Nanjing University of Chinese Medicine, China.
Jiangsu Province Hospital of TCM, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China.
J Ethnopharmacol. 2020 Jun 12;255:112738. doi: 10.1016/j.jep.2020.112738. Epub 2020 Mar 5.
Qian Yang Yu Yin Granule (QYYY) is a Chinese herbal formulation. It is used to treat hypertensive nephropathy for decades in China, but it is unknown that the exact mechanism of QYYY on hypertensive nephropathy.
The present study was to elucidate its epigenetic mechanism of QYYY on hypertensive nephropathy.
In the current study, HEK293T cells' proliferation induced by Ang II was chosen to observe epigenetic mechanisms of QYYY on renal damage. The cell proliferation was examined by MTT assays and ethynyldeoxyuridine analysis. Cell cycle analysis was performed. After treatment with QYYY, expression of Nicotinamide N-methyltransferase (NNMT), sirtuin1(SIRT1), S-adenosylhomocysteine(SAH), histone H3K4 methylation, and cortactin acetylation(acetyl-cortactin,ac-cortactin) were further investigated by western-blotting and real time PCR. DNA methylation was detected by ELISA. The study also observed the changes of SIRT1, SAH, H3K4 methylation, acetyl-cortactin when NNMT over-expressed by lentivirus transfection. Angiotensin II(Ang II) induced renal damage in spontaneously hypertensive rats(SHR). After eight weeks treatment of QYYY, blood pressure, serum and urine creatinine, and urinary microalbumin(mAlb) were assessed. The concentration of N1 -methylnicotinamide were detected by liquid chromatography with tandem mass spectrometry. The protein of NNMT, ac-cortactin, H3K3me3 were also assessed in vivo.
QYYY inhibited HEK293T cells' proliferation, down-regulated the expression of NNMT, SAH, acetyl-cortactin and DNA methylation, up-regulated the expression of SIRT1, histone H3K4 trimethylation(H3K4me3). Over-expression of NNMT increased the expression of SAH and acetyl-cortactin, and reduced the expression of SIRT1 and H3K4me3. The study also demonstrated that QYYY promoted urinary creatinine excretion and reduced serum creatinine and urinary mAlb in SHR. QYYY decreased the concentration of N1 -methylnicotinamide in Ang II group. QYYY decreased the protein of NNMT, ac-cortactin and increased H3K4me3 in vivo.
The results showed that QYYY alleviated renal impairment of SHR and inhibited HEK293T cells' proliferation induced by Ang II through the pathway of epigenetic mechanism linked to Nicotinamide N-Methyltransferase (NNMT) expression, including histone methylation, DNA methylation and acetyl-cortactin. This study unveiled a novel molecular mechanism by which QYYY controlled the progression of hypertensive nephropathy.
千阳育阴颗粒（QYYY）是一种中药配方。它在中国被用于治疗高血压性肾病已有几十年的历史，但目前尚不清楚 QYYY 治疗高血压性肾病的确切机制。
本研究旨在阐明 QYYY 治疗高血压性肾病的表观遗传机制。
在本研究中，选择用 Ang II 诱导的 HEK293T 细胞增殖来观察 QYYY 对肾损伤的表观遗传机制。通过 MTT 分析和乙炔脱氧尿苷分析来检测细胞增殖。进行细胞周期分析。用 QYYY 处理后，通过 Western blot 和实时 PCR 进一步研究烟酰胺 N-甲基转移酶（NNMT）、沉默调节蛋白 1（SIRT1）、S-腺苷同型半胱氨酸（SAH）、组蛋白 H3K4 甲基化和纽蛋白（cortactin）乙酰化（乙酰化-cortactin，ac-cortactin）的表达。用 ELISA 检测 DNA 甲基化。研究还观察了通过慢病毒转染过表达 NNMT 时 SIRT1、SAH、H3K4 甲基化、乙酰化-cortactin 的变化。用 Angiotensin II（Ang II）诱导自发性高血压大鼠（SHR）的肾损伤。用 QYYY 治疗 8 周后，评估血压、血清和尿液肌酐以及尿液微量白蛋白（mAlb）。用液相色谱-串联质谱法检测 N1 -甲基烟酰胺的浓度。体内还评估了 NNMT、ac-cortactin、H3K3me3 的蛋白表达。
QYYY 抑制 HEK293T 细胞增殖，下调 NNMT、SAH、乙酰化-cortactin 和 DNA 甲基化的表达，上调 SIRT1、组蛋白 H3K4 三甲基化（H3K4me3）的表达。过表达 NNMT 增加了 SAH 和乙酰化-cortactin 的表达，降低了 SIRT1 和 H3K4me3 的表达。该研究还表明，QYYY 促进了 SHR 的尿肌酐排泄，降低了血清肌酐和尿 mAlb。QYYY 降低了 Ang II 组中 N1 -甲基烟酰胺的浓度。QYYY 降低了体内 NNMT、ac-cortactin 的蛋白表达，增加了 H3K4me3 的蛋白表达。
结果表明，QYYY 通过与烟酰胺 N-甲基转移酶（NNMT）表达相关的表观遗传机制，包括组蛋白甲基化、DNA 甲基化和纽蛋白（cortactin）乙酰化，缓解了 SHR 的肾损伤，并抑制了 Ang II 诱导的 HEK293T 细胞增殖。本研究揭示了 QYYY 控制高血压性肾病进展的新分子机制。
