Study On Anti-inflammatory Effect Of ZHC116

Part1The effect of ZHC116on the proliferation of NIH3T3cell measured by the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetra-zolium Bromide] AssayBackground:Fibrosis is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. It can happen in the kidney, lung, liver, heart, skin and other organs, doing great harm to human health. Currently, there is few effective anti-fibrotic drug in clinical. Pirfenidone is a novel anti-fibrotic and anti-inflammatory drug, which was authorized for clinical application of idiopathic pulmonary fibrosis(IPF) in Japan and Europe recently. Fluorofenidone (AKF-PD) is an agent developed by our group independently, against inflammation and fibrosis, which has been applied for the Phase I clinical trials. ZHC116is another agent synthesized by our group and proved to be anti-fibrotic in previous studies of rat unilateral ureteral obstruction(UUO) model, which has better efficacy and smaller side effects, showing better clinical potential. The pathogenesis of fibrosis is complicated, in which fibroblast proliferation plays an important role. Fibroblasts are the major source of collagen producing cells (myofibroblasts), when activated, it can secrete large amounts of type Ⅰ and type Ⅲ collagen and fibronectin, leading to organ fibrosis.MTT assay is a method widely used to detect cell porliferation. N1H3T3(mouse embryonic fibroblasts) cell line is classical for MTT assay of anti-flbrotic efficacy.Objective:To find out the effect of ZHC116on the proliferation of NIH3T3cells, and search for the effctive concentrations.Method:We incubated NIH3T3cells with different concentrations of ZHC116(0.05mM、0.1mM、0.15mM、0.20mM), each for48or72hours. Then MTT assay was used to evaluate live cell number of each group.Results:In the concentration range of0.10-0.20mM, the inhibition effect of ZHC116on NIH3T3cells was growing with increase of the ZHC116concentration and duration of incubation.Conclusion:ZHC116could inhibit the proliferation of NIH3T3cells, in the concentration range of0.10-0.20mM.Part2The effect of ZHC116on lethal endotoxemia and IL-1βBackground:Previous studies showed that ZHC116could reduce inflammatory cell infiltration in the early stage of UUO, and protect from renal interstitial injury, which suggests potential anti-inflammation effect of ZHC116. However, the effect of ZHC116on the systemic inflammatory response is unrevealed. Endotoxemia is a classic model of inflammatory response syndrome (SIRS). With the stimulation of Lipopolysaccharides (LPS), massive inflammatory mediators overexpress, leading to over response of inflammation and immuno-disorder. NALP3inflammasome is a complex of apoptosis associated speck-like protein containing CARD (ASC), caspase-1and NOD-like receptor (NLRP3). It can activate caspase-1, which cut the precursor IL-1J3into the mature form. NALP3-/-or caspase-/-or ASC-/-mice have resistance on endotoxin. IL-1β is a classical early phase inflammatory factor as well as a marker of inflammasome activation. Our previous studies showed that AKF-PD could effectively improve the survival of endotoxic mice and decrease the level of IL-1β. It is unknown whether ZHC116has similar effects.Objective:To study the effects of ZHC116on the mortality of murine endotoxemia as well as serum IL-1β.Methods:64Balb/c mice were randomly devided into four groups: control group, LPS group, LPS+ZHC116(50mg/kg) or LPS+AKF-PD (200mg/kg) groups. LPS (15mg/kg) or PBS (control group) and drugs were administrated intraperitoneal spontaneously. Animal survival rate was monitored for144hours after injection. Another40Balb/c mice were grouped and treated similarly, blood was collected2h after LPS injection, and IL-1β was detected by ELSIA.Results:In144hours, the mortality rate in the LPS model group was87.5%, significantly greater than control group (0%), while ZHC116 (50mg/kg) or AKF-PD (200mg/kg) treatment significantly decreased the mortality rate,43.75%in the ZHC116group, and31.25%in the AKF-PD group, respectively. Two hours after administrated i.p. of LPS, the IL-1β level in the serum was significantly increased, ZHC116(50mg/kg) and AKF-PD (200mg/kg) treatment markedly decreased the IL-1β level with no statistical difference between each other.Conclusion:ZHC116had a protective effect on murine endotoxemia and can bring down the circulating IL-1β level, suggesting an anti-inflammatory effect. The effect of ZHC116(50mg/kg) was comparable with AKF-PD (200mg/kg)Part3The effect of ZHC116on IL-1β and NALP3inflammasomes in peritoneal macrophageBackground:Peritoneal macrophages are the main responsers in endotoxemia. It cleans bacteria as well as necrotic tissue, and releases inflammatory mediators. Inflammasomes are highly expressed in macrophages, and mediates cell death, resulting in immunosuppression. EL-1β is the marker of activated inflammasome. ZHC116could reduce circulating IL-1β level, with an unclear mechanism.Objective:To study the possible mechanisms of ZHC116on IL-1β and NALP3inflammasomes.Method:Primary mouse peritoneal macrophages were stimulated with LPS (1μg/ml) in the presence or absence of ZHC116(0.1mM).24hours after stimulation, supernatant was collected and the levels of IL-1β were determined by ELISA. Cell mRNA and cell proteins were obtained to process quantitative PCR.Results:ZHC116(0.1mM) can bring down the IL-1β level in the cell supernatants. ZHC116can also significantly decrease the expression of IL-1β mRNA and proIL-1β protein. ZHC116brought down the expression of NALP3and caspase-1mRNA.Conclusion:ZHC116could inhibit both the expression of proIL-1β and might inhibit the maturation process of IL-1β throgh NALP3inflammasome...