麦芽提取物调控NLRP3/Caspase-1/IL-1β通路抑制高催乳素血症大鼠垂体前叶细胞增殖及催乳素分泌

doi:10.11958/20221738

天津医药 ›› 2023, Vol. 51 ›› Issue (6): 618-623.doi: 10.11958/20221738

麦芽提取物调控NLRP3/Caspase-1/IL-1β通路抑制高催乳素血症大鼠垂体前叶细胞增殖及催乳素分泌

汪爱华¹(), 张小华¹, 张飞忠¹, 王雄², 赵勇³

1 武汉市第三医院首义院区体检中心（邮编430000）
2 武汉市第三医院首义院区药学部，（邮编430000）
3 武汉市第三医院首义院区检验科（邮编430000）

收稿日期:2022-10-25 修回日期:2022-11-29 出版日期:2023-06-15 发布日期:2023-06-20
作者简介:汪爱华（1978），女，主治医师，主要从事妇科疾病方面研究。E-mail：wangaihua1978w@163.com
基金资助:
武汉市中医药科研项目(WZ22C16)

Malt extract regulating the NLRP3/Caspase-1/IL-1β pathway to inhibit the proliferation and prolactin secretion of anterior pituitary cells in hyperprolactinemia rats

WANG Aihua¹(), ZHANG Xiaohua¹, ZHANG Feizhong¹, WANG Xiong², ZHAO Yong³

1 Physical Examination Center, Shouyi Hospital District of Wuhan Third Hospital, Wuhan 430000, China
2 Department of Pharmacy, Shouyi Hospital District of Wuhan Third Hospital, Wuhan 430000, China
3 Department of Clinical Laboratory, Shouyi Hospital District of Wuhan Third Hospital, Wuhan 430000, China

Received:2022-10-25 Revised:2022-11-29 Published:2023-06-15 Online:2023-06-20

汪爱华, 张小华, 张飞忠, 王雄, 赵勇. 麦芽提取物调控NLRP3/Caspase-1/IL-1β通路抑制高催乳素血症大鼠垂体前叶细胞增殖及催乳素分泌[J]. 天津医药, 2023, 51(6): 618-623.

WANG Aihua, ZHANG Xiaohua, ZHANG Feizhong, WANG Xiong, ZHAO Yong. Malt extract regulating the NLRP3/Caspase-1/IL-1β pathway to inhibit the proliferation and prolactin secretion of anterior pituitary cells in hyperprolactinemia rats[J]. Tianjin Medical Journal, 2023, 51(6): 618-623.

摘要/Abstract

摘要：

目的探讨麦芽提取物（ME）调控Nod样受体蛋白3（NLRP3）/胱天蛋白酶-1（Caspase-1）/白细胞介素-1β（IL-1β）通路对高催乳素血症（HPRL）大鼠垂体前叶细胞增殖及催乳素（PRL）分泌的影响。方法分别分离正常大鼠、HPRL大鼠的垂体前叶细胞，依次命名为NC组和Model组，免疫组织化学染色鉴定细胞中生长激素、PRL表达。将Model组细胞分别用0、25、50、100 mg/L ME，5 mmol/L腺苷三磷酸（ATP），100 mg/L ME+5 mmol/L ATP处理48 h，依次命名为空白组（Blank组）、ME低剂量组（ME-L组）、ME中剂量组（ME-M组）、ME高剂量组（ME-H组）、ATP组、ME-H+ATP组，光学显微镜观察细胞形态；CCK-8法检测细胞增殖；流式细胞术检测细胞凋亡；酶联免疫吸附试验检测上清液中PRL水平；Western blot检测增殖细胞核抗原（PCNA）、多巴胺受体D2（DRD2）、多巴胺转运体（DAT）、NLRP3、Caspase-1、IL-1β蛋白表达。结果成功分离大鼠垂体前叶细胞；与NC组比较，Blank组细胞体积变小，形状不规则，OD₄₅₀值、PRL水平、DAT、PCNA、NLRP3、Caspase-1、IL-1β蛋白表达升高，细胞凋亡率、DRD2蛋白表达降低（P<0.05）；与Blank组比较，ME-L组、ME-M组、ME-H组细胞形态有所改善，OD₄₅₀值、PRL水平、DAT、PCNA、NLRP3、Caspase-1、IL-1β蛋白表达降低，细胞凋亡率、DRD2蛋白表达升高，且呈剂量依赖性，而ATP组对应指标变化趋势与上述相反（P<0.05）；ATP减弱了高剂量ME对HPRL大鼠垂体前叶细胞增殖与PRL分泌的抑制作用。结论 ME可能通过下调NLRP3/Caspase-1/IL-1β通路蛋白表达抑制HPRL大鼠垂体前叶细胞增殖及PRL分泌。

关键词: 高催乳素血症, NLR家族，热蛋白结构域包含蛋白3, 胱天蛋白酶1, 白细胞介素1β, 受体，多巴胺D2, 麦芽提取物

Abstract:

Objective To investigate effects of malt extract (ME) on the proliferation and secretion of prolactin (PRL) in anterior pituitary cells of hyperprolactinemia (HPRL) rats by regulating the Nod-like receptor protein 3(NLRP3)/ cysteine aspartic specific protease-1 (Caspase-1)/ interleukin-1β (IL-1β) pathway. Methods The anterior pituitary cells of normal and HPRL rats were separated and named as the NC group and the model group. The expression levels of growth hormone and PRL in cells were identified by immunohistochemistry. Cells in the model group were treated with 0 mg/L ME, 25 mg/L ME, 50 mg/L ME, 100 mg/L ME, 5 mmol/L Adenosine 5'-triphosphate (ATP), 100 mg/L ME and 5 mmol/L ATP for 48 h, and named as the Blank group, the ME low dose group (ME-L group), the ME medium dose group (ME-M group), the ME high dose group (ME-H group), the ATP (NLRP3 activator) group and ME-H+ATP group. The morphology of cells in each group was observed by light microscope. Cell proliferation was detected by CCK-8 method. Cell apoptosis was detected by flow cytometry. The level of PRL in the supernatant of rat anterior pituitary cells was detected by ELISA. Western blot assay was applied to detect expression levels of proliferating cell nuclear antigen (PCNA), dopamine receptor D2 (DRD2), dopamine transporter (DAT), NLRP3, Caspase-1 and IL-1β in rat anterior pituitary cells. Results Rat anterior pituitary cells were isolated successfully. Compared with the NC group, the volume of anterior pituitary cells was smaller and the shape was irregular, the OD₄₅₀ value, prolactin level, DAT, PCNA, NLRP3, Caspase-1, IL-1β protein expression increased, and the apoptosis rate and DRD2 protein expression decreased in the blank group (P<0.05). Compared with the blank group, the morphology of anterior pituitary cells was improved in the ME-L group, the ME-M group and the ME-H group. The OD₄₅₀ value, prolactin level, DAT, PCNA, NLRP3, Caspase-1 and IL-1β protein expression levels decreased, and the apoptosis rate and DRD2 protein expression increased in a dose-dependent manner. The change trend of the corresponding indexes was contrary to the above in the ATP group (P<0.05). ATP attenuated the inhibitory effect of high-dose ME on the proliferation of anterior pituitary cells and PRL secretion in HPRL rats. Conclusion ME may inhibit the proliferation and PRL secretion of HPRL rat anterior pituitary cells by down-regulating the NLRP3/Caspase-1/IL-1β pathway.

Key words: hyperprolactinemia, NLR family, pyrin domain-containing 3 protein, Caspase 1, interleukin-1beta, receptors, dopamine D2, malt extract

中图分类号:

R584.1

图/表 8

图1 免疫组织化学鉴定大鼠垂体前叶细胞中生长激素和PRL的表达（×200）

Fig.1 Immunohistochemical identification of growth hormone and PRL secreted by anterior pituitary cells of rats (×200)

图2 各组大鼠垂体前叶细胞形态比较（结晶紫染色，×200）

Fig.2 Morphological comparison of anterior pituitary cells of rats between different groups (crystal violet staining, ×200)

表1 各组HPRL大鼠垂体前叶细胞OD450值、细胞凋亡率、PRL水平比较（n=6，$\bar{x}±s$）

Tab.1 Comparison of OD450 value, apoptosis rate and PRL level of anterior pituitary cells of HPRL rats between different groups

组别	OD₄₅₀值	细胞凋亡率（%）	PRL（mg/L）
NC组	0.39±0.03	12.64±0.31	193.36±4.12
Blank组	1.18±0.11^a	4.05±0.10^a	368.82±7.11^a
ME-L组	0.95±0.08^b	6.21±0.11^b	321.26±5.79^b
ME-M组	0.74±0.05^bc	8.36±0.12^bc	280.65±5.33^bc
ME-H组	0.45±0.04^bcd	11.12±0.13^bcd	221.27±4.34^bcd
ATP组	1.44±0.12^b	1.59±0.08^b	473.54±8.32^b
ME-H+ATP组	0.81±0.07^e	7.34±0.16^e	305.56±6.71^e
F	139.400^＊＊	3 416.000^＊＊	1 405.000^＊＊

图3 流式细胞术检测各组垂体前叶细胞凋亡

Fig.3 Apoptosis of anterior pituitary cells in each group detected by flow cytometry

图4 Western blot检测大鼠垂体前叶细胞中DRD2、DAT蛋白表达 A—G分别为NC组、Blank组、ME-L组、ME-M组、ME-H组、ATP组、ME-H+ATP组。

Fig.4 Western blot analysis of DRD2 and DAT protein expression in rat anterior pituitary cells

表2 各组大鼠垂体前叶细胞中DRD2、DAT蛋白表达的比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of expression levels of DRD2 and DAT proteins in anterior pituitary cells of rats in each group

组别	DRD2	DAT
NC组	0.83±0.07	0.54±0.04
Blank组	0.27±0.02^a	1.26±0.11^a
ME-L组	0.39±0.03^b	1.07±0.09^b
ME-M组	0.51±0.04^bc	0.83±0.07^bc
ME-H组	0.75±0.06^bcd	0.67±0.05^bcd
ATP组	0.11±0.01^b	1.62±0.13^b
ME-H+ATP组	0.43±0.03^e	0.92±0.06^e
F	218.600^＊＊	114.300^＊＊

图5 Western blot检测大鼠垂体前叶细胞中PCNA、NLRP3、Caspase-1、IL-1β蛋白表达 A—G分别为NC组、Blank组、ME-L组、ME-M组、ME-H组、ATP组、ME-H+ATP组。

Fig.5 Western blot analysis of PCNA, NLRP3, Caspase-1, IL-1β in rat anterior pituitary cells

表3 各组大鼠垂体前叶细胞中PCNA、NLRP3、Caspase-1、IL-1β蛋白表达水平比较（n=6，$\bar{x}±s$）

Tab.3 Comparison of expression levels of PCNA, NLRP3, Caspase-1, IL-1β in anterior pituitary cells of each group

组别	PCNA	NLRP3	Caspase-1	IL-1β
NC组	0.27±0.02	0.58±0.04	0.38±0.03	0.15±0.01
Blank组	0.88±0.07^a	1.57±0.12^a	1.42±0.11^a	1.23±0.11^a
ME-L组	0.73±0.06^b	1.32±0.10^b	1.24±0.12^b	1.06±0.11^b
ME-M组	0.55±0.05^bc	1.11±0.09^bc	1.01±0.08^bc	0.83±0.09^bc
ME-H组	0.36±0.02^bcd	0.73±0.06^bcd	0.54±0.04^bcd	0.32±0.03^bcd
ATP组	1.28±0.11^b	1.82±0.13^b	1.68±0.12^b	1.58±0.11^b
ME-H+ATP组	0.63±0.05^e	1.20±0.11^e	1.12±0.13^e	1.20±0.13^e
F	183.500^＊＊	120.500^＊＊	134.900^＊＊	178.700^＊＊

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麦芽提取物调控NLRP3/Caspase-1/IL-1β通路抑制高催乳素血症大鼠垂体前叶细胞增殖及催乳素分泌

Malt extract regulating the NLRP3/Caspase-1/IL-1β pathway to inhibit the proliferation and prolactin secretion of anterior pituitary cells in hyperprolactinemia rats

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