高糖抑制平滑肌细胞胶原合成参与糖尿病静脉病变发生

doi:10.3877/cma.j.issn.2095-5782.2020.01.013

目的

观察糖尿病大鼠大隐静脉（SV）的组织学改变，通过高糖处理静脉平滑肌细胞（SMCV）观察其活性及其Ⅰ型胶原（COL Ⅰ）、Ⅲ型胶原（COL Ⅲ）、基质金属蛋白酶2（MMP2）、基质金属蛋白酶组织抑制剂1(TIMP1)表达变化，探讨其表达异常的可能机制。

方法

用链脲佐菌素（STZ）腹腔注射构建糖尿病大鼠模型，对其SV进行HE染色、Masson染色观察其组织形态改变；体外培养SMCV，分别给与高糖、正常糖浓度培养，CCK8法检测其活性，RT-qPCR法、Western Blot（WB）法分别检测其细胞内COLⅠ、COLⅢ、MMP2、TIMP1 mRNA及蛋白表达，ELISA法检测细胞外COL Ⅰ、COLⅢ的表达。

结果

糖尿病大鼠SV管壁中膜变薄（P<0.05）、管腔面积增大（P>0.05）、胶原含量减少（P>0.05）。高糖刺激后SMCV细胞活性降低（P<0.05），细胞内COLⅠ mRNA无明显变化，COLⅢ mRNA表达下降（P<0.05），TIMP1 mRNA表达升高（P<0.05），MMP2 mRNA表达下降（P<0.05）。此外，高糖刺激后细胞内COLⅠ蛋白表达显著降低，COLⅢ蛋白表达无明显差异，TIMP1表达下降，MMP2表达升高，细胞外COLⅠ蛋白表达显著降低（P<0.05），COLⅢ表达降低但无统计学差异，COLⅠ/COLⅢ表达比例下降（P<0.05）。

结论

本研究证实糖尿病下肢静脉中膜变薄，管腔增大，胶原减少。高糖可能是通过转录后机制调控MMP2/TIMP1水平，影响SMCV细胞COLⅠ/COLⅢ的表达，抑制其胶原合成，参与静脉病变的发生。

关键词: 糖尿病, 慢性静脉疾病, 细胞外基质, 胶原

Objective

To determine the influence of diabetes on histological profile of lower limb veins, and to explore the mechanism of the effects of high-glucose medium in the viability and Collagen expression of venous smooth muscle cells (SMCV).

Methods

Diabetic rat model was established with intraperitoneal injection of streptozotocin. Hematoxylin-eosin (HE) and Masson staining were performed to observe the histological changes of great saphenous veins (SV). SMCVs were cultured in medium of high (DM group) or normal (NC group) levels of glucose, and the SMCVs viability was evaluated by cell counting kit-8 (CCK-8). The levels of TypeⅠ Collagen (COLⅠ), Type Ⅲ Collagen (COL Ⅲ), Metalloproteinase 2 (MMP2), Tissue inhibitor of metalloproteinase 1 (TIMP1) protein and mRNA expression in the cells were detected by Western Blot (WB) and Real-time quantitative Polymerase Chain Reaction (RT-qPCR). The contents of COLⅠ and COL Ⅲ in the cellular supernatant were detected using ELISA kit.

Results

Vein samples acquired from the diabetic rats showed lower medial thickness (P<0.05), larger luminal area (P>0.05) and lower collagen content (P>0.05). The viability of VSMCs was significantly declined in the DM group (P<0.05). There was no significant difference in the expression of COLⅠ mRNA between groups. The levels of COL Ⅲ mRNA was decreased in the DM group (P<0.05), followed by an ascending level of TIMP1 mRNA (P<0.05) and a declined level of MMP2 mRNA (P<0.05). WB analysis demonstrated that high-glucose treatment decreased the expression of COLⅠ and TIMP1 in SMCVs, accompanied with an increased expression of MMP2. ELISA showed that the expression of COLⅠ (P<0.05) and the ratio of COLⅠ / COL Ⅲ (P<0.05) in the cellular supernatant of DM group were also decreased.

Conclusions

The lower limb veins in diabetes presented histological changes as a lower medial thickness, larger luminal area and lower collagen content. High glucose treatment decreased the expression of COLⅠ and TIMP1 of SMCVs, and increased the expression of MMP2, which may suggest that high glucose inhibit the collagen expression of SMCVs via the regulation of MMP2/TIMP1 expression.

Key words: Diabetes mellitus, Chronic venous disease, Extracellular matrix, Collagen

图1 大鼠大隐静脉HE染色结果

表1 RT-qPCR引物序列

Gene	Sequence
COL1A1	GGCAACCTCAAGAAGTCCCT
	CCTGGTCTGGGGATACTCAC
COL3A1	AGGGCAGGGAACAACTGATG
	GGTCCCACATTGCACAAAGC
TIMP1	GGCACAGCTACAGGCTTTAC
	CCTGAGTCTCCCTAGAGCCAA
MMP2	ACCTTGACCAGAACACCATCGAG
	CAGGGTCCAGGTCAGGTGTGTA
β-actin	GGAGATTACTGCCCTGGCTCCTA
	GACTCATCGTACTCCTGCTTGCTG

表1 RT-qPCR引物序列

Gene	Sequence
COL1A1	GGCAACCTCAAGAAGTCCCT
	CCTGGTCTGGGGATACTCAC
COL3A1	AGGGCAGGGAACAACTGATG
	GGTCCCACATTGCACAAAGC
TIMP1	GGCACAGCTACAGGCTTTAC
	CCTGAGTCTCCCTAGAGCCAA
MMP2	ACCTTGACCAGAACACCATCGAG
	CAGGGTCCAGGTCAGGTGTGTA
β-actin	GGAGATTACTGCCCTGGCTCCTA
	GACTCATCGTACTCCTGCTTGCTG

图2 大鼠大隐静脉Masson染色结果

图3 SMCV a-SMA免疫荧光染色结果

图4 高糖处理后SMCV CCK8实验结果

图5 高糖培养72 h后SMCV细胞内mRNA检测结果（^*P<0.05)

图6 高糖培养后SMCV细胞内外相关蛋白表达情况

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High glucose inhibits the collagen expression of SMCVs in diabetes and participates in the development of diabetic venous disease

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High glucose inhibits the collagen expression of SMCVs in diabetes and participates in the development of diabetic venous disease