小檗碱调节AMPK/NF-κB信号通路对自身免疫性前列腺炎大鼠Th17/Treg平衡的影响

doi:10.11958/20221816

摘要/Abstract

摘要：

目的探讨小檗碱调节腺苷酸活化蛋白激酶（AMPK）/核因子-κB（NF-κB）信号通路对自身免疫性前列腺炎（EAP）大鼠辅助性T细胞17（Th17）/调节性T细胞（Treg）平衡的影响。方法将90只SD雄性大鼠随机分为对照组（正常饲养）、模型组、小檗碱低剂量组（25 mg/kg）、小檗碱高剂量组（50 mg/kg）、阳性对照组（40 mg/kg塞来昔布）、抑制剂组（50 mg/kg小檗碱+0.2 mg/kg AMPK抑制剂compound C），每组15只。除对照组外，其余组大鼠均采用盆腔区域及双侧肩胛皮下多点注射完全弗式佐剂与前列腺组织混合悬液构建EAP大鼠模型，建模成功后进行药物处理，每天灌胃给药1次，持续4周。HE染色观察各组大鼠前列腺组织病理情况；免疫组织化学法检测大鼠前列腺组织视黄酸相关核孤儿受体γt（RORγt）、叉头蛋白3（Foxp3）表达；酶联免疫吸附试验（ELISA）法检测大鼠血清中白细胞介素（IL）-17、IL-10水平；流式细胞仪检测大鼠外周血中Th17/Treg细胞比值；通过Western blot法检测大鼠前列腺组织中AMPK/NF-κB信号通路相关蛋白表达。结果与模型组比较，小檗碱低、高剂量组和阳性对照组大鼠前列腺组织腔内有粉色分泌物，腺上皮细胞排列整齐，炎症明显消退，血清IL-17水平、外周血Th17/Treg比值、前列腺组织RORγt和p-NF-κB p65/NF-κB p65表达水平降低，血清IL-10水平、前列腺组织Foxp3、p-AMPK/AMPK和IκBα表达水平升高（P<0.05）；加入AMPK抑制剂compound C后，可减弱小檗碱对EAP大鼠Th17/Treg平衡的促进作用。结论小檗碱可通过调节AMPK/NF-κB信号通路促进EAP大鼠Th17/Treg平衡。

关键词: 自身免疫性前列腺炎, 小檗碱, AMPK/NF-κB, Th17/Treg平衡

Abstract:

Objective To investigate the influence of berberine on T helper cell 17 (Th17)/regulatory T cell (Treg) balance in autoimmune prostatitis (EAP) rats by regulating adenylate-activated protein kinase (AMPK)/nuclear factor-κB (NF-κB) signaling pathway. Methods Ninety SD male rats were randomly divided into the control group (normal feeding), the model group, the low-dose berberine group (25 mg/kg), the high-dose berberine group (50 mg/kg), the positive control group (40 mg/kg celecoxib) and the inhibitor group (50 mg/kg berberine + 0.2 mg/kg AMPK inhibitor compound C), with 15 rats in each group. Except for the control group, rats in other groups were all injected with complete Freund's adjuvant and prostate tissue mixed suspension in pelvic region and bilateral scapula to construct the EAP rat model. After successful modeling, drug treatment was carried out by gavage, once a day for four weeks. HE staining was used to observe the pathological conditions of prostate tissue in each group. Immunohistochemical method was used to detect the expressions of retinoic acid-related nuclear orphan receptor γt (RORγt) and forkhead box protein 3 (Foxp3) in rat prostate tissue. ELISA method was used to detect serum levels of interleukin (IL)-17 and IL-10 in rats. Flow cytometry was used to detect the ratio of Th17/Treg cells in peripheral blood of rats. Western blot assay was used to detect the expression of AMPK/NF-κB signaling pathway-related proteins in rat prostate tissue. Results Compared with the model group, there were pink secretions in prostate tissue cavity of rats in the low-dose and the high-dose berberine groups and the positive control group. The glandular epithelial cells were neatly arranged, and the inflammation subsided obviously. Serum IL-17 levels, peripheral blood Th17/Treg ratio, and RORγt and p-NF-κB p65/NF-κB p65 expression levels in prostate tissue were decreased, and serum IL-10 level, Foxp3, p-AMPK/AMPK and IκBα expression levels in prostate tissue were increased (P<0.05). After adding AMPK inhibitor compound C, the promoting effect of berberine on Th17/Treg balance in EAP rats was attenuated. Conclusion Berberine can promote Th17/Treg balance in EAP rats by regulating AMPK/NF-κB signaling pathway.

Key words: autoimmune prostatitis, berberine, AMPK/NF-κB, Th17/Treg balance

中图分类号:

R285.5

图/表 8

图1 HE染色检测各组大鼠前列腺病理变化（×200）

Fig.1 Pathological changes of prostate of rats detected by HE staining in each group (×200)

表1 各组大鼠前列腺中RORγt、Foxp3阳性表达水平比较 (n=5,IOD,$\bar{x}±s$)

Tab.1 Comparison of positive expression levels of RORγt and Foxp3 in rat prostate tissue between the six groups

组别	RORγt	Foxp3
对照组	0.11±0.02	0.51±0.05
模型组	0.45±0.05^a	0.13±0.02^a
小檗碱低剂量组	0.36±0.04^b	0.22±0.03^b
小檗碱高剂量组	0.22±0.03^bc	0.37±0.04^bc
阳性对照组	0.20±0.02^bc	0.38±0.04^bc
抑制剂组	0.39±0.04^d	0.17±0.02^d
F	69.635^＊＊	87.189^＊＊

图2 免疫组织化学染色检测各组大鼠前列腺中RORγt的表达（×200）

Fig.2 Expression of RORγt in prostate of rats detected by immunohistochemistry satining in each group (×200)

图3 免疫组织化学染色检测各组大鼠前列腺中Foxp3的表达（×200）

Fig.3 Expression of Foxp3 in prostate of rats detected by immunohistochemistry satining in each group (×200)

表2 各组大鼠血清中IL-17、IL-10水平比较 (n=15,ng/L,$\bar{x}±s$)

Tab.2 Comparison of serum levels of IL-17 and IL-10 between the six groups

组别	IL-17	IL-10
对照组	12.56±1.04	25.38±2.24
模型组	38.47±3.25^a	9.85±1.12^a
小檗碱低剂量组	26.38±3.17^b	14.63±1.35^b
小檗碱高剂量组	14.25±2.23^bc	22.52±2.17^bc
阳性对照组	13.19±1.12^bc	23.47±2.21^bc
抑制剂组	24.64±2.16^d	16.74±1.23^d
F	288.985^＊＊	169.376^＊＊

图4 各组大鼠外周血Th17/Treg平衡情况

Fig.4 Th17/Treg balance in peripheral blood of rats in each group

图5 Western blot检测大鼠前列腺组织中AMPK/NF-кB通路相关蛋白表达 A：对照组；B：模型组；C：小檗碱低剂量组；D：小檗碱高剂量组；E：阳性对照组；F：抑制剂组。

Fig.5 Expression of AMPK/NF-κB pathway related proteins in rat prostate tissue detected by Western blot analysis

表3 各组大鼠前列腺组织中AMPK/NF-κB通路相关蛋白表达比较(n=5,$\bar{x}±s$)

Tab.3 Comparison of expressions of AMPK/NF-κB pathway related proteins in prostate tissue of rats between the six groups

组别	p-AMPK/ AMPK	p-NF-κB p65/ NF-κB p65	IκBα/ GAPDH
对照组	0.78±0.08	0.36±0.04	1.04±0.12
模型组	0.24±0.03^a	0.82±0.09^a	0.45±0.06^a
小檗碱低剂量组	0.46±0.05^b	0.67±0.08^b	0.62±0.08^b
小檗碱高剂量组	0.68±0.06^bc	0.43±0.04^bc	0.89±0.09^bc
阳性对照组	0.72±0.07^bc	0.41±0.05^bc	0.92±0.06^bc
抑制剂组	0.54±0.04^d	0.58±0.06^d	0.74±0.05^d
F	60.482^＊＊	38.417^＊＊	36.580^＊＊

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