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摘要: 钙联蛋白（Calnexin）是内质网上的凝集素样分子伴侣蛋白，也是线粒体结合内质网膜上的重要蛋白，在心脏疾病中发挥着重要作用，但其在心肌成纤维细胞活化中的作用和机制并未阐明。本研究通过小鼠胸主动脉缩窄（TAC）模型来观察体内心肌纤维化情况，并利用心脏超声，苏木精-伊红、Masson 和天狼星红染色，以及心脏纤维化标志物表达水平，鉴定模型是否成功。体外实验利用转化生长因子 TGFβ1 刺激建立心肌成纤维细胞活化模型，通过定量 PCR、Western blot、EdU 渗入法及划痕实验等方法检测其活化、细胞增殖、迁移的变化。利用腺病毒过表达和沉默 Calnexin，来观察 Calnexin 在体外心肌成纤维细胞活化中的作用。结果显示，Calnexin 在 TAC 模型和体外心肌成纤维细胞活化过程中均表达降低。过表达 Calnexin 能缓解心肌成纤维细胞活化；相反，沉默 Calnexin 导致其活化加重。进一步探究发现，内质网应激在心肌成纤维细胞活化过程中被激活，过表达 Calnexin 后内质网应激缓解，相反，敲低 Calnexin 后内质网应激加重，与活化表型变化一致。结果表明，过表达 Calnexin 可能通过缓解内质网应激从而减轻心肌成纤维细胞的活化程度。Abstract: Calnexin is a lectin-like molecular chaperone protein on the endoplasmic reticulum, mediating unfolded protein responses, the endoplasmic reticulum Ca2+ homeostasis, and Ca2+ signals conduction. In recent years, studies have found that calnexin plays a key role in the heart diseases. This study aims to explore the role of calnexin in the activation of cardiac fibroblasts. A transverse aortic constriction (TAC) mouse model was established to observe the activation of cardiac fibroblasts in vivo, and the in vitro cardiac fibroblasts activation model was established by transforming growth factor β1 (TGFβ1) stimulation. The adenovirus was respectively used to gene overexpression and silencing calnexin in cardiac fibroblasts to elucidate the relationship between calnexin and cardiac fibroblasts activation, as well as the possible underlying mechanism. We confirmed the establishment of TAC model by echocardiography, hematoxylin-eosin, Masson, and Sirius red staining, and detecting the expression of cardiac fibrosis markers in cardiac tissues. After TGFβ1 stimulation, markers of the activation of cardiac fibroblast, and proliferation and migration of cardiac fibroblast were detected by quantitative PCR, Western blot, EdU assay, and wound healing assay respectively. The results showed that the calnexin expression was reduced in both the TAC mice model and the activated cardiac fibroblasts. The overexpression of calnexin relieved cardiac fibroblasts activation, in contrast, the silencing of calnexin promoted cardiac fibroblasts activation. Furthermore, we found that the endoplasmic reticulum stress was activated during cardiac fibroblasts activation, and endoplasmic reticulum stress was relieved after overexpression of calnexin. Conversely, after the silencing of calnexin, endoplasmic reticulum stress was further aggravated, accompanying with the activation of cardiac fibroblasts. Our data suggest that the overexpression of calnexin may prevent cardiac fibroblasts against activation by alleviating endoplasmic reticulum stress.
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图 1 Calnexin 在小鼠 TAC 模型中的表达降低
a.心脏重量与体重比值；b.小鼠 TAC 术后超声心动图检测图像；c-g. 超声心动图检测心功能指标；h. 心脏组织病理切片；i. 纤维化指标 mRNA 相对表达量；j. Calnexin 的 mRNA 相对表达量；k. Calnexin 的蛋白相对表达量；l. Calnexin 在 TAC 术后不同时间的 mRNA 相对表达量。n = 6，与 sham 组相比，* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.000 1
Figure 1. The expression of calnexin was decreased in TAC model of mice
a. heart weight/body weight; b. representative images of an echocardiographic assessment of mice after TAC; c-g. the cardiac function indicators by echocardiogram; h. the stained sections of hearts; i. the mRNA levels of COL-1 and α-SMA; j. the mRNA level of calnexin; k. the protein level of calnexin; l. the mRNA levels of calnexin with time gradient. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, compared with sham group
图 2 Calnexin 在心肌成纤维细胞活化过程中的表达降低
a. EdU 渗入法检测心肌成纤维细胞的增殖能力；b. 划痕实验检测心肌成纤维细胞的迁移能力；c. 原代心肌成纤维细胞荧光染色；d. 心肌成纤维细胞活化标志物 mRNA 的相对表达量；e. Calnexin 的蛋白相对表达量。n = 6，与对照组相比，* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.000 1
Figure 2. The expression of calnexin was decreased in activated cardiac fibroblasts
a. the proliferation rate of cells by EdU staining; b. the migration capability of cardiac fibroblasts by wound healing assay; c. immunofluorescence staining of α-SMA in cardiac fibroblasts; d. the mRNA levels of fibrotic markers; e. the protein level of calnexin. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1, compared with control group
图 3 过表达 Calnexin 缓解 TGFβ1 诱导的心肌成纤维细胞活化
a. 腺病毒转导效率检测；b. 过表达 Calnexin 后心肌成纤维细胞活化指标 mRNA 水平变化；c. EdU 渗入法检测心肌成纤维细胞的增殖能力；d. 划痕实验检测心肌成纤维细胞的迁移能力；e. 心肌成纤维细胞荧光染色；f. 心肌成纤维细胞活化标志物 mRNA 的相对表达量。n = 6，* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.000 1
Figure 3. Calnexin overexpression alleviated the activation of cardiac fibroblasts induced by TGFβ1
a. the transduction effect of adenovirus; b. the mRNA levels of fibrosis markers; c. the proliferation rate of cells by EdU staining; d. the migration capability of cardiac fibroblasts by wound healing assay; e. immunofluorescence staining of α-SMA in cardiac fibroblasts; f. the mRNA levels of CTGF, α-SMA and TGF-β. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1
图 4 敲低 Calnexin 加重 TGFβ1 诱导的心肌成纤维细胞活化
a. 腺病毒转导效率检测；b. 沉默 Calnexin 后心肌成纤维细胞活化指标 mRNA 水平变化；c. EdU 渗入法检测心肌成纤维细胞的增殖能力；d. 划痕实验检测心肌成纤维细胞的迁移能力；e. 心肌成纤维细胞荧光染色；f. 心肌成纤维细胞活化标志物蛋白相对表达量。n = 6，* P < 0.05，** P < 0.01，*** P < 0.001，**** P < 0.000 1
Figure 4. Calnexin knockdown aggravated the activation of cardiac fibroblasts induced by TGFβ1
a. the transduction effect of adenovirus; b. the mRNA levels of fibrosis markers; c. the proliferation rate of cells by EdU staining; d. the migration capability of cardiac fibroblasts by wound healing assay; e. immunofluorescence staining of α-SMA in cardiac fibroblasts; f. the protein levels of CTGF, α-SMA and TGF-β. n = 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.000 1
图 5 Calnexin 影响 ER stress 来调节 TGFβ1 诱导的心肌成纤维细胞活化
a. TGFβ1 诱导的心肌成纤维细胞活化激活 ER stress；b. 病毒敲低 Calnexin 后，心肌成纤维细胞中 ER stress 通路蛋白的表达水平；c. 病毒过表达 Calnexin 后，心肌成纤维细胞中 ER stress 通路蛋白的表达水平。* P < 0.05，** P < 0.01，*** P < 0.001
Figure 5. Calnexin regulates ER stress in TGFβ1-induced cardiac fibroblasts activation
a. the protein expression of BIP, p-IRE1α, ATF4, and c-ATF6 with TGFβ1 treatment in cardiac fibroblasts; b. the protein levels of BIP, p-IRE1α, ATF4, and c-ATF6 with sh-adenovirus transduction in cardiac fibroblasts; c. the protein levels of BIP, P-IRE1α, ATF4, and c-ATF6 with overexpression-adenovirus transfection in cardiac fibroblasts. * P < 0.05, ** P < 0.01, *** P < 0.001
表 1 qPCR 反应使用的引物序列
Table 1. The sequences of primers in qPCR
基因 引物序列 Calnexin F: ATGGAAGGGAAGTGGTTACTGT R: GCTTTGTAGGTGACCTTTGGAG α-SMA F: ACAACTGGTATTGTGCTGGACT R: TCAGCAGTAGTCACGAAGGAAT TGF-β F: TGAGTGGCTGTCTTTTGACG R: ACTGAAGCGAAAGCCCTGTA COL-1 F: ACGTCCTGGTGAAGTTGGTC R: TCCAGCAATACCCTGAGGTC CTGF F: CTACCGACTGGAAGACACATTT R: TTCCTCTAGGTCAGCTTCACAG β-actin F: ACTATCGGCAATGAGCGGTTC R: ATGCCACAGGATTCCATACCC 
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