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河南大学国家食用菌中心对藏药囊距翠雀花抗炎作用研究方面取得重要进展

国家中心与巴基斯坦农业委员会Syed A. Rizvi博士合作,对藏药“恰羔贝”的研究成果“Two alkaloids from Delphinium brunonianum Royle, their anti-inflammatory and anti-oxidative stress activity via NF-κB signaling pathway”发表在中科院TOP 1区期刊Frontiers in NutritionIF6.576)。

囊距翠雀花Delphinium brunonianum Royle毛茛科翠雀属多年生草本植物,生于海拔4500~6000 m 的草地或多石处在我国主要分布于西藏南部,在巴基斯坦、尼泊尔阿富汗也有分布。其化学成分主要包括生物碱、黄酮等。作为一种民间用药,囊距翠雀花干燥的地上部分通常具有清热、解毒、消炎、镇痛的药理作用。但其抗炎活性成分和作用机制尚不清楚,因此本研究对提取分离得到的几种化合物进行抗炎活性筛选,并进一步展开对其抗炎机制的研究。

炎症的发生与发展

炎症是许多病理过程的基础。感染和组织损伤是引起炎症的主要原因,它们招募白细胞和血红蛋白向病变组织转移。巨噬细胞在炎症的发生发展过程中发挥重要作用巨噬细胞的两项基本功能是吞噬和适应性免疫应答。吞噬作用在机体对急性炎症的反应中起着至关重要的作用,它能迅速抵抗外来病原体的入侵除凋亡细胞巨噬细胞可以与树突状细胞一起呈递抗原。同时巨噬细胞还能分泌多种强大的炎症因子,如肿瘤坏死因子、白介素等。因此,选取巨噬细胞RAW264.7作为本研究的实验模型。

研究方法

本研究从囊距翠雀花干燥的地上部分用乙醇提取分离得到了4个化合物,包括Delbrunine (1), 4-O-α-D-Glucosyl benzoic acid (2), Kaempferol 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (3), Eldeline (4)。我们利用RAW264.7细胞对其进行了抗炎活性筛选,结果表明化合物1和化合物4具有较好的的抗炎效果。因此我们通过Western blot实验和PCR实验进一步阐明它们的抗炎作用机制,为囊距翠雀花植物开发新型抗炎药物提供依据。

化合物结构鉴定结果

Delbrunine (1), 4-O-α-D-Glucosyl benzoic acid (2), Kaempferol 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (3), Eldeline (4).


FIGURE 1 | Structures of compounds 1-4 from D. brunonianum

抗炎活性实验结果:

本研究通过LPS建立RAW264.7巨噬细胞炎症模型,通过NOELISA试剂盒检测发现,化合物1和化合物4能显著抑制NOIL-6TNF-α的分泌。PCR结果显示,化合物1和化合物4也可显著抑制相应mRNA的表达。Western blot结果显示,通过抑制NF-κB信号通路激活发挥抗炎作用,同时显著降低了iNOSCOX2蛋白的表达ROS的产生与炎症密切相关流式细胞术结果显示,这两种化合物均能够抑制ROS的产生。

MTT实验

FIGURE 2 | Effects of compound 1 (A), compound 4 (B) on the viability of RAW264.7 cells. The data were expressed as mean ± standard deviation, n = 6. ***P < 0.001, *P < 0.05 compared with the control group.

NO的检测及iNOSmRNA表达

FIGURE 3 | Effects of compound 1 (A), compound 4 (B) on NO production and effects of compound 1 (C), compound 4 (D) on iNOS mRNA expression in RAW264.7 cells. The data were expressed as mean ± standard deviation, n = 3. Compared with the control, ###P < 0.001. Compared with the LPS group, ***P < 0.001, **P < 0.01, *P < 0.05.

TNF-α, IL-6的检测

FIGURE 4 | Effects of compound 1 (A), (B) and compound 4 (C), (D) on the secretion of TNF-α, IL-6 in LPS-Induced RAW264.7 Cells. The data were expressed as mean ± standard deviation, n = 3. Compared with the control, ###P < 0.001. Compared with the LPS group, ***P < 0.001, **P < 0.01.

TNF-α, IL-6mRNA的表达

FIGURE 5 | Effects of compound 1 (A), (B) and compound 4 (C), (D) on the expression of TNF-α, IL-6 mRNA in LPS-Induced RAW264.7 cells. The data were expressed as mean ± standard deviation, n = 3. Compared with the control, ###P < 0.001. Compared with the LPS group, ***P < 0.001, **P < 0.01.

化合物1和化合物4LPS诱导的iNOSCOX-2蛋白表达的影响

FIGURE 6 | Effects of compound 1 (A) and compound 4 (B) on LPS-induced protein expression of iNOS and COX-2. The data were expressed as mean ± standard deviation, n = 3. Compared with the control, ###P < 0.001, ##P < 0.01. Compared with the LPS group, ***P < 0.001, **P < 0.01, *P < 0.05.

化合物1和化合物4对氧化应激的影响

FIGURE 7 | Effects of compound 1 at 50 μM (A), 25 μM (B), 12.5 μM (C) and compound 4 at 25 μM (D), 12.5 μM (E) and 6.25 μM (F) on LPS-induced oxidative stress.

化合物1和化合物4NF-κB 信号通路的影响

FIGURE 8 | Effects of compound 1 (A) and compound 4 (B) on NF-κB signaling pathway. The data were expressed as mean ± standard deviation, n = 3. Compared with the control, ##P < 0.01. Compared with the LPS group, ***P < 0.001, **P < 0.01, *P < 0.05.

结论:本研究从囊距翠雀花干燥地上部分提取分离得到4个化合物包括Delbrunine (1), 4-O-α-D-Glucosyl benzoic acid (2), Kaempferol 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (3), Eldeline (4)抗炎活性表明化合物1和化合物4具有较强的抗炎活性。其机制可能与抑制RAW264.7细胞中NF-κB信号通路的激活有关,从而减少炎症因子释放,抑制活性氧的产生,减轻炎症造成的损伤。

Two alkaloids from Delphinium brunonianum Royle, their anti-inflammatory and anti-oxidative stress activity via NF-κB signaling pathway

Qi Tanga1, Sitan Chena1, Syed Arif Hussain Rizvid1, Jiaojiao Qua,b, Li Wanga,b, Senye Wanga,b, Changyang Maa,b,c**, Lijun Liua,e**, Wenyi Kanga,b,c*

aNational R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng 475004, China

bFunctional Food Engineering Technology Research Center, Henan, Kaifeng, 475004, China

cJoint International Research Laboratory of Food & Medicine Resource Function, Henan Province, Kaifeng 475004, China

dPakistan agricultural research council, Islamabad 450000, Pakistan

e Huaihe Hospital, Henan University, Kaifeng 475004, China

1These authors contributed equally to this work.

**Corresponding authors to: Changyang Ma, Email: macaya1024@vip.henu.edu.cn; Lijun Liu, Tel: 13683781131; Wenyi Kang, E-mail: kangweny@hotmail.com.

ABSTRCT

In this study, we isolated and identified four compounds in Delphinium brunonianum Royle, and they were Delbrunine (1), 4-O-α-D-Glucosyl benzoic acid (2), Kaempferol 3-O-β-D-glucopyranoside 7-O-α-L-rhamnopyranoside (3) and Eldeline (4). Furthermore, the anti-inflammatory activity of these compounds was screened in RAW264.7 cells. The results showed that the anti-inflammatory activity of compound 2 and 3 was weak, and 1, 4 had good anti-inflammatory activity. The macrophage inflammation model was established by lipopolysaccharide (LPS). In this study, the levels of TNF-α and IL-6 were caried by ELISA kits, and the expression of cytokine mRNA was detected by qRT-PCR experiment. Then the effect of the compound 1 and 4 on reactive oxygen species was detected by flow cytometry. Following that, the western blot experiment was used to determine the expression of iNOS, COX-2 and the key proteins in the NF-κB signaling pathway. The results showed that compounds 1, 4 could significantly inhibit the elevation of inflammatory factors nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and also had obvious inhibitory effects on the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). In addition, compounds 1 and 4 could effectively inhibit the overexpression of reactive oxygen species (ROS) in RAW264.7 cells that activated by LPS. These results indicated that compound 1 and 4 may exert anti-inflammatory and anti-oxidative stress effects through the nuclear factor kappa-B (NF-κB) signaling pathway.

Reference: Tang Q, Chen S, Rizvi SA, Qu J, Wang L, Wang S, Ma C, Liu L, and Kang W (2022) Two alkaloids from Delphinium brunonianum Royle, their anti-inflammatory and anti-oxidative stress activity via NF-κB signaling pathway. Front. Nutr.