基于线粒体-内质网交互作用和钙稳态失衡探讨丁酸钠抗结直肠癌作用及其机制

Colorectal cancer(CRC) is one of the most common cancers. Sodium butyrate(NaB), a short-chain fatty acid salt derived from dietary fiber fermentation, can inhibit proliferation and induce apoptosis and autophagy in multiple cancer cells. Mitochondria-associated endoplasmic reticulum membrane(MAM) as an interactive platform of mitochondria and endoplasmic reticulum membrane, has attracted considerable attention because of its various biological effects generated by the regulation of calcium transportation. Our previous study suggested that NaB decreased the calcium concentration in endoplasmic reticulum while increasing the cellular concentration of calcium. This process could lead to the autophagy and apoptosis of colorectal cancer cells by inducing endoplasmic reticulum stress. However, it is still unclear whether the anti-CRC effects of NaB is mediated by the interaction between mitochondria and endoplasmic reticulum or calcium dyshomeostasis. Based on our previous results, this study purposes to explore whether calcium dyshomeostasis, which is induced by NaB and regulated by mitochondria and endoplasmic reticulum, is involved in the anti-CRC mechanism of NaB. Gradient centrifugation, transmission electron microscope, RNAi, intracellular calcium imaging, Co-Immunoprecipitation, western blotting and other biological techniques will be used to investigate this potential process. Relative results can provide scientific and theoretical basis for the clinical development of NaB treatment of colorectal cancer drugs.

结直肠癌是最常见的恶性肿瘤之一。丁酸钠(sodium butyrate, NaB)作为膳食纤维发酵产生的一种短链脂肪酸盐类，可抑制多种肿瘤细胞增殖、促进自噬和凋亡。线粒体-内质网交互作用及其促发的钙稳态失衡具有多种生物学作用，是近年来的研究热点。本课题组的前期结果显示，NaB可下调内质网中钙浓度、升高胞质中钙浓度，继而通过钙池调控胞外钙内流诱导细胞凋亡，NaB也可促发内质网应激诱导结直肠癌细胞自噬。那么，NaB是否促发线粒体和内质网交互作用，调控线粒体、内质网和胞质内钙稳态失衡，从而发挥其抗结直肠癌作用？本研究拟通过体外实验如梯度离心、透射电镜、RNAi、细胞内钙成像和免疫共沉淀等方法，以及体内裸鼠成瘤试验和炎性相关性结直肠癌模型，探讨NaB是否通过促发线粒体-内质网交互作用和钙稳态失衡，继而发挥其抗结直肠癌作用，为临床上开发NaB作为结直肠癌的治疗药物提供新靶点和理论依据。

结直肠癌是全球范围内最常见的恶性肿瘤之一。丁酸钠（sodium butyrate, NaB）作为膳食纤维发酵产生的一种短链脂肪酸盐类，可抑制多种肿瘤细胞增殖、促进自噬和凋亡。线粒体-内质网交互作用及其促发的钙稳态失衡具有多种生物学作用，是近年来的研究热点。本课题在前期研究结果，即NaB可促发胞质和内质网中钙稳态失衡、促发内质网应激并诱导结直肠癌自噬和凋亡的基础上，进一步探讨NaB是否促发线粒体和内质网交互作用，形成线粒体-内质网膜（mitochondrial-associated endoplasmic reticulum membrane，MAM）结构偶联，并探讨参与MAM偶联的相关分子及其抗结直肠癌机制，最后通过炎性相关性结直肠癌动物模型和裸鼠成瘤试验等动物模型进一步探讨NaB体内抗结直肠癌作用和机制。取得主要结果如下：（1）NaB处理结直肠癌细胞，透射电子显微镜检测到线粒体和内质网存在显著的共定位，MAM区线粒体膜蛋白VDAC1、内质网和线粒体连接蛋白GRP75和MFN2等蛋白表达量无显著变化，内质网膜蛋白IP3R1和线粒体钙离子通道MCU表达量显著上调，与NaB促发结直肠癌细胞内钙稳态失衡、线粒体自噬和凋亡等生物学效应密切相关。（2）炎症相关性结直肠癌动物模型结果显示，NaB可通过上调MCU、IP3R1分子等表达、线粒体自噬-NLRP3炎症小体，以及调节肠道菌群构成等途径显著逆转造模组小鼠体重下降、死亡率增加、肿瘤负荷增加和肿瘤组织增殖活性，发挥其预防和治疗结直肠癌作用。（3）体内裸鼠成瘤试验结果显示，丁酸前体物三丁酸甘油酯（Tributyrin, TB）可显著抑制裸鼠皮下肿瘤生长，对结直肠癌具有较好的治疗作用，相关机制尚在进一步探讨中。综上所述，NaB可通过上调MCU和IP3R1的表达促发MAM偶联形成，引起钙稳态失衡，进而诱导结直肠癌细胞线粒体自噬和凋亡等，发挥其抗结直肠癌作用；体内动物试验模型进一步证实NaB具有防治结直肠癌作用，为临床上开发NaB为结直肠癌的防治药物提供新靶点和理论依据，为指导我国居民合理摄入富含膳食纤维的食物提供指导。