This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1β, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1β in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1β, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1β. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.
Inflammasomes/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Interleukin-18/metabolism* ; Lipopolysaccharides/pharmacology* ; Macrophages ; Cytokines/metabolism* ; Caspase 1/metabolism* ; Autophagy ; Interleukin-1beta/metabolism*

Objective To investigate the neuroprotective effect of methylene blue on diabetic retinopathy in rats. Methods Thirty SD rats were randomly divided into blank, control and experimental groups. The control and experimental groups were induced with diabetes by streptozotocin (STZ) intraperitoneal injection. After 6 weeks of successful modeling, the experimental group received intravitreal injection of methylene blue at a dose of [0.2 mg/(kg.d)], while the control group received an equal amount of dimethyl sulfoxide (DMSO) intravitreal injection, both continuously injected for 7 days. ELISA was used to detect the levels of retinal superoxide dismutase (SOD), 8-iso-prostaglandin F2alpha (iPF2α) and interleukin-1β (IL-1β) in rats. Western blot analysis was used to detect the expression of retinal extracellular signal-regulated kinase 1/2 phosphorylation (p-ERK1/2) and phosphorylated protein kinase B (p-AKT), and PAS staining was used to detect retinal morphological changes. Results Compared with the blank group rats, the retinal SOD activity in the control and experimental group rats was significantly reduced. iPF2α, IL-1β and p-ERK1/2 level increased, while p-AKT level decreased. Compared with the control group, the SOD activity of the experimental group rats increased. iPF2α and IL-1β level went down, while p-ERK1/2 and p-AKT level went up significantly. The overall thickness of the retinal layer and the number of retinal ganglion cells were significantly reduced. Conclusion Methylene blue improves diabetic retinopathy in rats by reducing retinal oxidative stress and enhancing ERK1/2 and AKT phosphorylation.
Rats ; Animals ; Diabetic Retinopathy/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mitogen-Activated Protein Kinase 3/metabolism* ; Interleukin-1beta/metabolism* ; Methylene Blue/pharmacology* ; Phosphorylation ; Rats, Sprague-Dawley ; MAP Kinase Signaling System ; Diabetes Mellitus, Experimental/drug therapy* ; Superoxide Dismutase/metabolism*

The aim of this study is to investigate the effects of Gynostemma pentaphyllum dried with two different methods(air drying and heating) on inflammation in acute lung injury(ALI) mice in vivo and in vitro. Lipopolysaccharide(LPS) was sprayed into the airway of wild type C57BL/6J male mice to establish the model, and the drug was injected into the tail vein 24 h after modeling. Lung function, lung tissue wet/dry weight(W/D) ratio, the total protein concentration, interleukin 6(IL-6), IL-1β, and tumor necrosis factor-α(TNF-α) in the bronchoalveolar lavage fluid(BALF), and pathological changes of the lung tissue were used to evaluate the effects of different gypenosides on ALI mice. The results showed that total gypenosides(YGGPs) and the gypenosides substituted with one or two glycosyl(GPs_(1-2)) in the air-dried sample improved the lung function, significantly lowered the levels of IL-1β and TNF-α in BALF, and alleviated the lung inflammation of ALI mice. Moreover, GPs_(1-2) had a more significant effect on inhibiting NO release in RAW264.7 cells. This study showed that different drying methods affected the anti-inflammatory activity of G. pentaphyllum, and the rare saponins in the air-dried sample without heating had better anti-inflammatory activity.
Male ; Mice ; Animals ; Tumor Necrosis Factor-alpha/metabolism* ; Gynostemma ; Mice, Inbred C57BL ; Lung ; Anti-Inflammatory Agents/metabolism* ; Interleukin-6/metabolism* ; Interleukin-1beta/metabolism* ; Lipopolysaccharides/pharmacology*

10,11-Dehydrocurvularin (DCV) is a natural-product macrolide that has been shown to exert anti-inflammatory activity. However, the underlying mechanism of its anti-inflammatory activity remains poorly understood. Aberrant activation of the NLRP3 inflammasome is involved in diverse inflammation-related diseases, which should be controlled. The results showed that DCV specifically inhibited the activation of the NLRP3 inflammasome in association with reduced IL-1β secretion and caspase-1 activation, without effect on the NLRC4 and AIM2 inflammasomes. Furthermore, DCV disturbed the interaction between NEK7 and NLRP3, resulting in the inhibition of NLRP3 inflammasome activation. The C=C double bond of DCV was required for the NLRP3 inflammasome inhibition induced by DCV. Importantly, DCV ameliorated inflammation in vivo through inhibiting the NLRP3 inflammasome. Taken together, our study reveals a novel mechanism by which DCV suppresses inflammation, which indicates the potential role of DCV in NLRP3 inflammasome-driven inflammatory disorders.
Animals ; Mice ; Inflammasomes ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Inflammation/drug therapy* ; Anti-Inflammatory Agents/pharmacology* ; Interleukin-1beta/genetics* ; Mice, Inbred C57BL

OBJECTIVE: To investigate the effect of indirubin for relieving joint inflammation and injury in a rat model of osteoarthritis. METHODS: Articular cartilage chondrocytes were isolated from adult rat knee joint and cultured in the presence of interleukin-1β (IL-1β) and 0.1, 0.5, 1.0, or 2.0 μmol/L indirubin. The cells were transfected with NPAS2 siRNA or a non-specific siRNA, and the cell proliferation and apoptosis were evaluated using tetramethylthiazole blue staining and flow cytometry. The protein expression levels of Bax, Bcl-2, ACAN, COL2A1, MMP-13 and NPAS2 were detected with Western blotting, and the levels of NO, PGE2 and TNF-α in the culture supernatant were determined with ELISA. The mRNA expression levels of NPAS2, ACAN, COL2A1 and MMP-13 were detected using fluorescence quantitative PCR. In a C57BL/6 mouse model of osteoarthritis, the effect of indirubin on BAX, Bcl-2, ACAN and MMP-13 protein expressions in the bone and joint tissues were evaluated with Western blotting. RESULTS: Treatment with 0.1 μmol/L indirubin produced no significant changes in chondrocyte proliferation, apoptosis, caspase-3 activity, or BAX and Bcl-2 protein expressions. At higher doses (0.5, 1.0 and 2.0 μmol/L), indirubin significantly promoted cell proliferation, increased Bcl-2 protein expression, and lowered cell apoptosis rate, caspase-3 activity and Bax protein expression (P < 0.05). Indirubin treatment at 0.5 μmol/L up-regulated the protein and mRNA expressions of NPAS2, ACAN and COL2A1, and down-regulated the expressions of MMP-13, NO, PGE2 and TNF-α (P < 0.05). Interference of NPAS2 expression significantly attenuated the protective effect of 0.5 μmol/L indirubin against IL-1β-induced chondrocyte injury. The mouse model of osteoarthritis showed obviously increased protein levels of BAX and MMP-13 (P < 0.01) and decreased levels of Bcl-2 (P < 0.05) and ACAN (P < 0.01) in the knee joint, and indirubin treatment of the mouse models significantly inhibited the increase of BAX and MMP-13 protein expressions (P < 0.01) and up-regulated the protein expressions of Bcl-2 and ACAN (P < 0.05). CONCLUSION Indirubin has a protective effect on osteoarthritis tissue and alleviates inflammation and damage of osteoarthritis chondrocytes possibly through NPAS2.
Animals ; Apoptosis ; Caspase 3/metabolism* ; Cells, Cultured ; Chondrocytes ; Dinoprostone/pharmacology* ; Disease Models, Animal ; Indoles ; Inflammation/drug therapy* ; Interleukin-1beta/metabolism* ; Matrix Metalloproteinase 13/metabolism* ; Mice ; Mice, Inbred C57BL ; Osteoarthritis/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/metabolism* ; RNA, Small Interfering/metabolism* ; Rats ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism*

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

The rats were exposed to chronic unpredictable mild stress(CUMS) and kept in separate cages for inducing depressive disorder, which was judged by behavioral indicators. The number and morphology of neurons in hippocampal CA3 area and prefrontal cortex were observed by hematoxylin-eosin(HE) staining. The levels of brain-derived neurotrophic factor(BDNF), 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), norepinephrine(NE), glutamic acid(GLU), interleukin-1β(IL-1β), interleukin-18(IL-18), and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay(ELISA). Real-time polymerase chain reaction(RT-PCR) and Western blot were conducted to determine the mRNA and protein expression levels of related molecules in NLRP3 pathway. The results showed that compared with the model group, acidic polysaccharides from Poria at the low-, medium-, and high-doses(0.1, 0.3 and 0.5 g·kg~(-1)·d~(-1)) all improved the depression-like behavior of rats, increased the number of neurons and the levels of BDNF, 5-HT, 5-HIAA, DA, and NE in the hippocampus, and reduced GLU and serum IL-1β, IL-18, and TNF-α levels. The mRNA expression levels of ASC, caspase-1, IL-1β, and IL-18 and the protein expression levels of NLRP3, ASC, caspase-1, IL-1β, and IL-18 in each medication group were down-regulated, whereas the protein expression levels of pro-caspase-1, pro-IL-1β, and pro-IL-18 were up-regulated. All these have indicated that acidic polysaccharides from Poria exerted the antidepressant effect possibly by regulating neurotransmitters and NLRP3 inflammasome signaling pathway.
Animals ; Antidepressive Agents ; Depression/drug therapy* ; Interleukin-1beta ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Neurotransmitter Agents ; Polysaccharides/pharmacology* ; Poria ; Rats

BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.
Cell Line, Tumor ; Cells, Cultured ; Chondrocytes/metabolism* ; Glycogen Synthase Kinase 3 beta/genetics* ; Histone Deacetylases/genetics* ; Humans ; Interleukin-1beta/pharmacology* ; Matrix Metalloproteinase 13/metabolism* ; Matrix Metalloproteinase 3 ; Repressor Proteins ; Wnt Signaling Pathway ; Wnt3A Protein/genetics* ; beta Catenin/metabolism*

The apoptosis of nucleus pulposus cells (NPCs) is the main cellular process of intervertebral disc degeneration (IVDD). Our previous studies showed that 17β-estradiol (E
Animals ; Apoptosis ; Estradiol/pharmacology* ; Glycogen Synthase Kinase 3 beta ; Interleukin-1beta ; Nucleus Pulposus/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Signal Transduction ; TOR Serine-Threonine Kinases

To study the mechanism of polysaccharides from seeds of Vaccaria segetalis( PSV) in the treatment of bacterial cystitis through the NLRP3 inflammasome pathway. The rat model of urinary tract infection was used and treated with PSV,and the urine and bladders were collected. The level of interleukin-10( IL-10) in rat urine was detected by enzyme linked immunosorbent assay( ELISA). Western blot and immunofluorescence staining were used to detect the expressions of sonic hedgehog( SHH) and NLRP3 inflammasome [NOD-like receptor thermoprotein domain 3( NLRP3),apoptosis associated speck like protein( ASC) and pro-caspase-1]. The expression of Toll-like receptor pathway was detected by RT-PCR. The death of 5637 cells induced by uropathogenic Escherichia coli( UPEC) and lactate dehydrogenase( LDH) release were evaluated using live/dead staining. The results showed that in the rat bladder,the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors were significantly up-regulated,and NLRP3 inflammasomes were significantly activated by UPEC infection. The administration with PSV could significantly increase the concentration of IL-10 in urine,inhibit the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors in bladder,and inhibit the activation of NLRP3 inflammasomes. A large number of 5637 cells were dead after UPEC infection and caused LDH production. PSV could significantly inhibit the death of 5637 cells and the release of LDH. In conclusion,PSV could inhibit the expression and activation of NLRP3 inflammasomes by inhibiting the Toll-like receptor pathway,thereby mitigating the bladder injury.
Animals ; Hedgehog Proteins ; Inflammasomes/genetics* ; Interleukin-1beta ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Polysaccharides/pharmacology* ; Rats ; Seeds ; Urinary Bladder ; Urinary Tract Infections/drug therapy* ; Vaccaria