柴芩承气汤抑制急性坏死性胰腺炎巨噬细胞促炎因子释放的分子刹车蛋白机制

Timely recovery of the balance of pro-inflammatory and anti-inflammatory cytokine is the key to relieve the development of multiple organ failure from the systemic inflammatory response severe acute pancreatitis (SAP). Activation the Cholinergic anti-inflammatory pathway can inhibit the release of proinflammatory cytokine of macrophage, but does not affect the release of anti-inflammatory cytokine in the post-transcriptional level, and then restore the balance of pro-inflammatory and anti-inflammatory cytokine. The receptor for activated C kinase 1 (RACK1), a protein molecular brake, can regulate the protein synthesis in the level of post-transcriptionalion by action on the signaling pathway of unfolded protein response (UPR). Our previous studies have confirmed that the Chaiqinchengqi decoction (CQCQD),one of the prescription medicine, can elevate serum acetylcholine and increased expression of nicotinic acetylcholine receptors of macrophage of acute pancreatitis (AP) to reduce the release of pro-inflammatory cytokines in the post-transcriptional level. Whereby, We propose the hypothesis: the reducing of the release of pro-inflammatory cytokines of macrophage of AP by CQCQD was associated with the role of protein molecules brakes of RACK1 of the endoplasmic reticulum. In the present study, we intend to investigate the effects of the CQCQD on the signaling pathway of UPR of peritoneal macrophages through detection the changes of the expression of mRNA and protein of UPR- related proteins (such as RACK1 etc.), pro-inflammatory cytokines and anti-inflammatory cytokines in acute necrotizing pancreatitis mice intragastrically administered. This study is expected to enrich the theory of cholinergic anti-inflammatory pathway,and obtain the innovative discovery of further elucidation the mechanisms of the treatment of AP with traditional Chinese medicine.

恢复促炎/抗炎因子平衡是缓解重症急性胰腺炎由全身炎症反应向多器官功能衰竭发展的关键。激活胆碱能抗炎通路可在转录后水平抑制巨噬细胞促炎因子释放、不影响抗炎因子释放，而有助于该平衡的恢复。分子刹车蛋白--蛋白激酶C受体1作用于内质网未折叠蛋白响应信号通路，可在转录后水平调控蛋白合成。我们前期研究证实中药验方柴芩承气汤可升高急性胰腺炎血清乙酰胆碱、上调巨噬细胞烟碱型乙酰胆碱受体表达，在转录后水平减少促炎因子释放。据上,我们提出假说：柴芩承气汤减少急性胰腺炎巨噬细胞释放促炎因子与内质网蛋白激酶C受体1的分子刹车蛋白作用有关。本项目检测急性坏死性胰腺炎小鼠腹腔巨噬细胞蛋白激酶C受体1等内质网未折叠蛋白响应信号通路相关蛋白及促炎、抗炎因子基因和蛋白表达的变化，研究柴芩承气汤对该病腹腔巨噬细胞未折叠蛋白响应信号通路的影响。有望丰富胆碱能抗炎通路学说，为深入阐明中药防治急性胰腺炎的作用机制获得创新发现。

未折叠蛋白响应（UPR）作为内质网应激反应的一部分，可平衡细胞器的蛋白折叠能力和需求。UPR包含至少三条信号通路，主要通过IRE1α、ATF 6和PERK。UPR已经证实了参与包括急性胰腺炎（AP）在内的许多疾病。本研究探索柴芩承气汤对急性坏死性胰腺炎（ANP）炎性细胞内质网UPR信号通路影响及机制。1）、蛋白激酶C受体1（RACK1）作用于IRE1α介导的UPR信号通路，调控蛋白合成。前期研究已发现柴芩承气汤（CQCQD）可升高急性胰腺炎血清乙酰胆碱（Ach），减少促炎因子释放。本部分研究发现CQCQD升高犬黄胆酸钠ANP犬血清Ach；CQCQD和PNU282987降低ANP血清IL-6及TNF-α水平；CQCQD和PNU282987可显著升高ANP肺泡巨噬细胞RACK1表达，并降低IRE1α表达。说明CQCQD可作用于胆碱能抗炎通路，下调ANP犬肺泡巨噬细胞IRE1α基因及蛋白表达，上调RACK1基因及蛋白表达，降低血清IL-6及TNF-α水平。2）、为进一步验证RACK1的潜在作用，本部分使用不同病情严重程度的大鼠AP模型，发现大鼠外周血白细胞中RACK1的表达与TNF-a和IL-6的表达负相关。该结果同样在急性胰腺炎患者的外周血白细胞中得到验证，认为RACK1可能起AP炎症的负调控作用。3）、作用转录因子(ATF6)所介导的信号通路是UPR信号通路之一。CQCQD可降低L-精氨酸ANP大鼠模型肺泡巨噬细胞内ATF6表达、血清IL-6、TNF-α水平，且对 IL-10无影响。发现ATF6可能同样是CQCQD抗炎的作用靶点。本课题使用不同的动物不同的模型，阐述了CQCQD在急性胰腺炎UPR相关通路中的作用，进一步为CQCQD的临床使用提供可靠的理论依据。