绿原酸通过Nrf2/NLRP3通路减轻LPS致大鼠急性肾损伤的作用机制

Mechanism of chlorogenic acid attenuating LPS-induced acute kidney injury in rats through Nrf2/NLRP3 pathway

为了探究绿原酸(chlorogenic acid, CGA)对脓毒症致急性肾损伤(acute kidney injury, AKI)大鼠的保护作用及相关机制,试验选取32只健康雄性SD大鼠,随机分为4组,分别为对照组(CON组)、模型组(LPS组)、CGA干预组(CGA+LPS组)和CGA组。LPS组大鼠按体重腹腔注射10 mg/kg LPS,建立大鼠脓毒症模型;CGA+LPS组大鼠按体重腹腔注射20 mg/kg CGA,1 h后腹腔注射LPS;CGA组按体重腹腔注射20 mg/kg CGA。4 h后大鼠用异氟醚麻醉,打开腹腔,分别通过膀胱采集尿液、心脏采集血液用于肾脏功能和血清炎症因子检测;H.E.染色观察肾脏组织病理学变化;Western-blot法检测肾脏组织胞核(N)-核转录因子E2相关因子2(Nrf2)、总(T)-Nrf2、血红素加氧酶1(HO-1)、醌氧化还原酶1(NQO1)蛋白及NOD样受体家族pyrin域3(NLRP3)炎症小体相关蛋白(NLRP3、Caspase1 p20、IL-1β和IL-18)的相对表达量。结果表明:与CON组相比,LPS组血清尿素氮(BUN)和肌酐(Cre)水平极显著升高(P<0.01),尿液KIM-1和NGAL水平极显著升高(P<0.01);血清炎症细胞因子TNF-α、IL-6、IL-1β和IL-18水平极显著升高(P<0.01);肾脏组织病理学变化包括肾小管扩张或肿胀、空泡变性、炎性细胞浸润等;NLRP3炎症小体相关蛋白(NLRP3、Caspase1 p20、IL-1β和IL-18)相对表达量极显著升高(P<0.01),而Nrf2、HO-1和NQO1蛋白相对表达量差异不显著(P>0.05)。而与LPS组相比,CGA+LPS组血清BUN和Cre水平极显著降低(P<0.01),尿液KIM-1和NGAL水平极显著降低(P<0.01);血清炎症细胞因子TNF-α、IL-6、IL-1β和IL-18水平极显著降低(P<0.01);肾脏组织病理学变化仅可见轻度炎性细胞浸润和局灶性坏死;Nrf2、HO-1和NQO1蛋白表达量极显著升高(P<0.01),NLRP3炎症小体相关蛋白(NLRP3、Caspase1 p20、IL-1β和IL-18)表达量极显著降低(P<0.01)。说明CGA通过激活Nrf2信号通路促进下游HO-1和NQO1蛋白表达,抑制NLRP3炎症小体激活,减轻炎症级联瀑布反应,从而对LPS诱导的大鼠AKI起到保护作用。

In order to explore the protective effect and related mechanism of chlorogenic acid(CGA) on sepsis-induced acute kidney injury(AKI) in rats, thirty-two healthy male SD rats were selected and randomly divided into 4 groups, namely control group(CON group), model group(LPS group), CGA intervention group(CGA + LPS group) and CGA group. The rats in the LPS group were intraperitoneally injected with 10 mg/kg LPS according to their body weight to establish a rat model of sepsis;the rats in the CGA + LPS group were intraperitoneally injected with 20 mg/kg CGA according to their body weight, and LPS was injected intraperitoneally 1 hour later;the CGA group was intraperitoneally injected wih 20 mg/kg CGA according to their body weight. After 4 h, the rats were anesthetized with isoflurane;the abdominal cavity was opened;urine was collected from the bladder and blood was collected from the heart for the detection of renal function and serum inflammatory factors respectively. H.E. staining was used to observe the histopathological changes of the kidney. Western-blot method was used to detect the relative expression of nuclear factor E2 related factor 2(N-Nrf2), total(T)-Nrf2, Heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1) proteins and NLR family pyrin domain containing 3(NLRP3) inflammasome-related proteins(NLRP3, Caspase1 p20, IL-1β and IL-18) in renal tissue. The results showed that compared with the CON group, the serum urea nitrogen(BUN) and creatinine(Cre) levels in the LPS group were significantly increased(P<0.01);the urinary KIM-1 and NGAL levels were extremely significantly increased(P<0.01);the serum inflammatory cytokine levels of TNF-α, IL-6, IL-1β and IL-18 were significantly increased(P<0.01);renal histopathological changes included renal tubular dilatation or swelling, vacuolar degeneration, and inflammatory cell infiltration;the relative expression levels of NLRP3 inflammasome-related proteins(NLRP3, Caspase1 p20, IL-1β and IL-18) were significantly increased(P<0.01), while the relative expression levels of Nrf2, HO-1 and NQO1 proteins were not significantly different(P>0.05). Compared with the LPS group, the serum BUN and Cre levels of the CGA + LPS group were significantly decreased(P<0.01);the urinary KIM-1 and NGAL levels were extremely significantly decreased(P<0.01);the serum inflammatory cytokine levels of TNF-α, IL-6. The of IL-1β and IL-18 were significantly decreased(P<0.01);only mild inflammatory cell infiltration and focal necrosis were seen in renal histopathological changes;Nrf2, HO-1 and NQO1 protein expressions were extremely significantly increased(P<0.01);the expression of NLRP3 inflammasome-related proteins(NLRP3, Caspase1 p20, IL-1β and IL-18) was extremely significantly decreased(P<0.01). The results suggested that CGA promoted the expression of downstream HO-1 and NQO1 proteins by activating the Nrf2 signaling pathway, inhibited the activation of NLRP3 inflammasome, and alleviated the inflammatory cascade reaction, thereby protecting the LPS-induced rat AKI.