Therapeutic effect and mechanism of hordenine on ovalbumin-induced allergic rhinitis in rats
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摘要:
研究大麦芽碱对卵清蛋白（ovalbumin，OVA）诱导的大鼠变应性鼻炎（allergic rhinitis，AR）的治疗作用及相关机制。构建AR动物模型，采用HE染色和AB-PAS染色检测大麦芽碱对鼻黏膜病理损伤的改善情况。采用ELISA检测大麦芽碱对大鼠血清中OVA-sIgE及鼻黏膜组织上清液中IL-4的影响。采用免疫组化和Western blot实验检测大麦芽碱对Th1/Th2细胞平衡的影响。采用生物信息学进行通路预测，并通过体内及体外实验验证。实验结果表明，大麦芽碱可以减轻OVA诱导AR大鼠的行为学表现，缓解AR导致的鼻黏膜病理损伤，减少OVA-sIgE及IL-4的分泌。此外，大麦芽碱可以调节Th1/Th2平衡。生物信息学分析结果显示，大麦芽碱作用于AR的潜在作用通路为磷脂酰肌醇3（phosphoinositide 3 kinase，PI3K）/磷酸蛋白激酶（protein kinase B，Akt）信号通路。体内实验结果显示，模型组大鼠鼻黏膜中PI3K和p-Akt蛋白表达显著升高（P<0.01），经过大麦芽碱治疗后，其表达水平显著降低，体外细胞实验也验证了这一结果。本研究提示，大麦芽碱可能通过PI3K/Akt信号通路调节Th1/Th2细胞平衡，从而发挥缓解OVA诱导的变应性鼻炎的作用。
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关键词:
- 变应性鼻炎 /
- 大麦芽碱 /
- PI3K/Akt信号通路 /
- Th1/Th2细胞平衡
Abstract:To investigate the therapeutic effect and related mechanisms of hordenine on ovalbumin (OVA)-induced allergic rhinitis (AR) in rats, HE and AB-PAS staining were used to detect the improvement of pathological damage to the nasal mucosa induced by hordenine. ELISA was employed to detect the effect of hordenine on OVA-sIgE in serum and IL-4 in the nasal mucosa supernatant of rats. IHC and Western blot experiments were undertaken to examine the effect of hordenine on Th1/Th2 cell balance. Bioinformatics analysis was performed to predict pathways, which were verified by in vivo and in vitro experiments. The experimental results showed that hordenine could alleviate the behavioral manifestations of OVA-induced AR rats, alleviate nasal mucosal pathological damage caused by AR, and reduce the secretion of OVA-sIgE and IL-4. In addition, hordenine could regulate the Th1/Th2 balance. Bioinformatics analysis results showed that the potential pathway of action of hordenine on AR was the phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway. The in vivo experimental results showed that the expression of PI3K and p-Akt proteins in the nasal mucosa of the model group rats was significantly increased (P < 0.01), and that the protein expression level was significantly decreased after the administration of hordenine, which was also confirmed by an in vitro experiment. This study suggests that hordenine may regulate Th1/Th2 cell balance through the PI3K/Akt signaling pathway, thereby exerting an alleviating effect on OVA-induced AR.
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Keywords:
- allergic rhinitis /
- hordenine /
- PI3K/Akt pathway /
- Th1/Th2 cell balance
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Figure 1. Behavioral effects of hordenine on ovalbumin (OVA)-induced allergic rhinitis (AR) rats ($ \bar{x}\pm s $, n=6)
A: Scores of nasal symptoms within 30 min from the nasal stimulation stage to the last intragastric administration; B: Average number of rubbing; C: Average number of sneezing * P<0.05, ** P<0.01 vs control group; ## P<0.01 vs OVA group
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Figure 2. Safety evaluation of hordenine in the treatment of OVA-induced AR rats ($ \bar{x}\pm s $, n=6)
A: Organ coefficients of important organs (heart, liver, spleen, lungs, and kidneys) in rats; B: Result of MTT assay; C: HE staining of lung tissues ** P<0.01 vs control group; ## P<0.01 vs OVA group
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Figure 3. Effects of hordenine on histological alterations in OVA-induced AR rats ($ \bar{x}\pm s $, n=3)
A: HE staining results of the nasal mucosa, the black arrows indicated inflammatory cell infiltration; B: AB-PAS staining of the nasal mucosa; C: Goblet cells count of the nasal mucosa ** P<0.01 vs control group; ## P<0.01 vs OVA group
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Figure 4. Secretion level of OVA-sIgE in rats’ serum (A) and IL-4 in rats’ nasal mucosa tissue supernatant (B) determined by ELISA ($ \bar{x}\pm s $, n=3)
* P<0.05, ** P<0.01 vs control group; ## P<0.01 vs OVA group
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Figure 5. Expression of T-bet and GATA-3 in nasal mucosa of AR rats ($ \bar{x} $±s, n=3)
A: IHC results of GATA-3 and T-bet expression in the nasal mucosa tissues, the black arrows point to the positive expression of the target proteins; B: Western blot results of T-bet and GATA-3 expression in the nasal mucosa tissues * P<0.05 vs control group; # P<0.05 vs OVA group
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Figure 6. Network pharmacology analysis results of potential pathways of action of hordenine in the treatment of AR
A: PPI network diagram of AR-related target; B: PPI network diagram of hordenine-related target; C: Venn diagram showing the intersection between hordenine and AR; D: PPI interaction network of hordenine and AR intersection targets; E: KEGG analysis; F: GO analysis
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Figure 7. Diagram of molecular docking of hordenine with the following targets
A: AKT1-hordenine; B: EGFR-hordenine; C: CCL2-hordenine; D: PTGS1-hordenine; E: ADRB2-hordenine; F: HRH1-hordenine
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Figure 8. Expression of PI3K, p-Akt and Akt in nasal mucosa of OVA-induced AR rats ($ \bar{x}\pm s $, n=3)
A: IHC results of PI3K, p-Akt and Akt expression in nasal mucosa tissues, the black arrows point to the positive expression of the target proteins; B: Western blot of PI3K, p-Akt and Akt in the nasal mucosa tissues ** P<0.01 vs control group; # P<0.05, ## P<0.01 vs OVA group
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Figure 9. Effects of hordenine on the expression of PI3K, p-Akt and Akt in peritoneal macrophages ($ \bar{x} \pm s$, n=3)
* P<0.05 vs control group; # P<0.05, ## P<0.01 vs OVA group
Table 1 Topological analysis results of the main target network
Target Degree Betweenness Closeness AKT1 13 121.85238095 0.02325581 EGFR 12 97.38571428 0.02272727 CCL2 10 39.05714285 0.02083333 PTGS1 8 134.47619047 0.02222222 SLC6A4 7 52.48809523 0.01886792 ADRB2 7 117.69047619 0.02222222 HRH1 7 188.12619047 0.02 
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Table 2 Binding energy of hordenine for partial core targets
Target Binding energy/(kJ/mol) AKT1 − 29.677112 EGFR − 30.024384 CCL2 − 27.936568 PTGS1 − 35.250200 ADRB2 − 29.480464 HRH1 − 29.672928
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