微RNA-128-3p、沉默信息调节因子1（SIRT1）和AMP活化蛋白激酶（AMPK）对2型糖尿病合并非酒精性脂肪性肝病的诊断价值

Abstract

目的 分析2型糖尿病 (T2DM) 合并非酒精性脂肪性肝病 (NAFLD) 患者外周血中微RNA (miRNA) -128-3p、沉默信息调节因子1 (SIRT1) 和AMP活化蛋白激酶 (AMPK) 的表达情况，探讨miRNA-128-3p对T2DM患者发生NAFLD的预测作用。 方法 选取2022年9月—2023年8月在安徽中医药大学第一附属医院住院的80例T2DM患者，分为T2DM组 (40例) 和合并NAFLD组 (40例) ，并依据肝纤维化评分 (NFS) 分为T2DM合并进行性肝纤维化组 (16例) 和T2DM未合并进行性肝纤维化组 (64例) ，收集基本资料和生化指标，采用定量实时PCR方法检测外周血miRNA-128-3p、SIRT1、AMPK的mRNA表达水平，Western Blot方法检测SIRT1、AMPK蛋白表达水平。正态分布的数据两组间比较采用成组t检验，偏态分布的数据两组间比较采用Mann-Whitney U检验，计数资料两组间比较采用χ²检验；Logistic回归分析NAFLD及进行性肝纤维化的影响因素；使用受试者操作特征曲线 (ROC曲线) 以确定根据miRNA-128-3p水平判断发生NAFLD的最佳阈值。 结果 合并NAFLD组和T2DM组BMI、空腹血糖、糖化血红蛋白、空腹胰岛素、空腹C肽、ALT、AST、GGT、ALP、纤维连接蛋白、TG、HDL-C、总三碘甲状腺原氨酸 (TT3) 、胰岛素抵抗指数 (HOMA-IR) 、NFS比较差异均有统计学意义 (P值均<0.05) 。合并NAFLD组外周血miRNA-128-3p的mRNA表达水平高于T2DM组 (t=-8.765，P<0.001) ，而SIRT1和AMPK的mRNA及蛋白表达水平均明显降低 (P值均<0.001) 。T2DM合并进行性肝纤维化组与T2DM未合并进行性肝纤维化组的年龄、ALT、游离三碘甲状腺原氨酸、TT3、超氧化物歧化酶、miRNA-128-3p比较差异均有统计学意义 (P值均<0.05) 。Logistic回归分析表明，miRNA-128-3p是发生NAFLD和进行性肝纤维化的独立危险因素 (OR=8.221，95%CI：2.735～24.714，P<0.001；OR=1.493，95%CI：1.117～1.997，P=0.007) ；ROC曲线显示其曲线下面积为0.890 (95%CI：0.829～0.950) ，最佳截断值为13.165，敏感度89.3%，特异度72.7%。 结论 miRNA-128-3p在T2DM合并NAFLD患者外周血中表达增高，SIRT1、AMPK表达降低，miRNA-128-3p水平对识别NAFLD及肝纤维化具有一定诊断价值。

Alternate abstract:

Objective To investigate the expression levels of miR-128-3p, SIRT1, and AMPK in the peripheral blood of patients with type 2 diabetes mellitus (T2DM) comorbid with nonalcoholic fatty liver disease (NAFLD), as well as the role of miR-128-3p in predicting NAFLD in T2DM patients. Methods A total of 80 patients with T2DM who were hospitalized in The First Affiliated Hospital of Anhui University of Chinese Medicine from September 2022 to August 2023 were enrolled and divided into T2DM group with 40 patients and NAFLD group with 40 patients, and according to the NAFLD fibrosis score (NFS), the patients were further divided into progressive liver fibrosis group with 16 patients and non-progressive liver fibrosis group with 64 patients. General data and biochemical parameters were collected; quantitative real-time PCR was used to measure the mRNA expression levels of miR-128-3p, SIRT1, and AMPK in peripheral blood, and Western blot was used to measure the protein expression levels of SIRT1 and AMPK. The independent-samples t test was used for comparison of normally distributed data between two groups, and the Mann-Whitney U test was used for comparison of data with skewed distribution between two groups; the chi-square test was used for comparison of categorical data between two groups. The logistic regression analysis was used to identify the influencing factors for the presence of NAFLD and progressive liver fibrosis, and the receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off value of miR-128-3p for predicting NAFLD. Results There were significant differences between the NAFLD group and the non-NAFLD group in body mass index, fasting plasma glucose, glycated hemoglobin, fasting insulin, fasting C-peptide, alanine aminotransferase (ALT), aspartate aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, fibronectin, triglycerides, high-density lipoprotein cholesterol, total triiodothyronine (TT3), Homeostasis Model Assessment of Insulin Resistance (HOMA-IR), and NFS (all P<0.05). Compared with the non-NAFLD group, the NAFLD group had a significantly higher mRNA expression level of miR-128-3p in peripheral blood (t=-8.765, P<0.001) and significant reductions in the mRNA and proteins expression levels of SIRT1 and AMPK (P<0.001). There were significant differences between the progressive liver fibrosis group and the non-progressive liver fibrosis group in age, ALT, free triiodothyronine, TT3, superoxide dismutase, and miR-128-3p (all P<0.05). The logistic regression analysis showed that miR-128-3p was an independent risk factor for the development of NAFLD (odds ratio [OR]=8.221, 95% confidence interval [CI]: 2.735 — 24.714, P<0.001) and progressive liver fibrosis (OR=1.493, 95%CI: 1.117‍ ‍- ‍1.997, P=0.007). The ROC curve analysis showed that miR-128-3p had an area under the ROC curve of 0.890 (95%CI: 0.829 — 0.950), with an optimal cut-off value of 13.165, a sensitivity of 89.3%, and a specificity of 72.7%. Conclusion There is an increase in the expression of miR-128-3p in peripheral blood of T2DM patients with NAFLD, while there are reductions in the expression levels of SIRT1 and AMPK, suggesting that miR-128-3p has a certain diagnostic value in identifying NAFLD and liver fibrosis in such population.