We have previously reported that long-term treatment of beta cells with interleukin-6 (IL-6) is pro-apoptotic. However, little is known about the regulatory mechanisms that are involved. Therefore, we investigated pro-apoptotic changes in mRNA expression in beta cells in response to IL-6 treatment. We analyzed a microarray with RNA from INS-1 beta cells treated with IL-6, and found that TNF-α mRNA was significantly upregulated. Inhibition of TNF-α expression by neutralizing antibodies significantly decreased annexin V staining in cells compared with those treated with a control antibody. We identified three microRNAs that were differentially expressed in INS-1 cells incubated with IL-6. In particular, miR-181c was significantly downregulated in IL-6-treated cells compared with control cells and the decrease of miR-181c was attenuated by STAT-3 signaling inhibition. TNF-α mRNA was a direct target of miR-181c and upregulation of miR-181c by mimics, inhibited IL-6-induced increase in TNF-α mRNA expression. Consequently, reduction of TNF-α mRNA caused by miR-181c mimics enhanced cell viability in IL-6 treated INS-1 cells. These results demonstrated that miR-181c regulation of TNF-α expression plays a role in IL-6-induced beta cell apoptosis.

Atractylodin is one of the major constituents of the rhizome of Atractylodes lancea, which is widely used in Korean traditional medicine as a remedy for the treatment of gastritis and gastric ulcers. Despite of a major constituent of widely used botanical to treat inflammatory responses little is known about anti-inflammatory effect of atractylodin in the human mast cell (HMC-1). Hence, we evaluated the effect of atractylodin on the release of IL-6, the involvement of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) and mitogen-activated protein kinases (MAPKs) in phorbol-12-myristate-13-acetate and A23187-induced HMC-1. In addition, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), phospholipase C (PLC) gamma 1, and AKT phosphorylation relevant to NPM-ALK signal pathway were assessed. IL-6 levels in the HMC-1 stimulated by phorbol-12-myristate-13-acetate and A23187 were apparently decreased by the treatment of atractylodin. Concurrently, atractylodin not only inhibited the phosphorylation of NPM-ALK, but also suppressed the phosphorylation of JAK2, STAT3, PLC gamma 1, and AKT. Furthermore, the activated mitogen-activated protein kinases (MAPKs) by phorbol-12-myristate-13-acetate and A23187 were inhibited by atractylodin. These results suggested that atractylodin might have a potential regulatory effect on inflammatory mediator expression through blockade of both the phosphorylation of MAPKs and the NPM-ALK signaling pathway.

Interleukin-6 (IL-6) is involved in the pathogenesis of various inflammatory diseases, like rheumatoid arthritis (RA). In the present study, we investigated the role of IL-6 in osteoarthritis (OA) patients and the effects of the stilbenoids monomethyl pinosylvin and pinosylvin on the expression of the cartilage matrix components aggrecan and collagen II and the inflammatory cytokine IL-6 in human OA chondrocytes. Synovial fluid and plasma samples were obtained from 100 patients with severe OA [BMI 29.7 (8.3) kg/m², age 72 (14) years, median (IQR); 62/38 females/males] undergoing total knee replacement surgery. IL-6 and matrix metalloproteinase (MMP) concentrations in synovial fluid and plasma were measured by immunoassay. The effects of pinosylvin on the expression of IL-6, aggrecan, and collagen II were studied in primary cultures of human OA chondrocytes. IL-6 levels in synovial fluid from OA patients [119.8 (193.5) pg/mL, median (IQR)] were significantly increased as compared to the plasma levels [3.1 (2.7) pg/mL, median (IQR)] and IL-6 levels in synovial fluid were associated with MMPs and radiographic disease severity. Natural stilbenoids monomethyl pinosylvin and pinosylvin increased aggrecan expression and suppressed IL-6 production in OA chondrocytes. The results propose that IL-6 is produced within OA joints and has an important role in the pathogenesis of OA. Stilbenoid compounds monomethyl pinosylvin and pinosylvin appeared to have disease-modifying potential in OA chondrocytes.

In the past 25 years, a number of efforts have been made toward the development of small molecule interleukin-6 (IL-6) signaling inhibitors, but none have been approved to date. Monosaccharides are a diverse class of bioactive compounds, but thus far have been unexplored as a scaffold for small molecule IL-6-signaling inhibitor design. Therefore, in this present communication, we combined a structure-based drug design approach with carbohydrate building blocks to design and synthesize novel IL-6-signaling inhibitors targeting glycoprotein 130 (gp130). Of this series of compounds, LS-TG-2P and LS-TF-3P were the top lead compounds, displaying IC50 values of 6.9 and 16 µM against SUM159 cell lines, respectively, while still retaining preferential activity against the IL-6-signaling pathway. The carbohydrate moiety was found to improve activity, as N-unsubstituted triazole analogues of these compounds were found to be less active in vitro compared to the leads themselves. Thus, LS-TG-2P and LS-TF-3P are promising scaffolds for further development and study as IL-6-signaling inhibitors.

Development of small molecules that inhibit inflammatory cytokines is a desirable strategy for the treatment of inflammatory diseases such as rheumatoid arthritis (RA). Following up a previous study, we synthesized 10 novel compounds with a 2,5-diaminobenzoxazole moiety and evaluated their biological activities. Among them, compound 3e showed potent inhibitory activity on Interleukin 6 (IL-6)/Signal Transducer and Activator of Transcription 3 (STAT3) signaling inhibition (71.5%), and 3a showed excellent inhibitory activity on Interleukin 1 (IL-1β) (92.1%). To test in vivo anti-inflammatory activity, compounds 3a and 3e were administered by intraperitoneal (IP) injection after subcutaneous (SC) injection of zymosan A into the right footpad of mice. Inflammation on the footpad was reduced after administration of compounds 3a and 3e. Especially, compound 3a showed a significant ameliorative effect on zymosan-induced inflammation. From the in vivo and in vitro test results, we confirmed that our synthesized compounds are effective on the RA animal model through inhibition of the IL-6/STAT3 signaling pathway. Since drugs developed with small molecule inhibitors have several advantages over biological drugs, further study on these compounds is needed for the development of potent SMI drugs on RA.

Bioactivity-guided fractionation for the stems of leaves of Larrea nitida Cav., using interleukin-6 (IL-6) inhibitory assay in human mast cells (HMC-1), led to the isolation of three new compounds with an unprecedented skeleton in nature (1-3) and three known compounds (4-6). Their structures were elucidated through extensive spectroscopic analysis. The three new compounds were elucidated as two new spiroketones, nitidaones A (1), and B (2) and one new biphenyl analog, nitidaol (3). The known compounds were identified as nordihydroguaiaretic acid (4), 7,3',4'-tri-O-methylquercetin (5) and ayanin (6). All the isolates were tested for their inhibitory activity against IL-6 production in HMC-1 cells. Of them, compounds 1, 3-6 showed potent anti-inflammatory activity, with IC50 values of 12.8, 17.5, 14.9, 22.9, and 17.8 µM, respectively.

Akebia saponin D (ASD) is derived from the Dipsacus asper Wall. ex Henry, which is a traditional Chinese medicine commonly used to treat rheumatic arthritis (RA). However, the in-depth mechanism of the anti-inflammatory effect of ASD is still unclear. This study aimed to preliminarily explore the anti-inflammatory effect of ASD and the underlying mechanisms from the perspective of DNA methylation and inflammation-related pathways. We found that ASD significantly reduced the production of multiple inflammatory mediators, including nitric oxide (NO) and prostaglandin E2 (PGE2), in LPS-induced RAW264.7 cells. The expression of DNA methyltransferase (DNMT) 3b and inducible nitric oxide synthase (iNOS) was also obviously inhibited by the ASD treatment. The protein and mRNA levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also significantly inhibited by ASD. ASD inhibited the macrophage M1 phenotype, inhibited the high level of DNMT3b, and downregulated the signal transducer and activator of the transcription 3 (STAT3) pathway to exert its anti-inflammatory activity. Furthermore, DNMT3b siRNA and Nrf2 siRNA significantly promoted the anti-inflammatory effect of ASD. Our study demonstrates for the first time that ASD inhibits the IL-6-STAT3-DNMT3b axis and activates the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway to achieve its inhibitory effect on inflammatory reactions.

The activation of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome and/or its components is associated with the physio-pathogenesis of many respiratory diseases including asthma, COPD (chronic obstructive pulmonary disease), SARS Cov-2 (severe acute respiratory syndrome coronavirus 2), and in several autoimmune diseases. Hibiscus noldeae Baker f. has been widely reported to be traditionally used in the treatment of different ailments, some of which are of inflammatory background such as asthma, wounds, headache, etc. However, the claims have not been supported by evidence at the molecular and functional levels. Here, we report on the bio-guided fractionation of H. noldeae and assessment of the inhibitory properties of some fractions and purified compounds on NLRP3 inflammasome and Interleukin 6 (IL-6). The activation of the NLRP3 inflammasome was determined by detecting the activity of caspase-1 and the production of Interleukin 1β (IL-1β) in Lipopolysaccharide (LPS) and ATP-stimulated Tamm-Horsfall Protein 1 (THP-1) macrophages, while the production of IL-6 was studied in LPS-stimulated RAW264.7 mouse macrophages. It was observed that hexane and ethyl acetate fractions of the crude extract of the aerial parts of H. noldeae, as well as caffeic acid, isoquercetin, and ER2.4 and ER2.7 fractions revealed significant inhibitory effects on Caspase-1 activities, and on IL-1β and IL-6 production. The ER2.4 and ER2.7 fractions downregulated the production of IL-1β and IL-6, in a similar range as the caspase-1 inhibitor AC-YVAD-CHO and the drug Dexamethasone, both used as controls, respectively. Overall, our work does provide the very first scientific based evidence for Hibiscus noldeae anti-inflammatory effects and widespread use by traditional healers in Rwanda for a variety of ailments.

Experiments were conducted to evaluate the induction of apoptosis and the immunomodulatory activities of alkaloids and triterpenes of Alstonia scholaris (Linn.) R. Br. leaves (ASL). Importantly, their possible synergistic properties were also explored in this study. Human lung adenocarcinoma cell line A549 and Lewis tumor-bearing C57BL/6 mice were used for the evaluation of their activities. A MTT assay was used to determine the proliferation inhibition in A549 cells. Annexin-V/PI double staining as well as flow cytometry was performed to detect apoptosis and cell cycle status. Enzyme-linked immunosorbent assay (ELISA) was conducted to determine the levels of inflammatory mediators interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum. Furthermore, western blot analysis was applied to evaluate the expressions of proteins associated with cell death. Alkaloids or triterpenes showed a high anti-proliferative activity in A549 cells, with IC50 values of 14.4 µg/mL and 9.3 µg/mL, respectively. The alkaloids and triterpenes combination could significantly inhibit tumor growth in tumor-bearing C57BL/6 mice, compared with alkaloids or triterpenes alone (7.5, 15, 30 g raw material/kg). The immune organs indexes including spleen index and thymus index were increased remarkably by the combination of alkaloids and triterpenes, whereas the levels of IL-6 and TNF-α were up-regulated significantly. Moreover, Annexin-V/PI double staining and flow cytometry showed that the combination of alkaloids and triterpenes (1, 2 and 3 mg raw material/kg) could induce apoptosis and cause S cell cycle arrest in A549 cells. Western blot analysis also showed that their combination (2 mg raw material/kg) significantly down-regulated Bcl-2 expression and pro-casp8 level, whereas it remarkably increased the level of cleaved caspase-8 leading to apoptosis in A549 cells. These observations provide preliminary evidence that both alkaloids and triterpenes possess immune regulation and induction apoptosis activities. Their combination has a stronger activity than that of either class alone. Our findings suggested that these components might be beneficial for the prevention and treatment of NSCLC through a significant synergy effect.

Neuroinflammation has been well recognized as a key pathological event in acute glaucoma. The medical therapy of acute glaucoma mainly focuses on lowering intraocular pressure (IOP), while there are still scarce anti-inflammatory agents in the clinical treatment of acute glaucoma. Here we reported that β,3α,5α-trihydroxy-androst-6-one (sterone), a novel synthetic polyhydric steroid, blocked neuroinflammation mediated by microglia/macrophages and alleviated the loss of retinal ganglion cells (RGCs) caused by acute intraocular hypertension (AIH). The results showed that sterone significantly inhibited the morphological changes, the up-regulation of inflammatory biomarker ionized calcium-binding adapter molecule 1 (Iba-1), and the mRNA increase of proinflammatory tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) in BV2 microglia and RAW264.7 macrophages. Moreover, immunofluorescence and western blotting analysis revealed that sterone markedly abrogated the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB) p65 subunit. Furthermore, sterone significantly suppressed the inflammatory microglial activation and RGCs' reduction caused by retinal ischemia/reperfusion (I/R) injury in a rat AIH model. These results suggest sterone may be a potential candidate in the treatment of acute glaucoma caused by microglial activation-mediated neuroinflammatory injury.

The inhibitory effect of three chromones 1-3 and two coumarins 4-5 on the production of nitric oxide (NO) was evaluated in LPS-induced RAW 264.7 macrophage cells. Among the compounds tested heterocarpin (1), a furochromone, significantly inhibited its production in a dose-dependent manner. In addition, heterocarpin suppressed prostaglandin E2 (PGE2) production and expression of cytokines such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6).

Sceptridium ternatum is a herbaceous plant with significant potential for pharmaceutical and cosmetic applications. In this study, we established a spectrum-effect relationship-based strategy to investigate the bioactive basis and tissue distribution in S. ternatum. First, a phytochemical analysis on the ethanol extracts from roots, stems, and leaves of S. ternatum was performed using the colorimetric method, high-performance liquid chromatography-ultraviolet (HPLC-UV), and high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS/MS). Then, radical scavenging assays and the lipopolysaccharide-stimulated RAW 264.7 cell model were used to estimate the antioxidant and anti-inflammatory activities, respectively. Spectrum-effect relationship analysis and molecular docking were further employed to evaluate the correlation between the phytochemical profile and anti-inflammatory activity. Our results demonstrate that S. ternatum leaves contained the most abundant flavonoids and exerted the best biological activities. Their IC50 values for scavenging 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radicals were 2.43 ± 0.13 and 5.36 ± 0.54 mg/mL, respectively. In lipopolysaccharide-stimulated RAW 264.7 cells, the leaf extract caused the greatest reduction in nitric oxide production (38.15%) and interleukin-6 release (110.86%). Spectrum-effect relationship analysis and molecular docking indicated that quercetin 3-O-rhamnoside-7-O-glucoside possessed high anti-inflammatory activity by binding with interleukin-6. In conclusion, S. ternatum is a rich source of bioactive flavonoids with potential for exploitation in the prevention and treatment of oxidative stress and inflammation-related pathologies.

To investigate the anti-atherosclerosis related mechanism of blueberries, the phenolic acids (PAs) content, antioxidant and anti-inflammatory activities, as well as the microRNA (miRNA) regulation of polyphenol fractions in blueberry samples from China were studied. Sixteen batches of blueberries including 14 commercialized cultivars (Reka, Patriot, Brigitta, Bluecrop, Berkeley, Duke, Darrow, Northland, Northblue, Northcountry, Bluesource, Southgood, O'Neal, and Misty) were used in this study. Seven PAs in the polyphenol fractions from 16 blueberry samples in China were quantified by high performance liquid chromatography/tandem mass spectrometry (HPLC/MS²). The antioxidant activities of blueberry polyphenols were tested by (1,1-diphenyl-2-picrylhydrazyl [DPPH]) assay. The anti-inflammatory (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]) activities of the polyphenol fractions of the blueberries were investigated by using lipopolysaccharide (LPS) induced RAW 264.7 macrophages. The correlation analysis showed that the antioxidant (1,1-diphenyl-2-picrylhydrazyl [DPPH]) and anti-inflammatory (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]) activities of the polyphenol fractions of the blueberries were in accordance with their PA contents. Although the polyphenol-enriched fractions of blueberries could inhibit the microRNAs (miRNAs) (miR-21, miR-146a, and miR-125b) to different extents, no significant contribution from the PAs was observed. The inhibition of these miRNAs could mostly be attributed to the other compounds present in the polyphenol-enriched fraction of the blueberries. This is the first study to evaluate the PAs content, antioxidant and anti-inflammatory activities, and miRNA regulation of Chinese blueberries.

Amyloid is a complex pathologic matrix comprised principally of paracrystalline protein fibrils and heparan sulfate proteoglycans. Systemic amyloid diseases are rare, thus, routine diagnosis is often challenging. The glycosaminoglycans ubiquitously present in amyloid deposits are biochemically and electrochemically distinct from those found in the healthy tissues due to the high degree of sulfation. We have exploited this unique property and evaluated heparin-reactive peptides, such as p5+14, as novel agents for specifically targeting and imaging amyloid. Herein, we demonstrate that radiolabeled p5+14 effectively bound murine AA amyloid in vivo by using molecular imaging. Biotinylated peptide also reacted with the major forms of human amyloid in tissue sections as evidenced immunohistochemically. Furthermore, we have demonstrated that the peptide also binds synthetic amyloid fibrils that lack glycosaminoglycans implying that the dense anionic motif present on heparin is mimicked by the amyloid protein fibril itself. These biochemical and functional data support the translation of radiolabeled peptide p5+14 for the clinical imaging of amyloid in patients.

The anti-inflammatory effects and molecular mechanism of 6,8-diprenyl-7,4'-dihydroxyflavanone (DDF), one of the flavanones found in Sophora tonkinensis, were assessed in vitro through macrophage-mediated inflammation in the present study. The anti-inflammatory effects of DDF were not previously reported. DDF inhibited the production of nitric oxide and the expression of tumor necrosis factor α, interleukin-1β, and interleukin-6. Furthermore, the activation of nuclear factor-κB (NF-κB) and extracellular signal-regulated kinases (ERKs) in lipopolysaccharide-stimulated macrophages was suppressed by treatment with DDF. Therefore, DDF demonstrated potentially anti-inflammatory effects via the blockade of NF-κB and ERK activation in macrophages.

Seven novel derivatives of bis(2-aminoethyl)amine were synthesized. For compounds 1 and 7 single crystals were isolated and X-ray diffraction experiments were done. Lipophilicity and drug likeness were calculated in the initial stage of research. All compounds were screened for their in vitro cytotoxic activity against a panel of human cancer cell lines, which is contrary to normal (HaCaT) cell lines, by using the MTT method. Studies were followed by lactate dehydrogenase assay, apoptotic activity, and interleukin-6 assay. Within the studied group, compound 6 showed the most promising results in all biological studies. The strongest influence in A549 cells was denoted for derivative 4, which inhibited interleukin release almost tenfold, as compared to the control.

The consumption of plant-based food is important for health promotion, especially regarding the prevention and management of chronic diseases such as diabetes. We investigated the effects of a lemon extract (LE), containing ≥20.0% total flavanones and ≥1.0% total hydroxycinnamic acids, on insulin signaling in murine 3T3-L1 adipocytes treated with TNF-α, which was used to mimic in vitro the insulin resistance condition that characterizes diabetes mellitus. Our results showed LE increased PPARγ, GLUT4 and DGAT-1 levels, demonstrating the potential of this lemon extract in the management of insulin resistance conditions associated with TNF-α pathway activation. LE treatment further decreased the release of interleukin 6 (IL-6) and restored triglyceride synthesis, which is the main feature of a healthy adipocyte.

The effect of Sargassum pallidum (brown seaweed) aqueous extract on the immunity function and antioxidant activities in was studied gastric cancer rats. Treatment with Sargassum pallidum aqueous extract at oral doses 400, 600 or 800 mg/kg body weight was found to provide a dose-dependent protection against N-methyl-N′-nitro-Nnitrosoguanidine (MNNG)-induced immunity damage and oxidative injury by enhancing serum interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) levels, decreasing interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) levels, preserving normal antioxidant enzymes activities, and by inhibiting lipid peroxidation in gastric mucosa. It can be concluded that Sargassum pallidum aqueous extract may enhance the immunity and antioxidant activities in gastric cancer rats.

Neuroinflammation plays a crucial role in the progression of Alzheimer's disease and other neurodegenerative disorders. Overactivated microglia cause neurotoxicity and prolong the inflammatory response in many neuropathologies. In this study, we have synthesised a series of isatin derivatives to evaluate their anti-neuroinflammatory potential using lipopolysaccharide activated microglia as a cell model. We explored four different substitutions of the isatin moiety by testing their anti-neuroinflammatory activity on BV2 microglia cells. Based on the low cytotoxicity and the activity in reducing the release of nitric oxide, pro-inflammatory interleukin 6 and tumour necrosis factor α by microglial cells, the N1-alkylated compound 10 and the chlorinated 20 showed the best results at 25 µM. Taken together, the data suggest that 10 and 20 are promising lead compounds for developing new neuroprotective agents.

Catalpa ovata (Bignoniaceae) is widely distributed throughout Korea, China, and Japan. This study investigated the anti-inflammatory effects of catalpalactone isolated from C. ovata in lipopolysaccharide (LPS)-induced RAW264.7 cells. Catalpalactone significantly inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in LPS-induced RAW264.7 cells. The levels of cytokines such as interleukin-6 and tumor necrosis factor-α were reduced under catalpalactone exposure in LPS-induced RAW264.7 cells. Additionally, catalpalactone suppressed signal transducer and activator of transcription 1 (STAT-1) protein expression and interferon-β (IFN-β) production. Treatment with catalpalactone prevented interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB) activation. Taken together, these results suggest that the anti-inflammatory effects of catalpalactone are associated with the suppression of NO production and iNOS expression through the inhibition of IRF3, NF-κB, and IFN-β/STAT-1 activation.