Effect of Buyang Huanwu Decoction on Delaying Vascular Aging Based on miR-665/DRAM1 Signaling-Mediated Autophagy
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摘要:
目的 研究补阳还五汤对血管衰老的延缓作用, 探讨其机制是否与microRNA-665(miR-665)/DNA损伤调节自噬调控因子1(DNA damage-regulated autophagy modulator 1, DRAM1)信号介导的自噬有关。 方法 将自然衰老雄性SD大鼠随机分为衰老组, 补阳还五汤低、中、高(9.25、18.5、37.0 g·kg-1)剂量组和白藜芦醇组(80 mg·kg-1), 同时设立年轻组。分离胸主动脉, ELISA法测定血管组织衰老相关β-半乳糖苷酶(Senescence associated β-galactosidase, SA-β-Gal)活性和晚期糖基化终末产物(Advanced glycation end products, AGEs)水平; HE、Masson和EVG染色观察血管组织形态结构; qPCR检测血管组织miR-665表达; 生物信息学分析和双荧光素酶报告基因实验验证miR-665与DRAM1靶向关系; 透射电镜观察血管内自噬小体; Western blot法检测血管组织p16、DRAM1蛋白及自噬相关蛋白LC3、Beclin1和p62的表达; 免疫组织化学法检测血管组织DRAM1的蛋白表达。 结果 与年轻组相比，衰老组大鼠血管中SA-β-Gal活性、AGEs水平和p16蛋白表达增加(P＜0.01)；血管组织排列紊乱，中膜增厚，胶原纤维增加，弹力纤维出现断裂、紊乱；miR-665基因表达上调(P＜0.01)；自噬小体数量减少，Beclin1和LC3Ⅱ/Ⅰ蛋白表达降低(P＜0.01)，p62蛋白表达升高(P＜0.01)；DRAM1蛋白表达降低(P＜0.01)。与衰老组相比，补阳还五汤和白藜芦醇干预能够降低衰老大鼠血管中SA-β-Gal活性(P＜0.01)、AGEs水平和p16蛋白表达(P＜0.05，P＜0.01)；改善血管形态和弹力纤维结构，降低血管组织胶原纤维含量。高剂量补阳还五汤明显下调miR-665基因表达(P＜0.01)，增加血管内自噬小体数量；中剂量和高剂量补阳还五汤明显上调Beclin1和LC3Ⅱ/Ⅰ蛋白表达(P＜0.01)，下调p62蛋白表达(P＜0.05，P＜0.01)；高剂量补阳还五汤明显上调DRAM1蛋白表达(P＜0.05)。生物信息学分析显示，miR-665与DRAM1基因序列存在特异性互补结合位点，双荧光素酶报告实验证实miR-665靶向DRAM1基因并负调控DRAM1蛋白表达。 结论 补阳还五汤可能通过靶向抑制miR-665促进DRAM1蛋白表达, 进而促进血管自噬, 延缓血管衰老。 Abstract:OBJECTIVE This study aimed to investigate the effects of Buyang Huanwu Decoction (BYHWD) on delaying vascular aging and explore whether the underlying mechanism is associated with microRNA-665 (miR-665)/DNA damage-regulated autophagy modulator1 (DRAM1)-mediated autophagy. METHODS Male SD rats with natural aging were randomly divided into the aging group, BYHWD low, medium, high dosage groups (9.25, 18.5, 37.0 g·kg-1) and resveratrol group (80 mg·kg-1), with a young group set as well. The rats in each group were dissected and the blood vessels were collected. ELISA was used to assess senescence associated β-galactosidase (SA-β-Gal) activity and advanced glycation end products (AGEs) level in vascular tissues. HE, Masson, and EVG staining were performed to observe the morphological structure of the vascular tissues. The qPCR was performed to detect the expression of miR-665 in vascular tissues. Bioinformatics analysis and dual-luciferase reporter gene experiments were used to validate the targeting relationship between miR-665 and DRAM1. Transmission electron microscope was used to observe the autophagosome. Western blot was performed to determine the protein expression of p16, DRAM1 and autophagy-related proteins Beclin1, p62 and LC3. Immunohistochemistry was used to detect the protein expression of DRAM1 in vascular tissues. RESULTS Compared to the young group, the aging group showed increased SA-β-Gal activity, AGEs level and p16 protein expression (P < 0.01), disordered arrangement of vascular tissues, thickened media, increased collagen fibers, fractured and disorganized elastic fibers. The expression of miR-665 was upregulated (P < 0.01). The number of autophagosomes was reduced. The protein expression of Beclin1 and LC3Ⅱ/Ⅰ downregulated (P < 0.01), while the protein expression of p62 was upregulated (P < 0.01). In addition, the protein expression of DRAM1 was downregulated in vascular tissues (P < 0.01). Compared to the aging group, intervention with BYHWD and resveratrol reduced SA-β-Gal activity (P < 0.01), AGEs level and p16 protein expression (P < 0.05, P < 0.01), improved vascular morphology and elastic fiber structure, reduced collagen fibers. High dose BYHWD significantly downregulated miR-665 expression (P < 0.01), increased the number of autophagosomes. Medium and high dose of BYHWD significantly upregulated protein expression of Beclin1 and LC3Ⅱ/Ⅰ (P < 0.01), downregulated protein expression of p62 (P < 0.05, P < 0.01). High dose BYHWD significantly upregulated protein expression of DRAM1 in vascular tissues of aging rats (P < 0.05). Bioinformatics analysis revealed the presence of specific complementary binding sites between the sequences of miR-665 and DRAM1. Dual-luciferase reporter assays confirmed that miR-665 targeted DRAM1 gene and negatively regulated DRAM1 protein expression. CONCLUSION BYHWD may promote the protein expression of DRAM1 by inhibiting the expression of miR-665, thereby promoting vascular autophagy and delaying vascular aging. -
Key words:
- Buyang Huanwu Decoction /
- miR-665 /
- DRAM1 /
- autophagy /
- post-transcriptional regulation /
- vascular aging
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图 1 各组大鼠血管组织SA-β-Gal活性、AGEs水平(n=5)和p16蛋白表达(n=3)
注: 与年轻组比较, * *P＜0.01;与衰老组比较, #P＜0.05, ##P＜0.01。x±s。
Figure 1. SA-β-Gal activity, AGEs levels (n=5) and p16 protein expression (n=3) in vascular tissues of rats in each group
图 2 各组大鼠血管组织HE、Masson和EVG染色
注: 与年轻组比较, * *P＜0.01;与衰老组比较, #P＜0.05, ##P＜0.01。x±s，n=3。
Figure 2. HE, Masson, and EVG staining in vascular tissues of rats in each group
图 3 各组大鼠血管组织miR-665表达
注: 与年轻组比较, * *P＜0.01;与衰老组比较, ##P＜0.01。x±s，n=5。
Figure 3. Expression of miR-665 in vascular tissues of rats in each group
图 4 各组大鼠血管组织自噬小体数量及LC3、Beclin1、p62蛋白表达
注: 红色箭头代表自噬小体。与年轻组比较, * *P <0.01;与衰老组比较, #P <0.05, ##P <0.01。x±s，n=3。
Figure 4. The number of autophagosomes and the protein expressions of LC3, Beclin1 and p62 in vascular tissues of rats in each group
图 5 miR-665对下游自噬相关蛋白DRAM1的影响
注: 与miR-NC比较, *P＜0.05, * *P＜0.01。x±s，n=3。
Figure 5. Effect of miR-665 on the downstream autophagy-related protein DRAM1
图 6 各组大鼠血管组织DRAM1蛋白表达
注: 与年轻组比较, * *P＜0.01;与衰老组比较, #P＜0.05。x±s，n=3。
Figure 6. Expression of DRAM1 protein in vascular tissues of rats in each group
图 7 补阳还五汤延缓血管衰老的可能机制
Figure 7. Possible mechanism of Buyang Huanwu Decoction delaying vascular aging
表 1 引物序列
Table 1. Primer sequences
基因 序列(5'→3') miR-665 F: ACACTCCAGCTGGGACCAGGAGGCUGAGGUCC
R: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTAAGGGACU6 F: CTCGCTTCGGCAGCACA
R: AACGCTTCACGAATTTGCGTAll R: TCAACTGGTGTCGTGGA 
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