Shizukaol C alleviates trimethylamine oxide-induced inflammation through activating Keap1-Nrf2-GSTpi pathway in vascular smooth muscle cell.

BACKGROUND: Cardiovascular disease is one of the main causes of global mortality, and there is an urgent need for effective treatment strategies. Gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) promotes the development of cardiovascular diseases, and shizukaol C, a natural sesquiterpene isolated from Chloranthus multistachys with various biological activities, might exhibit beneficial role in preventing TMAO-induced vascular inflammation. PURPOSE: The purpose of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of shizukaol C on TMAO-induced vascular inflammation. METHODS: The effect and underlying mechanism of shizukaol C on TMAO-induced adhesion molecules expression, bone marrow-derived macrophages (BMDM) adhesion to VSMC were evaluated by western blot, cell adhesion assay, co-immunoprecipitation, immunofluorescence assay, and quantitative Real-Time PCR, respectively. To verify the role of shizukaol C in vivo, TMAO-induced vascular inflammation model were established using guidewire-induced injury on mice carotid artery. Changes in the intima area and the expression of GSTpi, VCAM-1, CD68 were examined using haematoxylin-eosin staining, and immunofluorescence assay. RESULTS: Our data demonstrated that shizukaol C significantly suppressed TMAO-induced adhesion molecule expression and the bone marrow-derived macrophages (BMDM) adhesion in vascular smooth muscle cells (VSMC). Mechanically, shizukaol C inhibited TMAO-induced c-Jun N-terminal kinase (JNK)-nuclear factor-kappa B (NF-κB)/p65 activation, and the JNK inhibition was dependent on the shizukaol C-mediated glutathione-S-transferase pi (GSTpi) expression. By further molecular docking and protein-binding analysis, we demonstrated that shizukaol C directly binds to Keap1 to induce Nrf2 nuclear translocation and upregulated GSTpi expression. Consistently, our in vivo experiment showed that shizukaol C elevated the expression level of GSTpi in carotid arteries and alleviates TMAO-induced vascular inflammation. CONCLUSION: Shizukaol C exerts anti-inflammatory effects in TMAO-treated VSMC by targeting Keap1 and activating Nrf2-GSTpi signaling and resultantly inhibits the downstream JNK-NF-κB/p65 activation and VSMC adhesion, and alleviates TMAO-induced vascular inflammation in vivo, suggesting that shizukaol C may be a potential drug for treating TMAO-induced vascular diseases.

Anti-inflammatory effects and related mechanisms in vitro and in vivo of Hedychium coccineum rhizome essential oil.

ETHNOPHARMACOLOGICAL RELEVANCE: Hedychium coccineum rhizome is an anti-inflammatory ethnomedicine used to remedy inflammation-related swelling and bronchial asthma. AIM OF THE STUDY: The study aimed to analyze the phytochemical constituents of H. coccineum rhizome essential oil (EO) and evaluate its in vitro and in vivo anti-inflammatory effects and underlying mechanisms. MATERIALS AND METHODS: Phytochemical constituents of H. coccineum rhizome EO were analyzed using GC-FID/MS. In RAW264.7 macrophages induced by LPS, blockade of PGE2, NO, IL-1ß, IL-6, and TNF-α secretion by H. coccineum rhizome EO was measured, and then Western blot, qRT-PCR, and immunofluorescent staining were used to evaluate its underlying mechanisms. Moreover, we used the xylene-induced ear edema model for testing anti-inflammatory potential in vivo and examined auricular swelling as well as tissue and serum contents of IL-1ß, IL-6, and TNF-α. RESULTS: EO's main components were E-nerolidol (40.5%), borneol acetate (24.8%), spathulenol (4.5%), linalool (3.8%), elemol (3.5%), and borneol (3.4%). In RAW264.7 cells stimulated by LPS, EO downregulated the expression of pro-inflammatory enzyme (iNOS and COX-2) genes and proteins, thereby suppressing pro-inflammatory mediators (NO and PGE2) secretion. Simultaneously, it reduced TNF-α, IL-1ß, and IL-6 release by downregulating their mRNA expression. Besides, H. coccineum EO attenuated LPS-stimulated activation of NF-κB (by reducing IκBα phosphorylation and degradation to inhibit NF-κB nuclear translocation) and MAPK (by downregulating JNK, p38, and ERK phosphorylation). In xylene-induced mouse ear edema, EO relieved auricular swelling and lowered serum and tissue levels of TNF-α, IL-1ß, and IL-6. CONCLUSIONS: H. coccineum EO had powerful in vivo and in vitro anti-inflammatory effects by inhibiting MAPK and NF-κB activation. Hence, H. coccineum EO should have great potential for application in the pharmaceutical field as a novel anti-inflammatory agent.

Omega-3 fatty acids for inflamed depression - A match/mismatch study.

Despite decades of research on the pathophysiology of depression, the development of new therapeutic interventions has been slow, and no biomarkers of treatment response have been clinically implemented. Several lines of evidence suggest that the clinical and biological heterogeneity among patients with major depressive disorder (MDD) has hampered progress in this field. MDD with low-grade inflammation - "inflamed depression" - is a subtype of depression that may be associated with a superior antidepressant treatment response to anti-inflammatory compounds. Omega-3 fatty acid eicosapentaenoic acid (EPA) has anti-inflammatory properties, and preliminary data suggest that it may be particularly efficacious in inflamed depression. In this study we tested the hypothesis that add-on EPA has greater antidepressant efficacy in MDD patients with high baseline high-sensitivity C-reactive protein (hs-CRP) compared to MDD patients with low hs-CRP. All subjects received 2.2 g EPA, 400 mg docosahexaenoic acid and 800 mg of other fatty acids daily for 8 weeks, added to stable ongoing antidepressant treatment. The primary outcome was change in the 17-item Hamilton Depression Rating Scale (HAMD-17). Patients and raters were blind to baseline hs-CRP status. In an intention-to-treat analysis including all subjects with at least one post baseline visit (n = 101), ahs-CRPcut-off of ≥1 mg/L, but not ≥3 mg/L, was associated with a greater improvement in HAMD-17 total score. In addition to a general antidepressant effect among patients with hs-CRP ≥ 1 mg/L, adjuvant EPA treatment improved symptoms putatively related to inflamed depression such as fatigue and sleep difficulties. This adds to the mounting evidence that delineation of MDD subgroups based on inflammation may be clinically relevant to predict treatment response to anti-inflammatory interventions.

A therapeutic strategy of parthenolide in improving imiquimod-induced psoriasis-like skin inflammation targeting IL-36/NETs through skin transdermal therapeutic system.

BACKGROUND: Psoriasis is an inflammatory skin disease that occurs repeatedly over time. The natural product of sesquiterpene lactones, Parthenolide (Par), is isolated from Tanacetum parthenium L. (feverfew) which has significant effects on anti-inflammatory. The therapeutic effect of the medication itself is crucial, but different routes of administration of the same drug can also produce different effects. PURPOSE: The aim of our research sought to investigate the ameliorating effects of Par in psoriasis-like skin inflammation and its related mechanism of action. RESULTS: In the IMQ-induced model, intragastric administration of Par reduced the Psoriasis Area and Severity Index (PASI) score, improved skin erythema, scaling, and other symptoms. And Par decreased the expression of Ki67, keratin14, keratin16 and keratin17, and increased the expression of keratin1. Par could reduce IL-36 protein expressions, meanwhile the expression of Il1b, Cxcl1 and Cxcl2 mRNA were also decreased. Par regulated the expression levels of F4/80, MPO and NE. However, skin transdermal administration of Par was more effective. Similarly, Par attenuated IL-36γ, IL-1ß and caspase-1 activated by Poly(I:C) in in vitro and ex vivo. In addition, Par also reduced NE, PR3, and Cathepsin G levels in explant skin tissues. CONCLUSION: Par ameliorated psoriasis-like skin inflammation in both in vivo and in vitro, especially after treatment with transdermal drug delivery, possibly by inhibiting neutrophil extracellular traps and thus by interfering IL-36 signaling pathway. It indicated that Par provides a new research strategy for the treatment of psoriasis-like skin inflammation and is expected to be a promising drug.

Investigation the mechanism of iron overload-induced colonic inflammation following ferric citrate exposure.

Iron overload occurs due to excessive iron intake compared to the body's demand, leading to iron deposition and impairment of multiple organ functions. Our previous study demonstrated that chronic oral administration of ferric citrate (FC) caused colonic inflammatory injury. However, the precise mechanism underlying this inflammatory response remains unclear. The current study aims to investigate the mechanism by which iron overload induced by FC exposure leads to colonic inflammation. To accomplish this, mice were orally exposed to three different concentrations of FC (71 mg/kg/bw (L), 143 mg/kg/bw (M) and 286 mg/kg/bw (H)) for continuous 16 weeks, with the control group receiving ultrapure water (C). Exposure to FC caused disturbances in the excretory system, altered colonic flora alpha diversity, and enriched pathogenic bacteria, such as Mucispirillum, Helicobacter, Desulfovibrio, and Shigella. These changes led to structural disorders of the colonic flora and an inflammatory response phenotype characterized by inflammatory cells infiltration, atrophy of intestinal glands, and irregular thickening of the intestinal wall. Mechanistic studies revealed that FC-exposure activated the NF-κB signaling pathway by up-regulating TLR4, MyD88, and NF-κB mRNA levels and protein expression. This activation resulted in increased production of pro-inflammatory cytokines, further contributing to the colonic inflammation. Additionally, in vitro experiments in SW480 cells confirmed the activation of NF-κB signaling pathway by FC exposure, consistent with the in vivo findings. The significance of this study lies in its elucidation of the mechanism by which iron overload caused by FC exposure leads to colonic inflammation. By identifying the role of pathogenic bacteria and the NF-κB signaling pathway, this study could potentially offer a crucial theoretical foundation for the research on iron overload, as well as provide valuable insights for clinical iron supplementation.

The effects of vitamin D supplementation on inflammatory biomarkers in patients with asthma: a systematic review and meta-analysis of randomized controlled trials.

Background: While the association between vitamin D and several inflammatory biomarkers in asthma patients has been extensively reported, it remains unclear whether supplementation modifies these biomarkers. This review aims to evaluate the impact of vitamin D supplementation on inflammatory biomarkers measured in vivo in individuals with asthma. Methods: We conducted a systematic review of randomized controlled trials (RCTs) published until November 2022 in six electronic databases evaluating the impact of vitamin D supplementation (any dose, form, administration route, frequency, or duration) compared to placebo in children or adults. The two co-primary outcomes were serum IgE and blood eosinophils reported at the endpoint. Secondary outcomes included other markers of type 2 inflammation (e.g., sputum eosinophils, fractional exhaled nitric oxide, etc.), anti-inflammatory biomarkers (e.g., interleukin (IL)-10, etc.), markers of non-type 2 inflammation (e.g., high-sensitivity C-reactive protein, etc.), and non-specific biomarkers (e.g., macrophages, etc.). Data were aggregated using fixed or random effect models. Results: Thirteen RCTs (5 in adults, 5 in pediatric patients, and 3 in mixed age groups) testing doses of vitamin D supplementation ranging from 800 to 400,000 IU over periods of 6 weeks to 12 months were included. Eight studies provided data on serum IgE and four on blood eosinophils. As secondary outcomes, three studies reported on sputum eosinophils, four on FeNO, five on serum IL-10, and two on airway IL-10. Compared to placebo, vitamin D supplementation had no significant effect on serum IgE (Mean difference [MD] [95% CI]: 0.06 [-0.13, 0.26] IU/mL), blood eosinophils (MD [95% CI]: - 0.02 [-0.11, 0.07] 103/µL), or FeNO (MD [95% CI]: -4.10 [-10.95, 2.75] ppb) at the endpoint. However, the vitamin D supplementation group showed higher serum IL-10 levels compared to placebo (MD [95% CI]: 18.85 [1.11, 36.59] pg/ml) at the endpoint. Although data could not be aggregated, narrative synthesis suggested no significant effect of supplementation on sputum eosinophils and IL-10 in both sputum and exhaled breath condensate, at the endpoint. Conclusion: Vitamin D supplementation in individuals with asthma was not associated with lower inflammatory biomarkers related to type 2 inflammation. However, it was significantly associated with higher serum IL-10 compared to placebo. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42022365666.

Agaro-oligosaccharides mitigate deoxynivalenol-induced intestinal inflammation by regulating gut microbiota and enhancing intestinal barrier function in mice.

Agarose-derived agaro-oligosaccharides (AgaroS) have been extensively studied in terms of structures and bioactivities; they reportedly possess antioxidant and anti-inflammatory activities that maintain intestinal homeostasis and host health. However, the protective effects of AgaroS on deoxynivalenol (DON)-induced intestinal dysfunction remain unclear. We investigated the effects of AgaroS on DON-induced intestinal dysfunction in mice and explored the underlying protective mechanisms. In total, 32 mice were randomly allocated to four treatments (n = 8 each) for 28 days. From day 1 to day 21, the control (CON) and DON groups received oral phosphate-buffered saline (200 µL per day); the AgaroS and AgaroS + DON groups received 200 mg AgaroS per kg body weight once daily by orogastric gavage. Experimental intestinal injury was induced by adding DON (4.8 mg per kg body weight) via gavage from day 21 to day 28. Phosphate-buffered saline was administered once daily by gavage in the CON and AgaroS groups. Herein, AgaroS supplementation led to a higher final body weight and smaller body weight loss and a lower concentration of plasma inflammatory cytokines, compared with the DON group. The DON group showed a significantly reduced ileal villus height and villus height/crypt depth, compared with the CON and AgaroS + DON groups. However, AgaroS supplementation improved DON-induced intestinal injury in mice. Compared with the DON group, ileal and colonic protein expression levels of claudin, occludin, Ki67, and mucin2 were significantly higher in the AgaroS supplementation group. Colonic levels of the anti-inflammatory cytokine IL-1ß tended to be higher in the DON group than in the AgaroS + DON group. AgaroS altered the gut microbiota composition, accompanied by increased production of short-chain fatty acids in mice. In conclusion, our findings highlight a promising anti-mycotoxin approach whereby AgaroS alleviate DON-induced intestinal inflammation by modulating intestinal barrier functional integrity and gut microbiota in mice.

Orally fast dissolving α-lipoic acid electrospun nanofibers mitigates lipopolysaccharide induced inflammation in RAW 264.7 macrophages.

α-Lipoic acid (LA), a dietary supplement known for its strong antioxidant and anti-inflammatory potential, faces challenges due to its poor aqueous solubility and thermal instability. To address these issues, herein methyl-beta-cyclodextrin (M-ß-CD) was utilized to create inclusion complex (IC) of LA in 1:1 M stoichiometric ratio of M-ß-CD to LA. The LA-M-ß-CD-IC was further combined with pullulan (PUL), a non-toxic and water-soluble biopolymer, for the development of electrospun nanofibers (NF) by green and sustainable approach. The resulting PUL/LA/M-ß-CD NF formed as a self-standing and flexible material with an average diameter of 569 ± 129 nm and encapsulation efficiency of ∼86.90 %. The developed NF demonstrated an accelerated release, quick dissolution, and disintegration when exposed to artificial saliva replicating the conditions of oral cavity. PUL/LA/M-ß-CD NF attenuated the production of ROS and NO by downregulating pro-inflammatory enzymes (iNOS and COX-2) in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Moreover, PUL/LA/M-ß-CD NF also significantly downregulated the expression of pro-inflammatory cytokines including TNF-α, IL-6, and IL-1ß along with suppression of NF-ĸB nuclear translocation in comparison to LA (at 250 µM). In nutshell, PUL/LA/M-ß-CD NF demonstrated great potential as a rapid disintegrating delivery system for oral anti-inflammatory treatment due to the enhanced physicochemical characteristics of LA.

Diosgenin protects against cationic bovine serum albumin-induced membranous glomerulonephritis by attenuating oxidative stress and renal inflammation via the NF-κB pathway.

CONTEXT: Membranous glomerulonephritis (MGN) is a leading cause of nephrotic syndrome in adults. Diosgenin (DG) has been reported to exert antioxidative and anti-inflammatory effects. OBJECTIVE: To investigate the renoprotective activity of DG in a cationic bovine serum albumin-induced rat model of MGN. MATERIALS AND METHODS: Fourty male Sprague-Dawley rats were randomized into four groups. The MGN model was established and treated with a DG dose (10 mg/kg) and a positive control (TPCA1, 10 mg/kg), while normal control and MGN groups received distilled water by gavage for four consecutive weeks. At the end of the experiment, 24 h urinary protein, biochemical indices, oxidation and antioxidant levels, inflammatory parameters, histopathological examination, immunohistochemistry and immunoblotting were evaluated. RESULTS: DG significantly ameliorated kidney dysfunction by decreasing urinary protein (0.56-fold), serum creatinine (SCr) (0.78-fold), BUN (0.71-fold), TC (0.66-fold) and TG (0.73-fold) levels, and increasing ALB (1.44-fold). DG also reduced MDA (0.82-fold) and NO (0.83-fold) levels while increasing the activity of SOD (1.56-fold), CAT (1.25-fold), glutathione peroxidase (GPx) (1.55-fold) and GSH (1.81-fold). Furthermore, DG reduced Keap1 (0.76-fold) expression, Nrf2 nuclear translocation (0.79-fold), and induced NQO1 (1.25-fold) and HO-1 (1.46-fold) expression. Additionally, DG decreased IL-2 (0.55-fold), TNF-α (0.80-fold) and IL-6 (0.75-fold) levels, and reduced protein expression of NF-κB p65 (0.80-fold), IKKß (0.93-fold), p-IKKß (0.89-fold), ICAM-1 (0.88-fold), VCAM-1 (0.91-fold), MCP-1 (0.88-fold) and E-selectin (0.87-fold), and also inhibited the nuclear translocation of NF-κB p65 (0.64-fold). DISCUSSION AND CONCLUSIONS: The results suggest a potential therapeutic benefit of DG against MGN due to the inhibition of the NF-κB pathway, supporting the need for further clinical trials.

Gentiopicroside ameliorates the lipopolysaccharide-induced inflammatory response and hypertrophy in chondrocytes.

PURPOSE: This study aimed to evaluate the protective effects of gentiopicroside against lipopolysaccharide-induced chondrocyte inflammation. METHODS: SW 1353 chondrosarcoma cells were stimulated with LPS (5 µg/ml) for 24 h and treated with different concentrations of gentiopicroside (GPS) for 24 h. The toxic effects of GPS on chondrocytes were determined using a CCK-8 assay and EdU staining. Western blotting, qPCR, and immunofluorescence analysis were used to examine the protective effect of GPS against the inflammatory response in chondrocytes induced by lipopolysaccharide (LPS). One-way ANOVA was used to compare the differences between the groups (significance level of 0.05). RESULTS: The CCK-8 results showed that 10, 20 and 40 µM GPS had no significant toxic effects on chondrocytes; GPS effectively reduced the production of IL-1ß and PGE2, reversed LPS-induced extracellular matrix degradation in cartilage by inhibiting the Stat3/Runx2 signaling pathway, and suppressed the hypertrophic transformation of SW 1353 chondrosarcoma cells. CONCLUSION: Our study demonstrated that GPS significantly inhibited the LPS-induced inflammatory response and hypertrophic cellular degeneration in SW 1353 chondrosarcoma cells and is a valuable traditional Chinese medicine for the treatment of knee osteoarthritis.

Oxyimperatorin attenuates LPS-induced microglial activation in vitro and in vivo via suppressing NF-κB p65 signaling.

BACKGROUND: Microglia-mediated neuroinflammation is an important pathological feature in many neurological diseases; thus, suppressing microglial activation is considered a possible therapeutic strategy for reducing neuronal damage. Oxyimperatorin (OIMP) is a member of furanocoumarin, isolated from the medicinal herb Glehnia littoralis. However, it is unknown whether OIMP can suppress the neuroinflammation. PURPOSE: To investigate the neuroprotective activity of oxyimperatorin (OIMP) in LPS-induced neuroinflammation in vitro and in vivo models. METHODS: In vitro inflammation-related assays were performed with OIMP in LPS-induced BV-2 microglia. In addition, intraperitoneal injection of LPS-induced microglial activation in the mouse brain was used to validate the anti-neuroinflammatory activity of OIMP. RESULTS: OIMP was found to suppress LPS-induced neuroinflammation in vitro and in vivo. OIMP significantly attenuated LPS-induced the production of free radicals, inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokines in BV-2 microglia without causing cytotoxicity. In addition, OIMP could reduce the M1 pro-inflammatory transition in LPS-stimulated BV-2 microglia. The mechanistic study revealed that OIMP inhibited LPS-induced NF-κB p65 phosphorylation and nuclear translocation. However, OIMP did not affect LPS-induced IκB phosphorylation and degradation. In addition, OIMP also was able to reduce LPS-induced microglial activation in mice brain. CONCLUSION: Our findings suggest that OIMP suppresses microglia activation and attenuates the production of pro-inflammatory mediators and cytokines via inhibition of NF-κB p65 signaling.

The azole biocide climbazole induces oxidative stress, inflammation, and apoptosis in fish gut.

Climbazole is an azole biocide that has been widely used in formulations of personal care products. Climbazole can cause developmental toxicity and endocrine disruption as well as gut disturbance in aquatic organisms. However, the mechanisms behind gut toxicity induced by climbazole still remain largely unclear in fish. Here, we evaluate the gut effects by exposing grass carp (Ctenopharyngodon idella) to climbazole at levels ranging from 0.2 to 20 µg/L for 42 days by evaluating gene transcription and expression, biochemical analyses, correlation network analysis, and molecular docking. Results showed that climbazole exposure increased cyp1a mRNA expression and ROS level in the three treatment groups. Climbazole also inhibited Nrf2 and Keap1 transcripts as well as proteins, and suppressed the transcript levels of their subordinate antioxidant molecules (cat, sod, and ho-1), increasing oxidative stress. Additionally, climbazole enhanced NF-κB and iκBα transcripts and proteins, and the transcripts of NF-κB downstream pro-inflammatory factors (tnfα, and il-1ß/6/8), leading to inflammation. Climbazole increased pro-apoptosis-related genes (fadd, bad1, and caspase3), and decreased anti-apoptosis-associated genes (bcl2, and bcl-xl), suggesting a direct reaction to apoptosis. The molecular docking data showed that climbazole could form stable hydrogen bonds with CYP1A. Mechanistically, our findings suggested that climbazole can induce inflammation and oxidative stress through CYP450s/ROS/Nrf2/NF-κB pathways, resulting in cell apoptosis in the gut of grass carp.

Electroacupuncture promotes macrophage/microglial M2 polarization and suppresses inflammatory pain through AMPK.

Inflammatory pain, the most prevalent disease globally, remains challenging to manage. Electroacupuncture emerges as an effective therapy, yet its underlying mechanisms are not fully understood. This study investigates whether adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-regulated silent information regulator 1 (SIRT1) contributes to electroacupuncture's antinociceptive effects by modulating macrophage/microglial polarization in the spinal dorsal horn of a mouse model of inflammatory pain. In this study, mice, introduced to inflammatory pain through subcutaneous injections of complete freund's adjuvant (CFA) in the plantar area, underwent electroacupuncture therapy every alternate day for 30-min sessions. The assessment of mechanical allodynia and thermal hyperalgesia in these subjects was carried out using paw withdrawal frequency and paw withdrawal latency measurements, respectively. Western blot analysis measured levels of AMPK, phosphorylation-adenosine 5'-monophosphate (AMP)-activated protein kinase, SIRT1, inducible nitric oxide synthase, cluster of differentiation 86, arginase 1, and interleukin 10. In contrast to the group treated solely with CFA, the cohort receiving both CFA and electroacupuncture demonstrated notable decreases in both thermal hyperalgesia and mechanical allodynia. This was accompanied by a marked enhancement in AMPK phosphorylation levels. AMPK knockdown reversed electroacupuncture's analgesic effects and reduced M2 macrophage/microglial polarization enhancement. Additionally, AMPK knockdown significantly weakened electroacupuncture-induced SIRT1 upregulation, and EX-527 injection attenuated electroacupuncture's facilitation of M2 macrophage/microglial polarization without affecting AMPK phosphorylation levels. Furthermore, combining electroacupuncture with SRT1720 enhanced the analgesic effect of SRT1720. Our findings suggest that AMPK regulation of SIRT1 plays a critical role in electroacupuncture's antinociceptive effect through the promotion of M2 macrophage/microglial polarization.

Effect and mechanism of Prunella vulgaris L. extract on alleviating lipopolysaccharide-induced acute mastitis in protecting the blood-milk barrier and reducing inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: According to ancient literature, Prunella vulgaris L. (P vulgaris) alleviates mastitis and has been used in China for many years; however, there are no relevant reports that confirm this or the mechanism of its efficacy. AIM OF THE STUDY: To explore the anti-acute mastitis effect and potential mechanism of P vulgaris extract. MATERIALS AND METHODS: First, the active ingredients and targets of P vulgaris against mastitis were predicted using network pharmacology. Next, the relevant active ingredients were enriched using macroporous resins and verified using UV and UPLC-Q-TOF-MS/MS. Lastly, a mouse model of acute mastitis was established by injecting lipopolysaccharides into the mammary gland and administering P vulgaris extract by oral gavage. The pathological changes in mammary tissue were observed by HE staining. Serum and tissue inflammatory factors were measured by ELISA method. MPO activity in mammary tissue was measured using colorimetry and MPO expression was detected by immunohistochemistry. The expression of tight junction proteins (ZO-1, claudin-3, and occludin) in mammary tissue was detected by immunofluorescence and Western blot. iNOS and COX-2 in mammary tissue were detected by Western blot. MAPK pathway and NF-κB pathway related proteins were also detected by Western blot. RESULTS: Network pharmacology predicted that phenolic acids and flavonoids in P vulgaris had anti-mastitis effects. The contents of total flavonoids and total phenolic acids in P vulgaris extract were 64.5% and 29.4%, respectively. UPLC-Q-TOF-MS/MS confirmed that P vulgaris extract contained phenolic acids and flavonoids. The results of animal experiments showed that P vulgaris extract reduced lipopolysaccharide-induced inflammatory edema, inflammatory cell infiltration, and interstitial congestion of mammary tissue. It also reduced the levels of serum and tissue inflammatory factors TNF-α, IL-6, and IL-1ß, and inhibited the activation of MPO. Furthermore, it downregulated the expression of MAPK and NF-κB pathway-related proteins. The expressions of ZO-1, occludin, and claudin-3 in mammary gland tissues were upregulated. CONCLUSIONS: P vulgaris extract can maintain the integrity of mammary connective tissue and reduce its inflammatory response to prevent acute mastitis. Its mechanism probably involves regulating NF-κB and MAPK pathways.

Zinc glycinate alleviates LPS-induced inflammation and intestinal barrier disruption in chicken embryos by regulating zinc homeostasis and TLR4/NF-κB pathway.

The effect of an immune challenge induced by a lipopolysaccharide (LPS) exposure on systemic zinc homeostasis and the modulation of zinc glycinate (Zn-Gly) was investigated using a chicken embryo model. 160 Arbor Acres broiler fertilized eggs were randomly divided into 4 groups: CON (control group, injected with saline), LPS (LPS group, injected with 32 µg of LPS saline solution), Zn-Gly (zinc glycinate group, injected with 80 µg of zinc glycinate saline solution) and Zn-Gly+LPS (zinc glycinate and LPS group, injected with the same content of zinc glycinate and LPS saline solution). Each treatment consisted of eight replicates of five eggs each. An in ovo feeding procedure was performed at 17.5 embryonic day and samples were collected after 12 hours. The results showed that Zn-Gly attenuated the effects of LPS challenge-induced upregulation of pro-inflammatory factor interleukin 1ß (IL-1ß) level (P =0.003). The LPS challenge mediated zinc transporter proteins and metallothionein (MT) to regulate systemic zinc homeostasis, with increased expression of the jejunum zinc export gene zinc transporter protein 1 (ZnT-1) and elevated expression of the import genes divalent metal transporter 1 (DMT1), Zrt- and Irt-like protein 3 (Zip3), Zip8 and Zip14 (P < 0.05). A similar trend could be observed for the zinc transporter genes in the liver, which for ZnT-1 mitigated by Zn-Gly supplementation (P =0.01). Liver MT gene expression was downregulated in response to the LPS challenge (P =0.004). These alterations caused by LPS resulted in decreased serum and liver zinc levels and increased small intestinal, muscle and tibial zinc levels. Zn-Gly reversed the elevated expression of the liver zinc finger protein A20 induced by the LPS challenge (P =0.025), while Zn-Gly reduced the gene expression of the pro-inflammatory factors IL-1ß and IL-6, decreased toll-like receptor 4 (TLR4) and nuclear factor kappa-B p65 (NF-κB p65) (P < 0.05). Zn-Gly also alleviated the LPS-induced downregulation of the intestinal barrier gene Claudin-1. Thus, LPS exposure prompted the mobilization of zinc transporter proteins and MT to perform the remodeling of systemic zinc homeostasis, Zn-Gly participated in the regulation of zinc homeostasis and inhibited the production of pro-inflammatory factors through the TLR4/NF-κB pathway, attenuating the inflammatory response and intestinal barrier damage caused by an immune challenge.

Hydroethanolic extract of Gentiana kurroo Royle rhizome ameliorates ethanol-induced liver injury by reducing oxidative stress, inflammation and fibrogenesis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo Royle is a medicinal plant mentioned as Traymana in Ayurveda. In the folklore, it is used to cure fever, stomach ache, skin diseases and liver disorders. However, limited reports are available on the therapeutic potential of Gentiana kurroo Royle against alcohol-induced liver damage. AIM OF THE STUDY: To assess the effectiveness of the hydroethanolic extract of Gentiana kurroo Royle rhizome (GKRE) against alcohol-induced liver injury and explore the mechanism of action. MATERIALS AND METHODS: GKRE was characterized using UHPLC-QTOF-MS/MS. The binding affinity of the identified compound was studied in silico. In vitro studies were performed in the Huh-7 cell line. An acute oral toxicity study (2 g/kg BW) of GKRE was done in rats following OECD 420 guidelines. In the efficacy study, rats were treated with 50% ethanol (5 mL/kg BW, orally) for 4 weeks, followed by a single intraperitoneal dose of CCl4 (30%; 1 mL/kg BW) to induce liver injury. After 4th week, the rats were treated with GKRE at 100, 200 and 400 mg/kg BW doses for the next fifteen days. The biochemical and antioxidant parameters were analyzed using commercial kits and a biochemistry analyzer. Histopathology, gene and protein expressions were studied using qRT PCR and western blotting. RESULTS: Thirteen compounds were detected in GKRE. Few compounds showed a strong interaction with the fibrotic and inflammatory proteins in silico. GKRE reduced (p < 0.05) the ethanol-induced ROS production and inflammation in Huh-7 cells. The acute oral toxicity study revealed no adverse effect of GKRE in rats at 2 g/kg BW. GKRE improved (p < 0.05) the body and liver weights in ethanol-treated rats. GKRE improved (p < 0.05) the mRNA levels of ADH, SREBP1c and mitochondrial biogenesis genes in the liver tissues. GKRE also improved (p < 0.05) the liver damage markers, lipid peroxidation and levels of antioxidant enzymes in the liver. A reduced severity (p < 0.05) of pathological changes, fibrotic tissue deposition and caspase 3/7 activity were observed in the liver tissues of GKRE-treated rats. Further, GKRE downregulated (p < 0.05) the expression of fibrotic (TGFß, αSMA and SMADs) and inflammatory markers (TNFα, IL6, IL1ß and NFκB) in the liver. CONCLUSION: GKRE showed efficacy against alcohol-induced liver damage by inhibiting oxidative stress, apoptosis, inflammation and fibrogenesis in the liver.

The water-soluble subfraction from Artemisia argyi alleviates LPS-induced inflammatory responses via multiple pathways and targets in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine, Artemisia argyi has been used medicinally and eaten for more than 2000 years in China. It is widely reported in treating inflammatory diseases such as eczema, dermatitis, arthritis, allergic asthma and colitis. Although several studies claim that its volatile oil and organic reagent extracts have certain anti-inflammatory effects, the water-soluble fractions and molecular mechanisms have not been studied. AIM OF THE STUDY: To evaluate the therapeutic effect of A. argyi water extract (AAWE) on lipopolysaccharide (LPS)-induced inflammatory responses and to identify the most effective water-soluble subfractions. Moreover, the relevant pharmacological and molecular mechanisms by which the active subfraction mitigates inflammation were further investigated. MATERIALS AND METHODS: Firstly, RAW 264.7 cells stimulated with LPS were treated with AAWE (50, 100, and 200 µg/mL) or the water-soluble subfractions separated by D101 macroporous resin (AAWE1-AAWE4, 100 µg/mL), and NO production and mRNA levels of inflammatory genes were evaluated to determine the most effective water-soluble subfractions. Secondly, the chemical components of the active subfraction (AAWE4) were analyzed by UPLC-QTOF-MS. Thirdly, transcriptome and network pharmacology analysis, RT-qPCR and Western blotting assays were conducted to explore the underlying anti-inflammatory mechanism and active compounds of AAWE4. Subsequently, the binding ability of the potential active components in AAWE4 to the core targets was further determined by molecular docking. Eventually, the in vivo anti-inflammatory activity of AAWE4 (1.17, 2.34 and 4.68 g/kg, administered per day for 7 d) was evaluated in mice with LPS-induced systemic inflammation. RESULTS: In this study, AAWE showed excellent anti-inflammatory effects, and its water-soluble subfraction AAWE4 exhibited the strongest inhibitory effect on NO concentration and inflammatory gene mRNA expression after LPS stimulation, indicating that it was the most effective subfraction. Thereafter, four main compounds in AAWE4 were confirmed or tentatively identified by UPLC-QTOF-MS, including three flavonoid glycosides and one phenolic acid. Furthermore, the transcriptome and network pharmacology analysis showed that AAWE4 inhibited inflammation via multiple pathways and multiple targets. Based on the RT-qPCR and Western blotting results, AAWE4 downregulated not only the p38, PI3K, CCL5, MMP9, AP-1, and BCL3 mRNA expression levels activated by LPS but also their upstream and downstream protein expression levels and protein phosphorylation (p-AKT/AKT, p-p38/p38, p-ERK/ERK, p-JNK/JNK). Moreover, four identified compounds (isochlorogenic acid A, vicenin-2, schaftoside and isoschaftoside) could significantly inhibit NO content and the overexpression of inflammatory factors TNF-α, IL-1ß, iNOS and COX-2 mRNA induced by LPS, and the molecular docking confirmed the high binding activity of four active compounds with selected core targets (p38, AKT1, MMP9, and CCL5). In addition, the mRNA expression and immunohistochemical analysis showed that AAWE44 could inhibit lung inflammation via multiple pathways and multiple targets in vivo. CONCLUSIONS: The findings of this study suggest that the water-soluble subfraction AAWE4 from A. argyi ameliorated the inflammation caused by LPS through multiple pathways and multiple targets in vitro and in vivo, providing scientific support for the medicinal use of A. argyi. Importantly, it shows that the A. argyi subfraction AAWE4 can be developed as an anti-inflammatory drug.

Therapeutic detoxification of quercetin for aflatoxin B1-related toxicity: Roles of oxidative stress, inflammation, and metabolic enzymes.

Aflatoxins (AFTs), a type of mycotoxin mainly produced by Aspergillus parasiticus and Aspergillus flavus, could be detected in food, feed, Chinese herbal medicine, grain crops and poses a great threat to public health security. Among them, aflatoxin B1 (AFB1) is the most toxic one. Exposure to AFB1 poses various health risks to both humans and animals, including the development of chronic inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, and cancer. The molecular mechanisms underlying these risks are intricate and dependent on specific contexts. This review primarily focuses on summarizing the protective effects of quercetin, a natural phenolic compound, in mitigating the toxic effects induced by AFB1 in both in vitro experiments and animal models. Additionally, the review explores the molecular mechanisms that underlie these protective effects. Quercetin has been demonstrated to not only have the direct inhibitory action on the production of AFTs from Aspergillus, both also possess potent ameliorative effects against AFB1-induced cytotoxicity, hepatotoxicity, and neurotoxicity. These effects are attributed to the inhibition of oxidative stress, mitochondrial dysfunction, mitochondrial apoptotic pathway, and inflammatory response. It could also directly target several metabolic enzymes (i.e., CYP3As and GSTA1) to reduce the production of toxic metabolites of AFB1 within cells, then reduce AFB1-induced cytotoxicity. In conclusion, this review highlights quercetin is a promising detoxification agent for AFB1. By advancing our understanding of the protective mechanisms offered by quercetin, we aim to contribute to the development of effective detoxification agents against AFB1, ultimately promoting better health outcomes.

Isolation, invitro, invivo anti-inflammatory, analgesic and antioxidant potential of Habenaria plantegania Lindl.

Habenaira plantaginea belong to orchid family which is native to Asia. Members of this family are commonly famous for the cure of pain and inflammation. To date, no research was found on isolation of compounds from this plant for the treatment of inflammation and analgesia nor has been published to our knowledge. The purpose of this study was to evaluate an analgesic, anti-inflammatory and anti-oxidant activity of the isolated compound from the most potent chloroform sub-fraction and the isolated compounds form the habenaria plantaginea. Anti-inflammatory analgesic and antioxidant potential of the various chloroform sub-fractions and isolated compounds from the most potent sub-fraction (HP-1 & HP-1) were screened for their in vitro enzymatic assays. Furthermore, prior to in-vivo investigation, the isolated compounds were subjected for their toxicity study. The potent compound was then examined for acetic acid-induced writhing, hot plate test, carrageenan-induced inflammation assays. Further various phlogistic agents were used for the evaluation of mechanism. In the COX-2 inhibitory assay the chloroform sub fraction Cf-4 demonstrated excellent activity as compared to the other sub-fraction with 92.15% inhibition. The COX-2 enzyme make prostaglandins which are directly involved in inflammation. Likewise against 5-LOX the Cf-4 was the most potent sub-fraction with IC50 3.77 µg/mL. The 5-LOX catalyzes the biosynthesis of leukotrienes which is a group of lipid mediators of inflammation derived from arachidonic acid. Free radicals can induce inflammation through cellular damage while chronic inflammation generates a large number of free radicals, whose eventually lead to inflammation. In antioxidant assays the Cf-4 fraction was displayed excellent results against ABTS, DPPH and H2O2 free radical with 88.88, 77.44, and 65.52% inhibition at highest concentration. Likewise, the compound HP-1 demonstrated 88.81, 89.34 and 80.43% inhibition while compound HP-2 displayed 84.34, 91.52 and 82.34% inhibition against ABTS, DPPH and H2O2 free radical which were comparable to the standard drug ascorbic acid respectively. This study's findings validate the use of this species as traditional use.

Ameliorative effects of Atriplex crassifolia (C.A.Mey) on pain and inflammation through modulation of inflammatory biomarkers and GC-MS-based metabolite profiling.

Atriplex crassifolia (A. crassifolia) is a locally occurring member of Chenopodiaceae family that has been used in folk medicine for the treatment of joint pain and inflammation. The present study was focused to determine the analgesic and anti-inflammatory potential of the plant. n-hexane (ACNH) and methanol (ACM) extracts of A. crassifolia were evaluated for in vitro anti-inflammatory potential using protein denaturation inhibition assay. In vivo anti-inflammatory potential was determined by oral administration of 250, 500, and 1000 mg/kg/day of extracts against carrageenan and formalin-induced paw edema models. Inflammatory mediators such as TNF-α, IL-10, IL-1ß, NF-kB, IL-4, and IL-6 were estimated in blood samples of animals subjected to formalin model of inflammation. Analgesic activity was determined using acetic acid-induced writhing and tail flick assay model. Phytochemical profiling was done by GC-mass spectrophotometer. The results of in vitro anti-inflammatory activity revealed that both ACNH and ACM displayed eminent inhibition of protein denaturation in concentration-dependent manner. In acute in vivo carrageenan-induced paw edema model, both extracts reduced inflammation at 5th and 6th hour of study (p < 0.05). A. crassifolia extracts exhibited significant inhibition against formalin-induced inflammation with maximum effect at 1000 mg/kg. ACNH and ACM significantly augmented the inflammatory mediators (p < 0.05). Levels of TNF-α, IL-6, IL-1ß, and NF-kB were reduced, while those of IL-4 and IL-10 were upregulated. ACNH displayed maximum analgesic effect at 1000 mg/kg, while ACM showed potent activity at 500 and 1000 mg/kg. The extracts restored the CBC, TLC and CRP toward normal. GC-MS analysis revealed the presence of compounds like n-hexadecanoic acid, Phytol, (9E,11E)-octadecadienoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester, 1-hexacosene, vitamin E, campesterol, stigmasterol, gamma sitosterol in both extracts. These compounds have been reported to suppress inflammation by inhibiting inflammatory cytokines. The current study concludes that A. crassifolia possesses significant anti-nociceptive and anti-inflammatory potential owing to the presence of phytochemicals.