To explore the effects of miR-101-3p on IL-1β-induced chondrocyte injury and its underlying mechanisms.
 Methods: Chondrocytes were divided into 4 groups: a control group (NC group), a IL-1β group, a negative control group (IL-1β+miR-NC group), and a miR-101-3p group (IL-1β+miR-101-3p group), which were treated with IL-1β after transfecting with miR-101-3p mimic or negative mimic. The expressions of miR-101-3p-5p and stanniocalcin 1 (STC1) at different concentrations of IL-1β (1, 5, 10 ng/mL)-induced chondrocytes were detected by Western blotting and real-time PCR. MTT assay was used to detect cell proliferation rate, while caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein, such as matrix metalloproteinase 9 (MMP9) and collagen Type II. In addition, 3'-untranslated regions (UTR) of wild-type STC1 (STC1-3'-UTR-WT) or 3'-UTR of mutant STC1 (STC1-3'-UTR-MUT) were co-transfected with miR-101-3p mimic or miR-NC, respectively, while luciferase reporter assay was used to examine the regulative role of miR-101-3p in STC1. In order to detect whether STC1 was involved in the effect of miR-101-3p on chondrocytes, miR-NC (miR-NC group), miR-101-3p (miR-101-3p group), anti-NC (anti-NC group) and anti-miR-101-3p (anti-miR-101-3p group) were respectively transfected into the cells, and the expression of STC1 protein was detected by Western blotting. Subsequently, the cells were randomly divided into a miR-101-3P group (IL-1β+miR-101-3p group), an over-expression control group (IL-1β+miR-101-3p+ad-GFP group), and an over-expression STC1 group (IL-1β+miR-101-3p+ad-STC1 group) to investigate whether STC1 was involved in the role of miR-101-3p in chondrocyte. Similarly, MTT assay was used to detect cell proliferation rate, caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein MMP9 and collagen Type II.
 Results: Compared with the 0 ng/mL IL-1β, the expression of miR-101-3p was decreased in chondrocyte at different concentration of IL-1β (1, 5, 10 ng/mL) (all P<0.05), while the level of STC1 was increased (P<0.05). Compared with the NC group, the chondrocyte proliferation rate was down-regulated (P<0.05), while the apoptosis rate, the levels of caspases, IL-6 and TNF-α were increased in the IL-1β group (P<0.05). Moreover, the MMP9 levels were increased obviously, and the protein levels of collagen Type II were decreased in the IL-1β group compared with the NC group (both P<0.05). Compared with the IL-1β+miR-NC group, the proliferation rate was increased (P<0.05), whereas the apoptosis rates, the caspase-3/9 levels, the IL-6 and TNF-α levels were increased in the IL-1β+miR-101-3p group (all P<0.05). Then MMP9 levels were decreased obviously (P<0.05), and the protein levels of collagen Type II were increased in IL-1β+miR-101-3p group compared with the IL-1β+miR-NC group (both P<0.05). In addition, the double luciferase assay showed that the STC1 levels could be inhibited in the miR-101-3p group compared with the miR-NC group (P<0.05). STC1 levels were decreased in the miR-101-3p group compared with the miR-NC group (P<0.05), and the STC1 levels were increased in the anti-miR-101-3p group compared with those in the anti-NC group (P<0.05). The results of miR-101-3p+ad-STC1 group showed that compared with the miR-101-3p+ad-GFP group, the STC1 could reverse the effects of miR-101-3p on IL-1β-induced proliferation, apoptosis, inflammatory responses and ECM protein of chondrocytes.
 Conclusion: The regulation of miR-101-3p/STC1 signal pathway may have a role in reducing the IL-1β-induced chondrocyte injury.
Cell Proliferation ; Chondrocytes ; Glycoproteins ; metabolism ; Interleukin-1beta ; metabolism ; MicroRNAs

OBJECTIVE: To investigate whether Salvianolic acid A (SAA) can restore cartilage endplate cell degeneration of intervertebral discs and to identify the mechanism via regulation of micro-RNA. METHODS: Cartilage endplate cells were isolated from lumbar intervertebral disc surgical samples and were treated with serum containing a series of concentrations of SAA (2, 5, and 10 ?M) for 24, 48, and 72 h to identify a proper dose and treatment time of SAA. The effect SAA on interlenkin-1β (IL-1β)-induced extracellular matrix degradation of cartilage endplate cells were analyzed by Alcian blue staining and assessment of the expression levels of ADAMTS-5, MMP3 and Col2a1. Further, the potential target miRNAs were preliminarily screened by micro-RNA sequencing combining qRT-PCR and Western blot, and then, the miRNAs mimics and inhibitors were used to verify the regulatory effect of SAA on potential target miRNAs. RESULTS: The 10 μM SAA treatment for 48 h significantly enhanced the viability of cartilage endplate cells, and increased Col2a1 expression and glycosaminoglycan accumulation that were repressed by IL-1β, and reduced the effect of IL-1β on ADAMTS-5, and MMP3. Screening analysis based on micro-RNA sequencing and Venny analysis identified the downstream micro-RNAs, including miR-940 and miR-576-5p. Then, the miR-940-mimic or miR-576-5p-mimic were transfected into CEPCs. Compared with the SAA group, the expression of ADAMTS-5 and MMP3 increased significantly and the expression of COL2A1 obviously decreased after overexpression of miR-940 or miR-576-5p in CEPCs. CONCLUSION Salvianolic acid A attenuated the IL-1β-induced extracellular matrix degradation of cartilage endplate cells by targeting regulate the miR-940 and the miR-576-5p.
Humans ; Apoptosis ; Cartilage/metabolism* ; Chondrocytes/metabolism* ; Interleukin-1beta/metabolism* ; Matrix Metalloproteinase 3/metabolism* ; MicroRNAs/metabolism*

BACKGROUNDInterleukin (IL)-1beta may effectively decrease introcular pressure (IOP) when administered by subconjunctival injection in normal rabbit. However, IL-1beta is a large molecular agent and an inflammation factor. The aim of this study was to evaluate the penetrability of IL-1beta, and the concentrations of both tumor necrosis factor (TNF)-alpha and IL-6 in the aqueous humor of normal rabbits treated with IL-1beta.

METHODSA total of 170 rabbits were used in the study and were assigned to several different treatment groups as follows: 125 of the rabbits were assigned to two groups. In one group, 33 rabbits were injected subconjunctivally with IL-1beta and 39 were injected with saline alone. In the other group, 27 rabbits were given eye drops containing IL-1beta (400 ng/ml) and 26 were given saline alone. Aqueous humor (AH) was drawn and the concentration of IL-1beta within the fluid measured. The IOP was measured in another six rabbits after administration of eye drops containing IL-1beta (400 ng/ml). A further 20 rabbits were assigned to 3 groups as follows: eight untreated normal controls; six injected subconjunctivally with IL-1beta; and six injected subconjunctivally with saline alone. AH was drawn and the concentration of TNF-alpha in the fluid was measured. Another 19 rabbits were assigned to 3 groups as follows: seven untreated normal controls; and six injected subconjunctivally with IL-1beta; and six injected subconjunctivally with saline alone. AH was drawn and the concentration of IL-6 in the fluid measured. Measurement of cytokine concentration was by radio-immunoassay in all cases.

RESULTSThe IL-1beta concentration in the AH was higher in those animals in which it had been administered subconjunctivally (P < 0.01). The IL-1beta concentration in the AH of the animals given eye drops was almost the same as that in the controls (P > 0.05). The administration of IL-1beta in the form of eye drops had little effect upon IOP reduction. Lower TNF-alpha concentrations were seen in the AH after the subconjunctival administration of IL-1beta, but the concentration of IL-6 was the same as in the normal controls.

CONCLUSIONSIL-1beta shows good corneal penetrability after subconjunctival injection into normal rabbit eyes. The IOP reduction induced by IL-1beta is unlikely be associated with an inflammatory response.

Animals ; Aqueous Humor ; drug effects ; metabolism ; Interleukin-1beta ; pharmacology ; Interleukin-6 ; metabolism ; Rabbits ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo evaluate the effect of different CO2 pneumoperitoneum on IL-1β and IL-6 in abdominal cavity.

METHODSFifty-six female SD rats were randomly divided into seven groups. One group was served as control and the others received CO2 pneumoperitoneum. Pneumoperitoneum was established at 0.67 kPa and 1.0 L/min gas flow for 1, 2 or 3 h with CO2 (group C1 h, C2 h, and C3 h, respectively). CO2 pneumoperitoneum was further established at 1.07 kPa and 1.0 L/min gas flow for 1 h (group C8p), at 0.67 kPa and 2.0 L/min gas flow for 1 h(group C2f), and at 0.67 kPa and 3.0 L/min gas flow for 1 h (group C3f). After the procedures, peritoneal fluid was collected to analyze the IL-1β and IL-6 level by ELISA method.

RESULTSCO2 pneumoperitoneum caused peritoneal inflammatory reaction. With the increasing of duration and gas flow in CO2 pneumoperitoneum, the concentrations of IL-1β and IL-6 in group C2 h, C3 h and C3f were higher than those in group C1 h (P<0.05). On the other hand, the concentrations of IL-1β and IL-6 in peritoneal fluid did not change significantly when pressure was increased (P>0.05).

CONCLUSIONSThe inflammatory reaction in abdominal cavity after CO2 pneumoperitoneum may be attributed to duration and gas flow instead of the pressure within the standard pneumoperitoneum working pressures. Surgeons should reduce surgical duration and adopt low-velocity gas flow within normal working pressures in clinical practice.

Abdominal Cavity ; Animals ; Carbon Dioxide ; Female ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Pneumoperitoneum, Artificial ; methods ; Rats ; Rats, Sprague-Dawley

Inflammatory bowel disease (IBD), the most important entities being ulcerative colitis and Crohn's disease, are chronic, relapsing and remitting inflammatory conditions that result from chronic dysregulation of the mucosal immune system in the intestinal tract. Although the precise pathogenesis of IBD is still incompletely understood, increased levels of proinflammatory cytokines, including interleukin (IL)-1beta, IL-18 and tumor necrosis factor-alpha, are detected in active IBD and correlate with the severity of inflammation, indicating that these cytokines may play a key role in the development of IBD. Recently, the intracellular nucleotide-binding oligomerization domain-like receptor (NLR) family members, including NLRP1, NLRP3, NLRC4 and NLRP6, are emerging as important regulators of intestinal homeostasis. Together, one of those aforementioned molecules or the DNA sensor absent in melanoma 2 (AIM2), apoptosis-associated speck-like protein containing 'a caspase recruitment domain (CARD)' (ASC) and caspase-1 form a large (>700 kDa) multi-protein complex called the inflammasome. Stimulation with specific microbial and endogenous molecules triggers inflammasome assembly and caspase-1 activation. Activated caspase-1 leads to the secretion of proinflammatory cytokines, including IL-1beta and IL-18, and the promotion of pyroptosis, a form of phagocyte cell death induced by bacterial pathogens, in an inflamed tissue. Therefore, inflammasomes are assumed to mediate host defense against microbial pathogens and gut homeostasis, so that their dysregulation might contribute to IBD pathogenesis. This review focuses on recent advances of the role of NLRP3 inflammasome signaling in IBD pathogenesis. Improving knowledge of the inflammasome could provide insights into potential therapeutic targets for patients with IBD.
CARD Signaling Adaptor Proteins/metabolism ; Carrier Proteins/metabolism/physiology ; Caspase 1/metabolism ; Humans ; Inflammasomes/*metabolism ; Inflammatory Bowel Diseases/metabolism/*pathology ; Interleukin-18/metabolism ; Interleukin-1beta/metabolism ; Signal Transduction

Astrocytes can regulate synaptic transmission by releasing gliotransmitter, and also can promote synaptogenesis and neurogenesis by releasing estrogen, thrombospondins, IL-1beta and IL-6. Astrocytes may play critical roles in neural nutrition and neuroprotection, so that it might be a new target for treatment of certain central nervous system diseases.
Astrocytes ; physiology ; Estrogens ; metabolism ; Humans ; Interleukin-1beta ; metabolism ; Neurogenesis ; physiology ; Neurons ; physiology ; Neurotransmitter Agents ; metabolism ; Synaptic Transmission ; physiology ; Thrombospondins ; metabolism

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*

BACKGROUNDChai Lai Prescription is a Chinese herbal compound which is used to sooth the liver, strengthen the spleen and harmonize the stomach for descending adverse Qi. We initiated the study to investigate its mechanism of treating in vitro rabbit reflux esophagitis models.

METHODSAdult male Japanese white rabbits, weighing 1.8-2.2 kg, were divided into five groups of three each, which were: normal control group (Krebs buffer, pH7.4), esophagitis model group (Krebs buffer, pH5.8), esophagitis model proup+low-dose Chinese herbal medicine protection group (0.6 mg × ml(-1)× kg(-1)), esophagitis model group+moderate-dose Chinese herbal medicine protection group (6 mg × ml(-1)× kg(-1)), esophagitis model group+high-dose Chinese herbal medicine protection group (60 mg × ml(-1)×kg(-1)). The RT-PCR method was used to test the influence of Chai Lai Prescription on IL-1 and IL-6 in in vitro rabbit models of esophagitis. We treated the in vitro models with different doses of Chinese herbal medicine.

RESULTSEsophageal mucosa were filled with various liquids. IL-6 and IL-1β mRNA expression was increased in rabbit esophageal mucosa stimulated with acid. Chinese herbal medicine significantly reduced the levels of IL-6 and IL-1β mRNA expression in the in vitro cultured rabbit esophageal mucosa. Using Chinese herbal medicine to treat in vitro models of RE, we found that the IL-6 and IL-1β mRNA expression levels went down, near to or lower than the normal control levels, compared with the group treated with acidified buffer solution.

CONCLUSIONSChai Lai Prescription lowered the IL-1β and IL-6 cytokine mRNA levels and protected the esophageal mucosa in the in vitro models of reflux esophagitis, suggesting that the traditional Chinese herbal compound may be able to treat reflux esophagitis by inhibiting the its inflammatory mediators.

Animals ; Drugs, Chinese Herbal ; therapeutic use ; Esophagitis, Peptic ; drug therapy ; metabolism ; Interleukin-1beta ; genetics ; Interleukin-6 ; genetics ; Male ; Rabbits

The innate immune system plays a crucial role in the rapid recognition and elimination of invading microbes. Detection of microbes relies on germ-line encoded pattern recognition receptors (PRRs) that recognize essential bacterial molecules, so-called pathogen-associated molecular patterns (PAMPs). A subset of PRRs, belonging to the nucleotide binding oligomerization domain (NOD)-like receptor (NLR) families, detects viral and bacterial pathogens in the cytosol of host cells and induces the assembly of a multi-protein signaling platform called the inflammasome. The inflammasome serves as an activation platform for the cysteine protease Caspase-1, a central mediator of innate immunity. Caspase-1 initiates a novel form of cell death called pyroptosis. Inflammasome activation by pathogen-associated signatures results in the autocatalytic cleavage of Caspase-1 and ultimately leads to the processing and thus secretion of pro-inflammatory cytokines, most importantly interleukin (IL)-1β and IL-18. Here, we review the recent advancements of negative regulatory functions and mechanisms leading to the activation of NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes.
Apoptosis ; Carrier Proteins ; Caspase 1 ; Humans ; Immunity, Innate ; Inflammasomes ; metabolism ; Inflammation ; metabolism ; Interleukin-18 ; metabolism ; Interleukin-1beta ; metabolism ; Nod Signaling Adaptor Proteins ; metabolism ; Signal Transduction

Inflammasomes are high-molecular-weight, multiprotein complexes in cells, which are assembled after cytoplasmic nucleotide-binding oligomerization domain like receptors (NLRs) sense pathogens and danger signals. The inflammasome can activate caspase-1, and later makes the pro-IL-1β, proIL-18 precursor mature by cleavaging, thereby mediates the innate immunity. Dysregulation of inflammasomes plays an important role in the development of sepsis and other immune inflammatory diseases, thus inflammasome may be a new target for prevention and treatment of sepsis.
Caspase 1 ; immunology ; metabolism ; Humans ; Inflammasomes ; chemistry ; immunology ; metabolism ; Interleukin-18 ; immunology ; metabolism ; Interleukin-1beta ; immunology ; metabolism ; Sepsis ; immunology ; metabolism ; Signal Transduction