were immediately administered the drug for a total of 5 days. Various methods such as hematoxylin and eosin (HE) staining, biochemical kits, qRT-PCR, and reactive oxygen species (ROS) were used for analysis. ResultsThe results demonstrated that Gyp-XLIX effectively mitigated the splenic histopathological damage, while reducing the malondialdehyde (MDA) lipid peroxidation index and enhancing the antioxidant activities of catalase (CAT), glutathione (GSH) and total antioxidant capacity (T-AOC). The utilization of Dihydroethidium (DHE) fluorescent probe revealed that Gyp-XLIX inhibited the acute splenic accumulation of ROS induced by CLP in septic mice. Further investigations revealed that Gyp-XLIX exhibited a down-regulatory effect on the protein levels of inflammatory mediators iNOS and COX-2, consequently leading to the suppression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. Additionally, it up-regulated the expression of anti-inflammatory factor IL-10. ConclusionIn conclusion, Gyp-XLIX was significantly effective in attenuating CLP-induced acute splenic inflammation and oxidative stress in septic mice.

Ginsenoside Rg1 Attenuates Eccentric Exercise-Induced Muscle Damage via the Modulation of Lipid Peroxidation and Inflammation

Eccentric exercise (EE) may lead to skeletal muscle injury, including oxidative stress and inflammation induction. Ginsenoside Rg1, a glycosylated triterpene present in the traditional Chinese medicine ginseng, was previously shown to prevent the development of multiple diseases through the attenuation of oxidative stress and inflammation. Therefore, this article hopes to investigate whether Rg1 exhibits anti-oxidant and anti-inflammatory effects in eccentric exercise-induced muscle damage (EEIMD). Additionally, Adult male Wistar rats were intraperitoneally injected with Rg1 (20 or 40 mg/kg) every day before EE for 5 consecutive days. The impact of Rg1 administration on levels of serum creatine kinase was evaluated, followed by observation of histological muscle damage through H&amp;E staining. To assess protein nitrotyrosylation, lipid peroxidation and leukocyte infiltration in rat skeletal muscles, the levels of nitrotyrosine, MDA and MPO protein were analysed through western blotting analysis. The inflammatory response was evaluated by detecting iNOS, COX-2, IL-1β, IL-6, MCP-1 and TNF-α mRNA and protein levels in rat skeletal muscles. The regulation of Rg1 on the NF-κB pathway was examined through the analysis of phosphorylated NF-κB p65 and IκBα protein levels. Result display, EE resulted in elevated serum creatine kinase levels, widespread leukocyte infiltration, and notable muscle cell vacuolization and fragmentation in muscles. Furthermore, EE increased nitrotyrosine, MDA, MPO, iNOS, COX-2, IL-1β, IL-6, MCP-1, and TNF-α levels in rats. However, these changes were reversed by Rg1 treatment. Furthermore, EE-induced upregulation in phosphorylated NF-κB p65 and IκBα levels was counteracted by Rg1. Overall, ginsenoside Rg1 plays an anti-oxidant and anti-inflammatory role in EEIMD through suppressing this NF-κB signaling pathway.

Synthesis of 9-Cinnamyl-9H-purine Derivatives as Novel TLR4/MyD88/NF-κB Pathway Inhibitors for Anti-inflammatory Effects.

The novel 9-cinnamyl-9H-purine skeleton, inspired by resveratrol and curcumin, was developed to avoid a pan-assay interference compound (PAINS) related to invalid metabolic pancreas activity (IMPS). It replaced the phenol group with purine analogues, the building blocks of DNA and RNA. Alterations to the hydroxyl group in the cinnamyl group, such as H, Me, or F substitutions, were made to impede its oxidation to a PAINS-associated quinone. Among the compounds tested, 5e significantly inhibited nitric oxide production in LPS-induced macrophages (IC50: 6.4 vs 26.4 μM for resveratrol). 5e also reduced pro-inflammatory cytokine levels (IL-6, TNF-α, IL-1β) and lowered iNOS and COX-2 protein levels. Mechanistically, 5e disrupted the TLR4-MyD88 protein interaction, leading to the suppression of the NF-κB signaling pathway suppression. In an atopic dermatitis mouse model, 5e reduced ear edema and inflammation. These findings indicate that the novel 9-cinnamyl-9H-purine skeleton provides therapeutic insight into treating various human diseases by regulating inflammation.

Akt, IL-4, and STAT Proteins Play Distinct Roles in Prostaglandin Production in Human Follicular Dendritic Cell-like Cells.

This study aimed to explore the role of Akt protein in the induction and inhibition of prostaglandin (PG) in human follicular dendritic cell (FDC)-like cells. FDC-like cells and B cells were isolated from human tonsils. PG production was assessed using enzyme immunoassay, while the upstream cyclooxygenase-2 (COX-2) protein levels were measured using immunoblotting with FDC-like cells transfected with Akt siRNA to analyze the impact of Akt knockdown. The COX-2 expression and PG production induced with IL-1β were significantly increased by Akt knockdown. However, IL-1β did not significantly alter either total or phosphorylated Akt protein levels. Akt knockdown resulted in the augmentation of COX-2 expression induced by B cells, although the addition of B cells did not significantly modulate both total and phosphorylated Akt proteins. In contrast, IL-4 specifically exhibited a potent inhibitory effect on COX-2 protein induction and PG production via STAT6. The inhibitory activity of IL-4 was not hampered by Akt knockdown. Interestingly, COX-2 expression levels induced with IL-1β were markedly modulated with STAT1 and STAT3 knockdown. STAT1 silencing resulted in further augmentation of COX-2, whereas STAT3 silencing prohibited IL-1β from stimulating COX-2 expression. The current results suggest that Akt, IL-4, and STAT1 play inhibitory roles in PG production in FDC-like cells and expand our knowledge of the immune inflammatory milieu.

Pathogenesis of chronic heart failure in rats based on ferroptosis-mediated oxidative stress and intervention effect of Shenfu Injection

This study aims to investigate the pathogenesis of chronic heart failure based on ferroptosis-mediated oxidative stress and predict the targets of Shenfu Injection in treating chronic heart failure. A rat model of chronic heart failure was established by the isoproterenol induction method. According to the random number table method, the modeled rats were assigned into three groups: a model group, a Shenfu Injection group, and a ferrostatin-1(ferroptosis inhibitor) group. In addition, a normal group was designed. After 15 days of intervention, the cardiac mass index and left ventricular mass index were determined. Echocardiography was employed to eva-luate the cardiac function. Hematoxylin-eosin staining and Masson staining were employed to reveal the pathological changes and fibrosis of the heart, and Prussian blue staining to detect the aggregation of iron ions in the myocardial tissue. Transmission electron microscopy was employed to observe the mitochondrion ultrastructure in the myocardial tissue. Colorimetry was adopted to measure the levels of iron metabolism, lipid peroxidation, and antioxidant indicators. Flow cytometry was employed to measure the content of lipid-reactive oxygen species(ROS) and the fluorescence intensity of ROS. Western blot and RT-qPCR were employed to determine the protein and mRNA levels, respectively, of ferroptosis-related factors in the myocardial tissue. The results showed that the rats in the model group had reduced cardiac function, elevated levels of total iron and Fe~(2+), lowered level of glutathione(GSH), increased malondialdehyde(MDA), decreased superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px), and rising levels of ROS and lipid-ROS. In addition, the model group showed fibrous tissue hyperplasia with inflammatory cell infiltration and myocardial fibrosis, iron ion aggregation, and characteristic mitochondrial changes specific for iron death. Moreover, the model group showcased upregulated protein and mRNA levels of p53 and COX2 and downregulated protein and mRNA levels of GPX4, FTH1, SLC7A11, and Nrf2 in the myocardial tissue. The intervention with Shenfu Injection significantly improved the cardiac function, recovered the iron metabolism, lipid peroxidation, and antioxidant indicators, decreased iron deposition, improved mitochondrial structure and function, and alleviated inflammatory cell infiltration and fibrosis. Furthermore, Shenfu Injection downregulated the mRNA and protein levels of p53 and COX2 and upregulated the mRNA and protein levels of GPX4, FTH1, SLC7A11, and Nrf2 in the myocardial tissue. Shenfu Injection can improve the cardiac function by regulating iron metabolism, inhibiting ferroptosis, and reducing oxidative stress injury.

"Nulichal" Barley Extract Suppresses Nitric Oxide and Pro-Inflammatory Cytokine Production by Lipopolysaccharides in RAW264.7 Macrophage Cell Line.

The cultivar "Nulichal," a type of naked waxy barley (Hordeum vulgare L.), was developed by the National Institute of Crop Science, Rural Development Administration, Korea, in 2010. In this study, we investigated the anti-inflammatory and antioxidant properties of the "Nulichal" ethanol extract (NRE) using various assays. The NRE exhibited a total phenolic content of 7.55±0.30 mg gallic acid equivalent/g and a flavonoid content of 1.74±0.08 mg rutin equivalent/g. Cell viability assays showed no toxicity of NRE on RAW264.7 macrophage cells up to concentrations of 500 μg/mL. The NRE (300 and 500 μg/mL) significantly reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). It also down-regulated the mRNA expression and protein levels of inducible NO synthase and cyclooxygenase-2 in a dose-dependent manner. Moreover, the NRE treatment significantly decreased the levels of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6, and their mRNA expression compared to LPS treatment alone. The NRE demonstrated strong free radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals in a dose-dependent manner. The ferric reducing antioxidant power assay also showed increased antioxidant activity with increasing NRE concentrations. These findings suggest that the NRE can be used as a functional food with anti-inflammatory and antioxidant properties.

Rumex crispus Leaf Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in BV-2 Microglia Cells

Background: Microglial cells are immune cells that operate within the central nervous system. Abnormally activated microglia cause neuroinflammation, which is linked with neurodegenerative disease. Previous research has revealed that Rumex crispus root extract exerts anti-inflammatory effects. However, it is not known whether Rumex crispus leaf extract (RLE) has anti-inflammatory effects on murine microglial cells, such as BV-2 cells. This study proposed to investigate the impact of RLE on inducing inflammation by LPS in BV-2 cells. Methods: LPS was used to induce inflammation in BV-2 cells, and then cell survival, changes in the levels of inflammation-related factors and pro-inflammatory cytokines, and NF-κB and MAPKs signaling pathway activity were evaluated in the presence or absence of RLE. Results: RLE treatment resulted in a reduction in nitric oxide (NO) production triggered by LPS without causing cytotoxic effects. In addition, both protein and mRNA expression levels of iNOS and COX-2, which were upregulated by LPS, were significantly decreased by RLE. Also, RLE effectively reduced the transcriptional expression and further suppressed the increased production of inflammatory cytokines by LPS stimulation. Additionally, RLE effectively suppressed the inflammatory response of BV-2 cells stimulated by LPS via interference with NF-κB and MAPK signaling pathways. Conclusions: Taken together, our results confirm the effective suppression of the inflammatory response induced by LPS in BV-2 cells by RLE. Consequently, we suggest that RLE holds promise as a preventive agent against diseases triggered by microglial inflammatory responses.

Bear bile powder alleviates Parkinson's disease-like behavior in mice by inhibiting astrocyte-mediated neuroinflammation.

Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. In particular, increasing evidence has showed that astrocyte-mediated neuroinflammation is involved in the pathogenesis of PD. As a precious traditional Chinese medicine, bear bile powder (BBP) has a long history of use in clinical practice. It has numerous activities, such as clearing heat, calming the liver wind and anti-inflammation, and also exhibits good therapeutic effect on convulsive epilepsy. However, whether BBP can prevent the development of PD has not been elucidated. Hence, this study was designed to explore the effect and mechanism of BBP on suppressing astrocyte-mediated neuroinflammation in a mouse model of PD. PD-like behavior was induced in the mice by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg·kg-1) for five days, followed by BBP (50, 100, and 200 mg·kg-1) treatment daily for ten days. LPS stimulated rat C6 astrocytic cells were used as a cell model of neuroinflammation. THe results indicated that BBP treatment significantly ameliorated dyskinesia, increased the levels of tyrosine hydroxylase (TH) and inhibited astrocyte hyperactivation in the substantia nigra (SN) of PD mice. Furthermore, BBP decreased the protein levels of glial fibrillary acidic protein (GFAP), cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS), and up-regulated the protein levels of takeda G protein-coupled receptor 5 (TGR5) in the SN. Moreover, BBP significantly activated TGR5 in a dose-dependent manner, and decreased the protein levels of GFAP, iNOS and COX2, as well as the mRNA levels of GFAP, iNOS, COX2, interleukin (IL) -1β, IL-6 and tumor necrosis factor-α (TNF-α) in LPS-stimulated C6 cells. Notably, BBP suppressed the phosphorylation of protein kinase B (AKT), inhibitor of NF-κB (IκBα) and nuclear factor-κB (NF-κB) proteins in vivo and in vitro. We also observed that TGR5 inhibitor triamterene attenuated the anti-neuroinflammatory effect of BBP on LPS-stimulated C6 cells. Taken together, BBP alleviates the progression of PD mice by suppressing astrocyte-mediated inflammation via TGR5.

Dietary emodin alleviates lipopolysaccharide-induced intestinal mucosal barrier injury by regulating gut microbiota in piglets.

This study was to determine the effects of dietary emodin (ED) on the intestinal mucosal barrier, nuclear factor kappa-B (NF-κB) pathways, and gut microbial flora in lipopolysaccharide (LPS)-induced piglets. Twenty-four weaned piglets were chosen and 4 treatments were created by randomly distributing piglets into CON, ED, LPS, and ED_LPS groups. Experiments were done in a 2×2 factorial arrangement and maintained for 21d. Dietary treatment (a basal diet or 300mg/kg ED) and immunological challenge (LPS or sterile saline) were 2 major factors. Intraperitoneal injections of LPS or sterilized saline were given to piglets on d 21. Six hours after the LPS challenge, all piglets were euthanized for sample collection and analysis. The results showed that piglets of the ED_LPS group had higher (P<0.05) villus height to crypt depth ratio (VCR), and lower (P<0.05) plasma D-lactate and diamine oxidase (DAO) than the LPS group. Furthermore, ED inhibited (P<0.05) the decrease of glutathione peroxidase (GSH-Px) and catalase (CAT) activities and increase of malonaldehyde level (P<0.05) in jejunal mucosa induced by LPS. The mRNA levels of pro-inflammatory cytokine genes (IL-6, IL-1β, and TNF-α) were significantly reduced (P<0.05), and the mRNA levels of antioxidant enzyme genes (GPX-1, SOD2 and CAT), as well as protein and mRNA levels of tight junction proteins (occludin, claudin-1, and ZO-1), were also significantly increased (P<0.05) by ED addition in LPS-induced piglets. Meanwhile, ED supplementation significantly decreased the LPS-induced protein levels of cyclooxygenase-2 and phosphorylation levels of NF-κB p65 and IκBα in jejunal mucosa. Emodin had a significant effect on the composition of gut microbial flora at various taxonomic positions as indicated by 16S RNA sequencing. The acetic acid, isobutyric acid, valeric acid, and isovaleric acid concentrations in the cecum were also increased by ED addition in pigs (P<0.05). Furthermore, the correlation analysis revealed that some intestinal microbiota had a potential relationship with jejunal VCR, plasma D-lactate and DAO, jejunal mucosa GSH-Px and CAT activity, and cecal short-chain fatty acid concentration. These data suggest that ED is effective in alleviating LPS-induced intestinal mucosal barrier injury by modulating gut microbiota in piglets.

Cocktail of three isoquinoline alkaloids derived from Glaucium grandiflorum Boiss. & A. Huet subsp. refractum (Nábelek) Mory inhibits the production of LPS-induced ROS, pro-inflammatory cytokines, and mediators through the down-regulation of p38 MAPK in BV-2 cells

Glaucium grandiflorum extracts have traditionally been used to treat brain-related disorders. G. grandiflorum extracts also exhibited inhibitory effects on cholinesterase enzymes, as well as antigenotoxic activity. However, no research has been done on the effect of G. grandiflorum alkaloid extracts on the anti-oxidative and anti-inflammatory mechanisms. In this study we aimed to evaluate the anti-oxidative and anti-inflammatory activities of the alkaloid extract obtained from G. grandiflorum as well as the mechanisms responsible for their neuroprotective effects in neuronal damage caused by LPS in BV2 cells. We used LC-MS/MS and 1H, 13C NMR analysis to determine the presence of major alkaloids (allocryptopine, tetrahydropalmatine, and tetrahydroberberine N-oxide (trans-cannadine-N-oxide) in the alkaloid extracts. We used flow cytometry to study the alkaloid extracts' effects on ROS production; we also employed qRT-PCR and Western Blot to analyze the effects of oxidative stress and inflammation-related genes and proteins. ROS production within the cell was inhibited by chloroform alkaloid extract (CAE). There occurred marked CAE-induced reductions in IL-1β, Cox-2, and iNOS mRNA expressions. We also observed marked reductions in IL-6 and TNF-α mRNA expressions with methanol alkaloid extract (MAE). CAE effectively suppressed IL-1β and iNOS protein levels, especially as in qRT-PCR studies, while MAE effectively reduced IL-6 and TNF-α protein levels. Additionally, MAE was found to be prominent in suppressing the levels of Cox-2 protein, unlike qRT-PCR studies. According to our study findings, oxidative stress brought about by inflammation was suppressed by alkaloid extracts from G. grandiflorum which can be attributed to their suppressor effects on the pro-inflammatory cytokines-mediators, and p38 MAPK. As a result, a drug active substance that suppresses oxidative stress and inflammation has been brought to the neuropharmacological field.

Anti-Inflammatory Effects of Serotonin Receptor and Transient Receptor Potential Channel Ligands in Human Small Intestinal Epithelial Cells.

Intestinal inflammation and dysbiosis can lead to inflammatory bowel diseases (IBD) and systemic inflammation, affecting multiple organs. Developing novel anti-inflammatory therapeutics is crucial for preventing IBD progression. Serotonin receptor type 2A (5-HT2A) ligands, including psilocybin (Psi), 4-Acetoxy-N,N-dimethyltryptamine (4-AcO-DMT), and ketanserin (Ket), along with transient receptor potential (TRP) channel ligands like capsaicin (Cap), curcumin (Cur), and eugenol (Eug), show promise as anti-inflammatory agents. In this study, we investigated the cytotoxic and anti-inflammatory effects of Psi, 4-AcO-DMT, Ket, Cap, Cur, and Eug on human small intestinal epithelial cells (HSEIC). HSEIC were exposed to tumor necrosis factor (TNF)-α and interferon (IFN)-γ for 24 h to induce an inflammatory response, followed by treatment with each compound at varying doses (0-800 μM) for 24 to 96 h. The cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and protein expression by Western blot (WB) analysis. As single treatments, Psi (40 μM), Cur (0.5 μM), and Eug (50 μM) significantly reduced COX-2 levels without cytotoxic effects. When combined, Psi (40 μM) and Cur (0.5 μM) exhibited synergy, resulting in a substantial decrease in COX-2 protein levels (-28× fold change), although the reduction in IL-6 was less pronounced (-1.6× fold change). Psi (20 μM) and Eug (25 μM) demonstrated the most favorable outcomes, with significant decreases in COX-2 (-19× fold change) and IL-6 (-10× fold change) protein levels. Moreover, the combination of Psi and Eug did not induce cytotoxic effects in vitro at any tested doses. This study is the first to explore the anti-inflammatory potential of psilocybin and 4-AcO-DMT in the intestines while highlighting the potential for synergy between the 5-HT2A and TRP channel ligands, specifically Psi and Eug, in alleviating the TNF-α/IFN-γ-induced inflammatory response in HSEIC. Further investigations should evaluate if the Psi and Eug combination has the therapeutic potential to treat IBD in vivo.

Growth hormone-releasing hormone antagonist MIA-602 inhibits inflammation induced by SARS-CoV-2 spike protein and bacterial lipopolysaccharide synergism in macrophages and human peripheral blood mononuclear cells.

COVID-19 is characterized by an excessive inflammatory response and macrophage hyperactivation, leading, in severe cases, to alveolar epithelial injury and acute respiratory distress syndrome. Recent studies have reported that SARS-CoV-2 spike (S) protein interacts with bacterial lipopolysaccharide (LPS) to boost inflammatory responses in vitro, in macrophages and peripheral blood mononuclear cells (PBMCs), and in vivo. The hypothalamic hormone growth hormone-releasing hormone (GHRH), in addition to promoting pituitary GH release, exerts many peripheral functions, acting as a growth factor in both malignant and non-malignant cells. GHRH antagonists, in turn, display potent antitumor effects and antinflammatory activities in different cell types, including lung and endothelial cells. However, to date, the antinflammatory role of GHRH antagonists in COVID-19 remains unexplored. Here, we examined the ability of GHRH antagonist MIA-602 to reduce inflammation in human THP-1-derived macrophages and PBMCs stimulated with S protein and LPS combination. Western blot and immunofluorescence analysis revealed the presence of GHRH receptor and its splice variant SV1 in both THP-1 cells and PBMCs. Exposure of THP-1 cells to S protein and LPS combination increased the mRNA levels and protein secretion of TNF-α and IL-1β, as well as IL-8 and MCP-1 gene expression, an effect hampered by MIA-602. Similarly, MIA-602 hindered TNF-α and IL-1β secretion in PBMCs and reduced MCP-1 mRNA levels. Mechanistically, MIA-602 blunted the S protein and LPS-induced activation of inflammatory pathways in THP-1 cells, such as NF-κB, STAT3, MAPK ERK1/2 and JNK. MIA-602 also attenuated oxidative stress in PBMCs, by decreasing ROS production, iNOS and COX-2 protein levels, and MMP9 activity. Finally, MIA-602 prevented the effect of S protein and LPS synergism on NF-кB nuclear translocation and activity. Overall, these findings demonstrate a novel antinflammatory role for GHRH antagonists of MIA class and suggest their potential development for the treatment of inflammatory diseases, such as COVID-19 and related comorbidities.

Isolation and Identification of a Novel Anti-Dry Eye Peptide from Tilapia Skin Peptides Based on In Silico, In Vitro, and In Vivo Approaches.

Tilapia skin is a great source of collagen. Here, we aimed to isolate and identify the peptides responsible for combating dry eye disease (DED) in tilapia skin peptides (TSP). In vitro cell DED model was used to screen anti-DED peptides from TSP via Sephadex G-25 chromatography, LC/MS/MS, and in silico methods. The anti-DED activity of the screened peptide was further verified in the mice DED model. TSP was divided into five fractions (TSP-I, TSP-II, TSP-III, TSP-IV, and TSP-V), and TSP-II exerted an effective effect for anti-DED. A total of 131 peptides were identified using LC/MS/MS in TSP-II, and NGGPSGPR (NGG) was screened as a potential anti-DED fragment in TSP-II via in silico methods. In vitro, NGG restored cell viability and inhibited the expression level of Cyclooxygenase-2 (COX-2) protein in Human corneal epithelial cells (HCECs) induced by NaCl. In vivo, NGG increased tear production, decreased tear ferning score, prevented corneal epithelial thinning, alleviated conjunctival goblet cell loss, and inhibited the apoptosis of corneal epithelial cells in DED mice. Overall, NGG, as an anti-DED peptide, was successfully identified from TSP, and it may be devoted to functional food ingredients or medicine for DED.