Objective: To investigate the relationship between gut microbiota structure and biochemical changes in patients with different types of nonalcoholic fatty liver disease (NAFLD), in order to provide evidence for clinical diagnosis and prevention of NAFLD. Methods: Forty-eight NAFLD cases (NAFLD group), 40 NAFLD cases with type 2 diabetes mellitus (NAFLD combined with type 2 diabetes mellitus group) and 30 healthy cases (healthy group) were randomly enrolled, and their body mass index, serum alanine aminotransferase, aspartate aminotransferase, total bilirubin, total cholesterol, triglyceride, high density lipoprotein, low density lipoprotein and uric acid were measured. Serum levels of TNF-alpha and fasting insulin were measured using ELISA, and then insulin resistance index was calculated. The gut microbiota of three groups of subjects was detected using 16S rDNA-based high-throughput sequencing. Lastly, the correlations between the various factors were analyzed. The comparison among groups was conducted by 2 test, and one-way ANOVA was used for comparison among groups with normal distribution and homogeneity of variance. Furthermore, the LSD method was used to compare the two groups. K-W rank sum test was used for comparison among groups without normal distribution or homogeneity of variance. Results: Body mass index, aspartate aminotransferase, triglyceride, total cholesterol, low density lipoprotein, uric acid, tumor necrosis factor-alpha, fasting insulin and insulin resistance index of NAFLD group were higher than healthy group, while the high-density lipoprotein was lower in the healthy group, and the difference was statistically significant (P< 0.05). Compared with NAFLD group, the life expectancy, fasting blood glucose and insulin resistance index of NAFLD combined with type 2 diabetes mellitus group were higher, while the body mass index, aspartic acid aminotransferase, total cholesterol and HDL levels were decreased, and the difference was statistically significant (P< 0.05). NAFLD group (P= 0.016) had decreased abundance of firmicutes than healthy group, and the abundancy of the firmicutes in the NAFLD combined with type 2 diabetes group was significantly lower (P< 0.001). The abundance of bacteroidetes in NAFLD combined with type 2 diabetes group was higher than healthy group, and the difference was statistically significant (P= 0.006). At the "genus level," the abundance of Roseburia and Subdoligranulum in the NAFLD group was decreased, while the Roseburia in the NAFLD group with type 2 diabetes group was significantly lower (P< 0.05). In addition, the abundance of Faecalibacterium, Blautia, Anaerostipes and Fusicatenibacter in NAFLD combined with type 2 diabetes group was lower than healthy group, and the difference was statistically significant (P< 0.001). Fusicatenibacter, Blautia, Anaerostipes, Faecalibacterium, and Roseburia were negatively correlated with fasting blood glucose and insulin resistance index levels (r< 0,P< 0.05), and positively correlated with high-density lipoprotein levels (r> 0,P< 0.05). Fusicatenibacter was negatively correlated with tumor necrosis factor-alpha (r= -0.211,P= 0.044), and Lachnoclostridium was positively correlated with body mass index, alanine aminotransferase, aspartate aminotransferase levels (r> 0,P< 0.05). Fusobacterium was positively correlated with aspartate aminotransferase level (r= 0.245,P= 0.019). Escherichia-shigella was positively correlated with fasting blood glucose, low-density lipoprotein, alanine aminotransferase, aspartate aminotransferase levels (r > 0,P< 0.05). Megamonas was negatively correlated with high-density lipoprotein levels (r= -0.231,P= 0.027). Conclusion: A structural change of gut microbiota had occurred in patients with NAFLD, suggesting changes in some of these bacterial genuses had relation to insulin resistance and inflammatory response, which may become a new target for the treatment of NAFLD.
目的： 探讨不同类型非酒精性脂肪性肝病(NAFLD)患者肠道菌群结构与生物化学指标改变的相关性，以期为NAFLD临床诊断和防治提供依据。 方法： 随机纳入NAFLD患者（NAFLD组）48例、NAFLD合并2型糖尿病患者（NAFLD合并2型糖尿病组）40例和体检健康者（健康组）30例，检测其体质量指数及血清中丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总胆红素、总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白、尿酸和空腹血糖，用酶联免疫吸附法检测血清中肿瘤坏死因子-α和空腹胰岛素并计算胰岛素抵抗指数。用16S rDNA高通量测序检测3组对象肠道菌群，最后综合分析各研究因素间的相关性。各组之间的比较采用χ(2)检验，符合正态分布且方差齐的多组间比较采用单因素方差分析，进一步两两比较用LSD法，不符合正态分布或方差齐性的多组间比较采用K-W秩和检验。 结果： NAFLD组体质量指数、天冬氨酸氨基转移酶、甘油三酯、总胆固醇、低密度脂蛋白、尿酸、肿瘤坏死因子-α、空腹胰岛素、胰岛素抵抗指数均高于健康组，高密度脂蛋白低于健康组，差异均有统计学意义(P值均< 0.05)。与NAFLD组比较，NAFLD合并2型糖尿病组年龄、空腹血糖和胰岛素抵抗指数升高，体质量指数、天冬氨酸氨基转移酶、总胆固醇和高密度脂蛋白降低，差异均有统计学意义(P值均< 0.05)。与健康组比较，NAFLD组厚壁菌门丰度降低（P = 0.016），NAFLD合并2型糖尿病组厚壁菌门丰度降低（P < 0.001)。NAFLD合并2型糖尿病组拟杆菌门丰度高于健康组，差异有统计学意义（P = 0.006)。在"属"水平，NAFLD组Roseburia、Subdoligranulum的丰度降低，且NAFLD合并2型糖尿病组Roseburia明显降低，差异均有统计学意义（P值均< 0.05)；此外，NAFLD合并2型糖尿病组Faecalibacterium、Blautia、Anaerostipes、Fusicatenibacter丰度均低于健康组，差异均有统计学意义（P值均< 0.001）。Fusicatenibacter、Blautia、Anaerostipes、Faecalibacterium、Roseburia均与空腹血糖、胰岛素抵抗指数水平负相关（r < 0,P < 0.05)，与高密度脂蛋白水平正相关（r > 0，P < 0.05)；Fusicatenibacter与肿瘤坏死因子-α水平负相关（r = -0.211，P = 0.044）；Lachnoclostridium与体质量指数、丙氨酸氨基转移酶、天冬氨酸氨基转移酶水平正相关（r > 0，P < 0.05）；Fusobacterium与天冬氨酸氨基转移酶水平正相关（r = 0.245,P = 0.019)；Escherichia-shigella与空腹血糖、低密度脂蛋白、丙氨酸氨基转移酶、天冬氨酸氨基转移酶水平正相关（r > 0，P < 0.05）；Megamonas与高密度脂蛋白水平负相关（r = -0.231，P = 0.027）。 结论： NAFLD患者肠道菌群结构发生了改变，部分菌属的改变与胰岛素抵抗和炎症反应有关，这可能成为治疗NAFLD的新"靶点"。.
Keywords: Fatty liver, non-alcoholic; Gut microbiota; Inflammatory reaction; Insulin resistance.