Scutellarin Alleviates LPS-induced Renal Injury Via Mediating CCN1 Expression To Inhibit NF-κB Signaling Pathway

OBJECTIVE:To explore the protective effect and mechanism of scutellarin（Scu）on sepsis associated acute kidney injury（SA-AKI）.METHODS:（1）In vivo experiment:male C57BL/6 mice were divided into control group,lipopolysaccharide（LPS）model group,20 mg/kg Scu group,Scu pretreatment groups（Scu 5,Scu 10,Scu 20+LPS groups）by random number table with 6 mice in each group.The AKI model was reproduced by intraperitoneal injection of 10 mg/kg LPS.The control group was injected with normal saline intraperitoneally;the LPS model group was injected with 10 mg/kg LPS;20 mg/kg Scu group was intraperitoneally injected with 20mg/kg Scu for 1 week.The pretreatment groups were intraperitoneally injected with different doses of Scu every day before LPS injection for 1 week.After 24 hours of LPS treatment,mice in each group were sacrificed,kidney tissues were collected,and kidney injury was detected by hematoxylin-eosin（HE）staining.Western Blot was used to detect the protein expression levels of KIM-1,NGAL,IL-18 and Cys C,NF-κB signaling pathway related molecules（IL-1β,TNF-α,MCP-1,p65 and IκBα）,apoptosis-related proteins（Bax and Bcl-2）and CCN1.（2）In vitro experiment:In the cells without LPS treatment and after 100 g/L LPS treatment,pc DNA3.1-CCN1 and small interfering RNA（si RNA）CCN1 sequence were transfected to overexpress and inhibit CCN1 expression,respectively.The m RNA and protein levels of IL-1β,TNF-a,MCP-1,and the expression levels of NF-κB signaling pathway related molecules,apoptosis-related proteins,and CCN1 were detected,to cbserve whether CCN1 is involved in NF-κB signaling pathway activation and apoptosis.In addition,100g/L LPS and 20μmol/L Scu were added into HK-2 cells transfected with and without CCN1 si RNA to investigate the protective effect of Scu on LPS-induced HK-2 cells.RESULTS:（1）In vivo experiment:the renal function of AKI mice induced by LPS was significantly decreased,and had kidney histological damage and severely damaged renal tubules.Scu could alleviate renal function and histological damage of AKI in a dose-dependent manner.In terms of m RNA levels,compared with LPS group,the expression levels of IL-1β,TNF-α,MCP-1 in LPS+Scu 20 group were significantly down-regulated[IL-1βm RNA(2^-ΔΔCT):2.68±0.44 vs.4.04±0.49,TNF-αm RNA(2^-ΔΔCT):2.02±0.25 vs.3.56±0.82,MCP-1 m RNA(2^-ΔΔCT):2.09±0.38 vs.4.21±0.70,all P<0.05].Western Blot results showed Scu could reduce the protein expression markers of renal injury,NF-κB signaling pathway related molecules（2）In vitro experiment:In HK-2 cells treated with LPS,overexpression of CCN1 significantly inhibited the expression of IL-1β,TNF-αand MCP-1[IL-1βm RNA(2^-ΔΔCT):3.20±0.57 vs.4.88±0.69,TNF-αm RNA(2^-ΔΔCT):2.98±0.44 vs.5.00±0.81,MCP-1 m RNA(2^-ΔΔCT):2.81±0.50 vs.5.41±0.75,P<0.05],and the apoptosis-related proteins were significantly down-regulated.However,when si RNA was used to inhibit the expression of CCN1,the expression of IL-1β,TNF-αand MCP-1 were significantly increased[IL-1βm RNA(2^-ΔΔCT):6.01±1.13 vs.4.88±0.69,TNF-αm RNA(2^-ΔΔCT):5.15±0.86 vs.5.00±0.81,P<0.05],and apoptosis-related proteins were significantly up-regulated.In the LPS-induced HK-2 cells,the m RNA levels of IL-1β,TNF-αand MCP-1 were significantly down-regulated after Scu treatment[IL-1βm RNA(2^-ΔΔCT):2.55±0.50 vs.6.15±1.04,TNF-αm RNA(2^-ΔΔCT):2.58±0.40 vs.3.95±0.52,MCP-1 m RNA(2^-ΔΔCT):2.64±0.44 vs.6.21±0.96,P<0.05],and the levels of p-p65 and p-IκBαproteins were also significantly reduced.Compared with LPS+Scu group,the m RNA levels of IL-1β,TNF-α,and MCP-1 were significantly up-regulated in LPS+CCN1 si RNA+Scu group[IL-1βm RNA(2^-ΔΔCT):5.34±0.76 vs.2.55±0.50,TNF-αm RNA(2^-ΔΔCT):3.66±0.54 vs.2.58±0.40,MCP-1 m RNA(2^-ΔΔCT):5.15±0.79 vs.2.64±0.44,P<0.05].CONCLUSION:Scu could protect the renal function in AKI mice,and the protective effect is associated with NF-κB signaling pathway and CCN1.Thus,Scu could alleviate LPS-induced kidney injury.