文献学习097--CB1 antagonist genistei

marijuana与很多心血管疾病的发生相关。其主要成分Δ⁹-tetrahydrocannabinol (Δ⁹-THC)的受体是cannabinoid receptor 1 (CB1/CNR1) 和 cannabinoid receptor 2 (CB2/CNR2)（两者都属于GPCR家族）。

1. UK Biobank analysis reveals an association between cannabis use and myocardial infarction

Fig 1a： 作者首先使用UKB的数据探究了cannabis use, MI, 和其他 cardiovascular events的关系。在控制了性别年龄和BMI后，作者使用logistic回归模型发现cannabis use was a statistically significant positive predictor for MI.
Fig 1b： 炎症反应是atherosclerosis的核心pathogenesis。所以接下来作者使用olink蛋白组分析探究了recreational smokers (marijuana cigarette) 血浆中的炎症分子水平。结果显示多种与atherosclerosis相关的炎症因子水平显著上调。

2. Genistein binds to the CB1 receptor and inhibits CB1 activity

Fig 2a： 随后作者使用了ROCS software suite，在SWEETLEAD chemical database中对4 selective CB1 antagonists进行了筛选。
Fig 2b： Ligand-based high-throughput virtual screening with a chemical database was used to discover new selective CB1 antagonists. We found 62 chemical compounds that were structurally homologous with the 4 selective CB1 antagonists.
Fig 2c-d： 随后作者通过molecular docking 探究了筛选出的62个化合物和CB1 receptor的结合（selective CB1 antagonist AM6538做为阳性对照）。结果发现genistein, a natural soybean flavonoid, 与CB1受体结合。The Schrödinger (Portland, OR, United States of America) suite with glide protein-ligand docking functionality was used to validate that genistein was a ligand for the CB1 receptor。

药物筛选及设计软件——Schrodinger

Fig 2e： Genistein adopted a similar shape to the selective CB1 antagonist query structures.
Fig 2f： An in vitro GTPase assay found that genistein functions as a neutral antagonist.

作者还使用Radioligand binding assays验证了genistein binds human CB1 with an IC50 of 150 nM.

3. Δ⁹-THC induces cytotoxicity in human endothelial cells

随后作者在3种细胞中评估了Δ⁹-THC cytotoxicity：(1) human endothelial cells (human umbilical vein endothelial cells [HUVECs] and human coronary artery endothelial cells [HCAECs]), (2) human embryonic stem cell-derived cardiomyocytes (H7 hESC-CMs), and (3) normal human cardiac fibroblasts-ventricular (NHCF-V) cells.
Fig 3a： 结果显示Δ⁹-THC引起了人内皮细胞的毒性，而对心肌和成纤维没有显著副作用。
Fig 3b-c： Δ⁹-THC引起了内皮细胞多种炎症相关基因的表达，而氧化应激保护性基因(antioxidant-related gene)的表达则出现下降。
Fig 3d： 在来自4 healthy individuals 的 hiPSC-EC 中也得到了一致的结果。

4. Δ⁹-THC induces inflammation and NF-kB activation in hiPSC-ECs

Fig 4a： 此前有文献报道cannabis和Δ⁹-THC具有抗炎作用，但作者发现Δ⁹-THC可以引起hi-PSC-ECs的多种炎症相关基因的表达。而 I kappa B (NFKBIA)，一个NF-kB转录因子的特异性抑制分子，在Δ⁹-THC的诱导下出现下调。
Fig 4b： 此前文献报道过TNF-a可以引起血管功能失代偿。作者发现Δ⁹-THC诱导了hiPSC-ECs的TNF-a表达。
Fig 4c-d： 单核和内皮的粘附与动脉粥样硬化对的发生有关。作者发现Δ⁹-THC处理后，hiPSC-ECs对单核细胞的粘附增加。
Fig 4e： 随后作者使用wash-out 实验评估了Δ⁹-THC引起的hiPSC-ECs炎症反应的时长。在使用Δ⁹-THC处理48h后，对hiPSC-ECs进行换液，随后作者每2天检测一次验证基因的表达，一直到2w。结果显示Δ⁹-THC引起的hiPSC-ECs炎症基因的表达可以持续8-10天。
Fig 4f-h： 随后作者想要去探究Δ⁹-THC通过CB1引起的血管氧化应激反应的机制。免疫荧光结果显示Δ⁹-THC诱导了hiPSC-ECs中NFkB的核转位和NFkB磷酸化。选择性 NF-kB inhibitor BAY11-7082可以抑制Δ⁹-THC诱导的 hiPSC-ECs细胞NFkB磷酸化。

5. Genistein reverses Δ⁹-THC-induced oxidative stress and inflammation in hiPSC-ECs by inhibiting CB1

The CB1 receptor is implicated in the pathological effects of Δ⁹-THC on the vasculature. We next employed pharmacologic and genetic inactivation of the CB1 receptor to determine whether the CB1 receptor plays a role in Δ⁹-THC-induced effects in hiPSC-ECs.
作者首先使用了selective CB1 antagonists AM6545, AM251 和 NESS-0327，发现它们阻断了Δ⁹-THC诱导的hiPSC-ECs细胞炎症基因和氧化应激相关基因的表达（附件），使用CB1的siRNA也得到了一致的结果（附件）。
随后作者使用了
Fig 5a-b： CRISPR interference (CRISPRi) of CB1 expression reversed Δ⁹-THC-induced inflammation and oxidative stress in hiPSC-ECs

随后作者探究了CB1 antagonist genistein对Δ⁹-THC引起的血管功能失代偿的保护作用。
Fig 5c： 作者筛选了一系列的抗氧化剂，发现genistein阻断Δ⁹-THC引起的hiPSC-ECs氧化应激效果最好。
Fig 5d： 在hiPSC-ECs中，genistein除了上调了Δ⁹-THC引起的hiPSC-ECs抗氧化基因的下调，还影响了NOS2和NOX1的表达。
Fig 5e-f： genistein没有阻断Δ⁹-THC引起的hiPSC-ECs和monocyte的粘附增加，但降低了NFKB的磷酸化。此外，genistein还阻断了Δ⁹-THC引起的hiPSC-ECs的TLR4的表达。
Fig 5g： Cotreatment with genistein also shortened the recovery time of Δ⁹-THC- induced inflammation from 8–10 days to 4–6 days.

These experimental results indicated that genistein attenuated Δ⁹-THC-induced oxidative stress and inflammation in hiPSC-ECs.

6. Genistein reverses Δ⁹-THC-induced endothelial dysfunction, oxidative stress, and inflammation in a mouse model

Fig 6a： 为了验证genistein in vivo的效果，作者对雄性C57小鼠给予了vehicle, Δ⁹-THC, or Δ⁹-THC+genistein治疗。
Fig 6b： Wire myograph显示Δ⁹-THC诱导了小鼠对的内皮损伤，而genistein则减轻了这种效应。
Fig 6c-d： 对胸主动脉的组织检测结果显示Genistein和其他已知的CB1 antagonist降低了Δ⁹-THC诱导的炎症基因表达。上调了Δ⁹-THC引起的oxidative stress protective-related gene的下调。
Fig 6e-f： NF-kB磷酸化和小鼠血浆中SOD的表达在治疗后也出现下降。
Fig 6g-j： Δ⁹-THC administration decreased circulating levels of the antioxidant glutathione, which was ameliorated with genistein cotreatment.

Collectively, these results indicated that genistein could reverse D9-THC-induced effects in vivo.

7. Effect of THC and genistein on atherosclerosis

作者在动脉粥样硬化模型鼠上也进行了治疗，得到了很好的治疗效果。

8. Genistein binds to the CB1 receptor and does not attenuate D⁹-THC neurobehavioral effects in vivo

Fig 7a-b： After finding that genistein attenuated the vascular dysfunction caused by Δ⁹-THC in both in vitro and in vivo studies, we found direct evidence of genistein-binding CB1 using fluorescently labeled genistein (BODIPY517/547-genistein)
Fig 7c： In CB1 radioligand binding assays, BODIPY517/547-genistein had an IC50 of 375 nM on human CB1 receptors and labeled nearly all hiPSC-ECs cells after 12 h incubation.
Fig 7d-g： In vivo binding was investigated using intravenously injected BODIPY517/547-genistein in C57BL/6J mice. After 48 h, BODIPY517/547-genistein was detected in the abdominal viscera, thoracic aorta, heart, and lungs but was minimally detected in the brain.

Fig 7h-i： confocal显示Alexa Fluor-488-labeled CB1 receptor与BOD- IPY517/547-genistein在aortic vascular cells中存在共定位。
Fig 7j： The specificity of genistein binding was interrogated by injecting the selective CB1 antagonist rimonabant before BOD- IPY517/547-genistein. Rimonabant has a stronger binding affinity for the CB1 receptor and reduced BODIPY517/547-genistein binding and fluorescence significantly (0.78 ± 0.08 versus 0.13 ± 0.04, p < 0.00001)

Genistein alone did not affect tetrad effects, and genistein could not attenuate the Δ⁹-THC neurobehavioral effects in cotreatment.

缺陷：iPSC-ECs are immature, having low eNOS expression and are a mixture of arterial, venous, and lymphatic ECs. Therefore, it was imperative to validate the effects observed in the iPSC-ECs with primary endothelial cells and murine models.