C1q/tumour necrosis factor-related protein-9 aggravates lipopolysaccharide-induced inflammation via promoting NLRP3 inflammasome activation.

The NLRP3 inflammasome plays a vital role in inflammation by increasing the maturation of interleukin-1ß (IL-1ß) and promoting pyroptosis. Given that C1q/tumour necrosis factor-related protein-9 (CTRP9) has been shown to be involved in diverse inflammatory diseases, we sought to assess the underlying impact of CTRP9 on NLRP3 inflammasome activation. In vitro, macrophages isolated from murine peritonea were stimulated with exogenous CTRP9, followed by lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP). We demonstrated that CTRP9 markedly augmented the activation of the NLRP3 inflammasome, as shown by increased mature IL-1ß secretion, triggering ASC speck formation and promoting pyroptosis. Mechanistically, CTRP9 increased the levels of NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). Suppressing ROS with N-acetylcysteine (NAC) or interfering with NOX2 by small interfering RNA weakened the promoting effect of CTRP9 on the NLRP3 inflammasome. Furthermore, NLRP3 inflammasome activation, pyroptosis and secretion of mature IL-1ß were significantly decreased in macrophages from CTRP9-KO mice compared to those from WT mice with the same treatment. In vivo, we established a sepsis model by intraperitoneal injection of LPS into WT and CTRP9-KO mice. CTRP9 knockout improved the survival rates of the septic mice and attenuated NLRP3 inflammasome-mediated inflammation. In conclusion, our study indicates that CTRP9 aggravates LPS-induced inflammation by promoting NLRP3 inflammasome activation via the NOX2/ROS pathway. CTRP9 could be a promising target for NLRP3 inflammasome-driven inflammatory diseases.

CTRP9 alleviates foam cells apoptosis by enhancing cholesterol efflux.

The apoptosis of foam cells leads to instability of atherosclerotic plaques. This study was designed to explore the protective role of CTRP9 in foam cell apoptosis. In our experiment, CTRP9 alleviated foam cell apoptosis. Meanwhile, CTRP9 upregulated the expression of proteins important for cholesterol efflux, such as LXRα, CYP27A1, ABCG1 and ABCA1, and improved cholesterol efflux in foam cells. Moreover, CTRP9 inhibited Wnt3a and ß-catenin expression and ß-catenin nuclear translocation in foam cells. In addition, adenovirus overexpression of Wnt3a abolished the effect of CTRP9 on macrophage apoptosis. Mechanistically, the AMPK inhibitor abolished the effect of CTRP9 on foam cell apoptosis, and downregulation of AdipoR1 by siRNA abrogated the activation of AMPK and the effect of CTRP9 on foam cell apoptosis. We concluded that CTRP9 achieved these protective effects on foam cells through the AdipoR1/AMPK pathway.

CTRP9 induces macrophages polarization into M1 phenotype through activating JNK pathway and enhances VSMCs apoptosis in macrophages and VSMCs co-culture system.

Inflammation plays a critical role in the development of atherosclerosis (AS), which has been identified as a major predisposing factor for stroke. Macrophages and VSMCs are associated with plaque formation and progression. Macrophages can dynamically change into two main functional phenotypes, namely M1 and M2, they can produce either pro-inflammatory or anti-inflammatory factors which may affect the outcome of inflammation. As a member of CTRPs family, CTRP9 has been reported play important protective roles in the cardiovascular system. However, whether CTRP9 can regulate macrophage activation status in inflammatory responses and have effect on VSMCs behaviors in co-culture system have not been fully investigated. In the present study, using peritoneal macrophages treated with CTRP9, we found that CTRP9 facilitated macrophages towards M1 phenotype, promoted TNF-α secretion and MMPs expression. CTRP9 showed synergistic effect with LPS in inducing M1 macrophages. In macrophages-VSMCs co-culture system, apoptosis and down-regulated proliferation of VSMCs were accelerated with CTRP9-treated macrophages. Then we attempted to explore the underlying molecular mechanisms of CTRP9 resulting in M1 activation. The c-Jun NH2-terminal kinases (JNK) are members of the mitogen activated protein kinases (MAPK) family, plays a central role in the cell stress response, with outcomes ranging from cell death to cell proliferation and survival. We found JNK expression was upregulated following CTRP9 stimulation, and inhibiting JNK phosphorylation level was associated with decreased expression of M1 markers and TNF-α concentration. Moreover, VSMCs apoptosis were ameliorated after inhibition of JNK. These results suggested that CTRP9 may promote macrophage towards M1 activation status through JNK signaling pathway activation.

CTRP9 induces iNOS expression through JAK2/STAT3 pathway in Raw 264.7 and peritoneal macrophages.

The C1q tumor necrosis factor (TNF)-related proteins 9 (CTRP9), an adipocyte-derived cytokine, affects a number of physiological processes, including immune function and inflammation. We investigated whether CTRP9 affects the expression of inflammation-related genes in Raw 264.7 and peritoneal macrophages. The CTRP9-induced expression of iNOS increased in a time- and dose-dependent manner. LPS and CTRP9 promote the expression of iNOS jointly in Raw 264.7 and peritoneal macrophages. CTRP9 induced the phosphorylation of JAK2 and STAT3 in Raw 264.7 and peritoneal macrophages. VX509 (JAK2 inhibitor) reduced the CTRP9-induced iNOS protein production. In addition, the CTRP9-induced phosphorylation of JAK2 and STAT3 was dramatically reduced by VX509. Collectively, these results suggest that JAK2/STAT3 signaling is involved in the CTRP9-induced expression of iNOS.