甘草多糖对LPS诱导小鼠睾丸炎的调节作用

doi:10.11843/j.issn.0366-6964.2024.06.044

摘要/Abstract

摘要：

本研究旨在探讨甘草多糖(glycyrrhiza polysaccharide，GPS)对LPS诱导小鼠睾丸炎的调节作用及其可能机制。将60只8周龄的雄性C57BL/6J小鼠，随机分为6组(每组10只)：空白对照组、模型组(LPS组)、阳性对照组(地塞米松组)、GPS低中高剂量组(100、200、400 mg·kg^-1)。试验前20 d GPS低中高剂量组灌胃给予相应剂量的GPS，其他组灌胃同等剂量的生理盐水，第21天模型组腹腔注射5 mg·kg^-1 LPS，其他组腹腔注射等量的生理盐水，2 h后，阳性对照组灌胃给予5 mg·kg^-1的地塞米松，12 h后，按照动物伦理学标准处死小鼠，收集小鼠血液及睾丸组织。运用HE染色法观察小鼠睾丸组织结构形态学变化，酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测血清中睾酮激素(testosterone, T)含量及乳酸脱氢酶(lactate dehydrogenase, LDH)水平，南京建成试剂盒检测氧化损伤指标(T-SOD、CAT、GSH)变化，实时荧光定量PCR法检测睾丸组织中紧密连接蛋白(Occludin、ZO-1)、炎症因子(IL-18、IL-1β、IL-6、TNF-α)及细胞焦亡关键因子(caspase-1、NLRP3、GSDMD)mRNA表达变化，Western blot法检测IL-18、IL-1β、NLRP3、GSDMD的蛋白表达水平。结果显示，阳性对照组与GPS中高剂量组可以显著缓解LPS诱导的小鼠睾丸组织结构损伤，增加血清睾酮含量，改善氧化损伤，降低促炎因子mRNA表达水平，并抑制细胞焦亡发生。综上所述，GPS可以有效缓解LPS诱导的睾丸炎损伤，其作用机制可能与改善睾丸组织氧化损伤，抑制细胞焦亡，阻止炎症反应相关。

关键词: 甘草多糖, LPS, 睾丸炎, 细胞焦亡

Abstract:

The present study aimed to investigate the regulation and its possible mechanism of glycyrrhiza polysaccharide (GPS) on LPS-induced orchitis in mice. Sixty 8-week-old male C57BL/6J mice were randomly divided into 6 groups (10 mice per group): Blank control group, model group (LPS group), positive control group (dexamethasone group) and GPS low, medium, high dose (100, 200, 400 mg·kg^-1) group. On the first 20 days, GPS low, medium, high dose group was given the corresponding dose of GPS by intragastric, and other groups were given the same dose of physiological saline by intragastric. On the 21st day, model group was intraperitoneally injected with 5 mg·kg^-1 LPS, and other groups were intraperitoneally injected with the same amount of physiological saline, 2 h later, the positive control group was given 5 mg·kg^-1 dexamethasone by intragastric, 12 h later, the mice were sacrificed according to animal ethical standards, blood and testicular tissues were collected. The morphological changes of testicular tissue were observed by HE staining, the content of testosterone (T) and the level of lactate dehydrogenase (LDH) in serum were detected by enzyme-linked immunosorbent assay (ELISA), and the changes of oxidative damage indexes (T-SOD, CAT, GSH) were detected by Nanjing Jiancheng Kit. Real-time fluorescence quantitative PCR was used to detect mRNA expression changes of tight junction proteins (Occludin, ZO-1), inflammatory factors (IL-18, IL-1β, IL-6, TNF-α) and key cytokines of pyroptosis (caspase-1, NLRP3, GSDMD) in testis, western blot was used to detect the protein expression of IL-18, IL-1β, NLRP3, GSDMD. The results showed that dexamethasone group and GPS medium, high dose groups could significantly relieve the LPS-induced testicular damage, increase the serum testosterone content, reduce oxidative damage, decrease the mRNA expressions of pro-inflammatory cytokines, and inhibit the occurrence of pyroptosis. In summary, GPS could effectively alleviate LPS-induced orchitis injury, which may related to reducing oxidative damage, inhibiting pyroptosis and preventing inflammatory response.

Key words: glycyrrhizol polysaccharide, LPS, orchitis, pyroptosis

中图分类号:

S853.74

李美艳, 邢晓莹, 李娜, 张椰莉, 乔宏萍, 武晓英. 甘草多糖对LPS诱导小鼠睾丸炎的调节作用[J]. 畜牧兽医学报, 2024, 55(6): 2741-2750.

Meiyan LI, Xiaoying XING, Na LI, Yeli ZHANG, Hongping QIAO, Xiaoying WU. Regulation of Glycyrrhizol Polysaccharide Alleviate LPS-induced Orchitis in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2741-2750.

图/表 7

表 1

图 1

图 2

图 3

图 4

图 5

图 6

参考文献 31

1	FONTAINE E . Acute orchitis in a male breeding dog[J]. Summa Animali Da Compagnia, 2010, 40 (298): 49.
2	BONAPARTE A , PAGE C , BEELER E . Orchitis and balanoposthitis in a dog with Leptospira interrogans serovar Canicola in Southern California[J]. Vet Rec Case Rep, 2018, 6 (3): e000463.
3	赵民, 温伟, 陈显魁, 等. 种公猪睾丸炎的中药治疗[J]. 中国兽医杂志, 2014, 50 (4): 99- 100.
	ZHAO M , WEN W , CHEN X K , et al. Chinese medicine treatment of orchitis in boar[J]. Chinese Journal of Veterinary Medicine, 2014, 50 (4): 99- 100.
4	李秀娟. 一例羊睾丸炎的治疗经验[J]. 吉林畜牧兽医, 2017, 38 (5): 47, 49.
	LI X J . Treatment experience of a case of sheep orchitis[J]. Jilin Animal Husbandry and Veterinary Medicine, 2017, 38 (5): 47, 49.
5	OK F , KAPLAN H M , KIZILGOK B , et al. Protective effect of alpha-linolenic acid on lipopolysaccharide-induced orchitis in mice[J]. Andrologia, 2020, 52 (9): e13667.
6	WANG H , YANG L L , HU Y F , et al. Maternal LPS exposure during pregnancy impairs testicular development, steroidogenesis and spermatogenesis in male offspring[J]. PLoS One, 2014, 9 (9): e106786. doi: 10.1371/journal.pone.0106786
7	WU Y , YI L , LI E T , et al. Optimization of glycyrrhiza polysaccharide liposome by response surface methodology and its immune activities[J]. Int J Biol Macromol, 2017, 102, 68- 75. doi: 10.1016/j.ijbiomac.2017.04.006
8	ZHAO Y Y , LI C X , WANG X Y , et al. Effects of Glycyrrhiza polysaccharide on growth performance, appetite, and hypothalamic inflammation in broilers[J]. J Anim Sci, 2023, 101, skad027. doi: 10.1093/jas/skad027
9	DU J L , CAO L P , JIA R , et al. Hepatoprotective and antioxidant effects of dietary glycyrrhiza polysaccharide against TCDD-induced hepatic injury and RT-PCR quantification of AHR2, ARNT2, CYP1A mRNA in Jian Carp (Cyprinus carpio var. Jian)[J]. J Environ Sci (China), 2017, 51, 181- 190. doi: 10.1016/j.jes.2016.06.026
10	武晓英, 刘地, 李娜, 等. 甘草多糖对小鼠肺组织炎症及氧化损伤的修复机制[J]. 甘肃农业大学学报, 2020, 55 (5): 8-14, 30.
	WU X Y , LIU D , LI N , et al. Effects of glycyrrhiza polysaccharide on inflammation and oxidative damage of lung tissue in mice[J]. Journal of Gansu Agricultural University, 2020, 55 (5): 8-14, 30.
11	WEI X X , LI N , WU X Y , et al. The preventive effect of glycyrrhiza polysaccharide on lipopolysaccharide-induced acute colitis in mice by modulating gut microbial communities[J]. Int J Biol Macromol, 2023, 239, 124199. doi: 10.1016/j.ijbiomac.2023.124199
12	PAN Y Q , LIU Y , WANG L , et al. MKP-1 attenuates LPS-induced blood-testis barrier dysfunction and inflammatory response through p38 and IκBα pathways[J]. Oncotarget, 2016, 7 (51): 84907- 84923. doi: 10.18632/oncotarget.12823
13	张逢, 陈指龙, 易思亮, 等. 细菌脂多糖急性暴露通过炎症反应诱导小鼠睾丸损伤[J]. 畜牧兽医学报, 2019, 50 (5): 1099- 1105. doi: 10.11843/j.issn.0366-6964.2019.05.021
	ZHANG F , CHEN Z L , YI S L , et al. Acute exposure of bacterial lipopolysaccharide induces testicular injury in mice through inflammatory response[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50 (5): 1099- 1105. doi: 10.11843/j.issn.0366-6964.2019.05.021
14	赵欣, 王莹, 李春亭, 等. 蒲公英提取物对LPS诱导小鼠乳腺炎的减轻效应及其机制分析[J]. 畜牧兽医学报, 2022, 53 (8): 2773- 2781. doi: 10.11843/j.issn.0366-6964.2022.08.034
	ZHAO X , WANG Y , LI C T , et al. Alleviating effect and mechanism of dandelion extract on LPS-induced mastitis in mice[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53 (8): 2773- 2781. doi: 10.11843/j.issn.0366-6964.2022.08.034
15	李宏军. 睾丸炎会引起男人不育[J]. 黄河. 黄土. 黄种人, 2016, (2): 43.
	LI H J . Orchitis can cause infertility in men[J]. The Yellow River. Loess. The Yellow Man, 2016, (2): 43.
16	SHEN P L , JI S Q , LI X L , et al. LPS-induced systemic inflammation caused mPOA-FSH/LH disturbance and impaired testicular function[J]. Front Endocrinol (Lausanne), 2022, 13, 886085.
17	刘阳, 乔萍, 朱玉琢. 甘草对小鼠雄性生殖细胞遗传损伤的防护作用[J]. 中国公共卫生, 2005, 21 (10): 1203- 1204.
	LIU Y , QIAO P , ZHU Y Z . Preventive effect of glycyrrhiza uralensis fisch on damage of genetic materials in male mouse reproductive cells[J]. Chinese Journal of Public Health, 2005, 21 (10): 1203- 1204.
18	HUANG C Y , LUO X Q , LI L L , et al. Glycyrrhiza polysaccharide alleviates dextran sulfate sodium-induced ulcerative colitis in mice[J]. Evid Based Complement Alternat Med, 2022, 2022, 1345852.
19	BISHT S , FAIQ M , TOLAHUNASE M , et al. Oxidative stress and male infertility[J]. Nat Rev Urol, 2017, 14 (8): 470- 485.
20	CHEN Y H , JIN S , TENG X , et al. Hydrogen sulfide attenuates LPS-induced acute kidney injury by inhibiting inflammation and oxidative stress[J]. Oxid Med Cell Longev, 2018, 2018, 6717212.
21	KUMAR J , HALDAR C , VERMA R . Melatonin ameliorates LPS-induced testicular nitro-oxidative stress (iNOS/TNFα) and inflammation (NF-kB/COX-2) via modulation of SIRT-1[J]. Reprod Sci, 2021, 28 (12): 3417- 3430.
22	王晴, 洪叶, 柳振宇, 等. 甘草多糖的抗氧化及降血糖作用研究[J]. 食品工业科技, 2023, 44 (1): 398- 404.
	WANG Q , HONG Y , LIU Z Y , et al. Study on the antioxidant and hypoglycemic effects of glycyrrhiza polysaccharide[J]. Science and Technology of Food Industry, 2023, 44 (1): 398- 404.
23	廖成水, 王晓利, 牛晓婉, 等. 甘草多糖对鼠伤寒沙门菌诱导的巨噬细胞氧化-抗氧化平衡紊乱的调解[J]. 中国兽医学报, 2015, 35 (6): 941- 945.
	LIAO C S , WANG X L , NIU X W , et al. Effect of radix glycyrrhiza polysaccharide on the imbalance of oxidation/antioxidation induced by Salmonella Typhimurium in murine peritoneal macrophages[J]. Chinese Journal of Veterinary Science, 2015, 35 (6): 941- 945.
24	MCCABE M J , TARULLI G A , LAVEN-LAW G , et al. Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction[J]. Hum Reprod, 2016, 31 (4): 875- 886.
25	FENG R , ADENIRAN S O , HUANG F S , et al. The ameliorative effect of melatonin on LPS-induced Sertoli cells inflammatory and tight junctions damage via suppression of the TLR4/MyD88/NF-κB signaling pathway in newborn calf[J]. Theriogenology, 2022, 179, 103- 116.
26	SHI G X , KONG J R , WANG Y L , et al. Glycyrrhiza uralensis fisch. alleviates dextran sulfate sodium-induced colitis in mice through inhibiting of NF-κB signaling pathways and modulating intestinal microbiota[J]. J Ethnopharmacol, 2022, 298, 115640.
27	SHI J J , GAO W Q , SHAO F . Pyroptosis: gasdermin-mediated programmed necrotic cell death[J]. Trends Biochem Sci, 2017, 42 (4): 245- 254.
28	KANG J Y , XU M M , SUN Y , et al. Melatonin attenuates LPS-induced pyroptosis in acute lung injury by inhibiting NLRP3-GSDMD pathway via activating Nrf2/HO-1 signaling axis[J]. Int Immunopharmacol, 2022, 109, 108782.
29	QIU Z , HE Y H , MING H , et al. Lipopolysaccharide (LPS) aggravates high glucose- and hypoxia/reoxygenation-induced injury through activating ROS-dependent NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes[J]. J Diabetes Res, 2019, 2019, 8151836.
30	HUANG C C , ZHANG C T , YANG P P , et al. Eldecalcitol inhibits LPS-induced NLRP3 inflammasome-dependent pyroptosis in human gingival fibroblasts by activating the Nrf2/HO-1 signaling pathway[J]. Drug Des Devel Ther, 2020, 14, 4901- 4913.
31	喻浩恒. 甘草苷通过雌激素受体抑制小胶质细胞焦亡的作用和机制[D]. 新乡: 新乡医学院, 2020.
	YU H H. The effect and mechanism of liquiritin on microglial pyroptosis via estrogen receptor[D]. Xinxiang: Xinxiang Medical University, 2020. (in Chinese)

引物名称 Name	引物序列(5′→3′) Sequence	引物大小/bp Product size
β-actin	F：TACCCAGGCATTGCTGACAG R：ACTCCTGCTTGCTGATCCAC	169
IL-1β	F：TCAGCACCTCACAAGCAGAG R：TTCTTGTGACCCTGAGCGAC	230
IL-18	F：ATGCTTTCTGGACTCCTGCC R：AGTCTTCTGACATGGCAGCC	149
IL-6	F：TTGGGACTGATGCTGGTGAC R：AGACAGGTCTGTTGGGAGTG	94
TNF-α	F：TGTCCCTTTCACTCACTGGC R：TCTTCTGCCAGTTCCACGTC	98
ZO-1	F：AAACAGCCCTACCAACCTCG R：TTCGAGGCAGCTGCTCATAG	163
Occludin	F：TTGACTGGGCTGAACACTCC R：ACATCACAGCTCACACCAGG	122
Caspase-1	F：AGTTGCTGCTGGAGGATCTG R：AGAAGTCTTGTGCTCTGGGC	113
NLRP3	F：TCTGCACCCGGACTGTAAAC R：CATTGTTGCCCAGGTTCAGC	131
GSDMD	F：TGCGTGTGACTCAGAAGACC R：AAACAGGTCATCCCCACGAC	107

[1]	季小禹, 王永伟, 邱妍, 张才. 甘草多糖的生理功能及其在畜禽生产中的应用[J]. 畜牧兽医学报, 2024, 55(6): 2379-2387.
[2]	吴开慧, 张留光, 汪超, 许师源, 刘松奇, 原开敏, 王栋, 庞云渭. 褪黑素缓解白消安损伤小鼠睾丸的机理研究[J]. 畜牧兽医学报, 2024, 55(6): 2486-2497.
[3]	孙雯莉, 王浩奇, 泽里磋, 高雨樊, 张非凡, 张健, 段梦琪, 商鹏, 强巴央宗. 藏猪促炎因子(IL-1β、IL-6、TNF-α)多态性及其表达与免疫性状的关联分析[J]. 畜牧兽医学报, 2024, 55(5): 1958-1969.
[4]	杨小峰, 秦小伟, 吕丽华. MNQ的一种衍生物对LPS体外诱导的牛卵巢卵泡颗粒细胞炎性损伤的保护作用[J]. 畜牧兽医学报, 2024, 55(5): 2032-2041.
[5]	苏依曼, 叶嘉莉, 邱文粤, 章心婷, 庞晓玥, 王荣梅, 唐兆新, 苏荣胜. 积雪草酸通过抑制HMGB1/TLR4/NF-κB通路减轻脂多糖诱导肉鸡肾细胞焦亡[J]. 畜牧兽医学报, 2024, 55(4): 1777-1786.
[6]	马亚军, 焦智慧, 刘笑凝, 陆翔羽, 刘涛, 王月, 朴晨曦, 王洪斌. 脂肪间充质干细胞对小型猪肝缺血再灌注合并肝切除组织细胞焦亡的影响[J]. 畜牧兽医学报, 2024, 55(1): 355-364.
[7]	樊磊, 莘余, 尤留超, 田欣宇, 罗皓, 王辛, 张婷婷, 沈留红. 脂多糖致奶牛糖脂代谢异常研究进展[J]. 畜牧兽医学报, 2023, 54(2): 484-493.
[8]	孟梅娟, 汪艳, 霍然, 李雪睿, 常广军, 沈向真. 抑制PERK对LPS诱导的奶牛乳腺上皮细胞自噬的影响[J]. 畜牧兽医学报, 2023, 54(1): 351-360.
[9]	赵欣, 王莹, 李春亭, 王巍, 桑锐, 李海涛, 张雪梅. 蒲公英提取物对LPS诱导小鼠乳腺炎的减轻效应及其机制分析[J]. 畜牧兽医学报, 2022, 53(8): 2773-2781.
[10]	梁文清, 刘忠华, 常晓月, 何婷, 陈嘉, 马晓莉, 唐兆新, 余文兰. 长期高铜暴露通过影响线粒体自噬和细胞焦亡诱导大鼠肝组织损伤[J]. 畜牧兽医学报, 2022, 53(12): 4490-4500.
[11]	贺尚文, 王月明, 张慧, 侯思鲁, 刘晓晔, 董虹. 白头翁汤通过抑制TLR4-ERK1/2信号通路缓解LPS诱导的微血管内皮细胞炎性反应[J]. 畜牧兽医学报, 2021, 52(8): 2317-2325.
[12]	石玉华, 傅心雨, 郑梦洁, 吕倩, 师福山. 非经典细胞焦亡体外模型的构建[J]. 畜牧兽医学报, 2021, 52(8): 2284-2290.
[13]	聂雪伊, 刘蕾, 郑雪迪, 杨易, 徐金瑞, 王玉炯. BCG感染巨噬细胞后内质网应激对细胞焦亡的调控作用[J]. 畜牧兽医学报, 2021, 52(10): 2842-2851.
[14]	张海洋, 陆翔宇, 张家华, 刘欣, 韩政轩, 崔安, 赵元, 宋曼玉, 刘涛, 范宏刚. 褪黑素对LPS致大鼠海马炎性损伤的保护作用[J]. 畜牧兽医学报, 2021, 52(1): 226-234.
[15]	邵丹, 宋朋杰, 闫宝琪, 武小虎, 王东升, 李亚娟, 张世栋, 严作廷. 百里香酚对脂多糖诱导的子宫内膜上皮细胞炎性反应的抑制作用研究[J]. 畜牧兽医学报, 2019, 50(7): 1493-1499.