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

doi:10.11843/j.issn.0366-6964.2024.05.021

摘要/Abstract

摘要： 旨在采用凤仙花(Impatiens balsamina L)提取物2-甲氧基-1,4-萘醌(MNQ)的衍生物D₁₉消除体外暴露于脂多糖(LPS)中牛卵巢卵泡颗粒细胞(GCs)的炎症反应，并缓减由LPS引起的卵泡GCs功能性紊乱。本研究采集性成熟且健康的荷斯坦牛卵巢组织，分离并培养卵泡GCs。MTT法测定D₁₉和LPS对卵泡GCs存活率的影响。试验分为3组：对照组、LPS处理组和D₁₉联合LPS处理组，每组3个重复。qRT-PCR测定炎性因子和类固醇合成相关基因的相对表达量。TEM观察D₁₉对细胞炎性损伤的保护作用。ELISA检测培养液上清中雌二醇(E₂)和孕酮(P₄)的含量。结果表明，D₁₉对卵泡GCs作用12 h的最大安全浓度为64 μmol·L^-1。LPS浓度为10 μg·mL^-1时，作用12 h对卵泡GCs存活率影响不大，但炎性因子IL-6、IL-1β和TNF-α的相对表达量显著升高(P<0.01)，D₁₉+L组与LPS组比较炎性因子的相对表达量却显著降低(P<0.01)。通过TEM观察表明D₁₉对LPS引起的细胞器结构性损伤具有保护作用。ELISA结果表明，LPS处理后培养液中E₂和P₄的含量显著降低(P<0.01)，qRT-PCR检测到类固醇合成相关基因CYP19A1、CYP11A1、3β-HSD和STAR的相对表达量也显著降低(P<0.01)。但D₁₉联合LPS处理后，E₂和P₄分泌量以及类固醇合成相关基因的相对表达量均比LPS组显著升高(P<0.01)。本研究证明MNQ的衍生物D₁₉具有消除LPS体外诱导卵泡GCs炎症反应的功能，并能一定程度缓减LPS导致的卵泡GCs功能性紊乱。

关键词: MNQ, D₁₉, LPS, GCs, 炎症反应

Abstract: The aim of this experiment was to eliminate the inflammatory response of bovine ovarian follicular granulosa cells (GCs) exposed to lipopolysaccharide (LPS) in vitro and to attenuate LPS-induced functional disorders of follicular GCs using D₁₉, a derivative of 2-methoxy-1,4-naphthoquinone (MNQ) from the extract of Impatiens balsamina L. Ovarian tissues were collected from sexually mature and healthy Holstein cattle, and follicular GCs were isolated and cultured. The effect of D₁₉ and LPS on the survival of follicular GCs was determined by MTT assay. The experiment was divided into 3 groups: control, LPS-treated and D₁₉-combined LPS-treated groups, with 3 replicates in each group. The relative expression of inflammatory factors and genes related to steroid synthesis was determined by qRT-PCR. TEM was performed to observe the protective effect of D₁₉ against cellular inflammatory injury. ELISA was performed to measure the levels of estradiol (E₂) and progesterone (P₄) in the supernatant of the culture fluid. The results showed that the maximum no-cytotoxic concentration of D₁₉ acting on follicular GCs for 12 h was 64 μmol·L^-1. LPS concentration of 10 μg·mL^-1 had little effect on the survival of follicular GCs after 12 h of action, but the relative expression of the inflammatory factors IL-6, IL-1β, and TNF-α was significantly higher (P<0.01), whereas the relative expression of the inflammatory factors in the D₁₉+L group was significantly lower compared with the LPS group (P<0.01). The protective effect of D₁₉ against LPS-induced structural damage to organelles was demonstrated by TEM observation. ELISA results showed that the levels of E₂ and P₄ in the culture broth were significantly reduced after LPS treatment (P<0.01), and the relative expression of steroid synthesis-related genes CYP19A1, CYP11A1, 3β-HSD, and STAR as detected by qRT-PCR was also significantly reduced (P<0.01). However, the amount of E₂ and P₄ secretion as well as the relative expression of steroid synthesis-related genes were significantly higher in the D₁₉ combined with LPS treatment than that in the LPS group (P<0.01). In the present study, we demonstrated that D₁₉, a derivative of MNQ, has the function of eliminating the inflammatory response of follicular GCs induced by LPS in vitro, and it can alleviate the functional disorders of follicular GCs caused by LPS to a certain extent.

Key words: MNQ, D₁₉, LPS, GCs, inflammatory response

中图分类号:

S823.3

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.

YANG Xiaofeng, QIN Xiaowei, LÜ Lihua. Protective Effect of a Derivative of MNQ Against LPS-Induced Inflammatory Injury in Bovine Ovarian Follicular Granulosa Cells in Vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2032-2041.

参考文献

[1] DEO P,CHOW S H,HAN M L,et al.Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation[J].Nat Microbiol,2020,5(11):1418-1427.
[2] PRICE J C,BROMFIELD J J,SHELDON I M.Pathogen-associated molecular patterns initiate inflammation and perturb the endocrine function of bovine granulosa cells from ovarian dominant follicles via TLR2 and TLR4 pathways[J].Endocrinology, 2013,154(9):3377-3386.
[3] ROSALES E B,AMETAJ B N.Reproductive tract infections in dairy cows:can probiotics curb down the incidence rate?[J].Dairy,2021,2(1):40-64.
[4] SABRY R.The effects of bisphenol a on the expression of microRNA-21 and miR-21 mediated pathways including epigenetic regulation and apoptosis in bovine granulosa cells[D].Guelph:University of Guelph,2023.
[5] SHEN Q Z,LIU Y,LI H G,et al.Effect of mitophagy in oocytes and granulosa cells on oocyte quality[J].Biol Reprod,2021,104(2):294-304.
[6] TABANDEH M R,JOZAIE S,GHOTBEDIN Z,et al.Dimethyl itaconic acid improves viability and steroidogenesis and suppresses cytokine production in LPS-treated bovine ovarian granulosa cells by regulating TLR4/nfkβ,NLRP3,JNK signaling pathways[J].Res Vet Sci,2022,152:89-98.
[7] SHEN J,ZHAO W M,CHENG J R,et al.Lipopolysaccharide accelerates tryptophan degradation in the ovary and the derivative kynurenine disturbs hormone biosynthesis and reproductive performance[J].J Hazard Mater,2023,458:131988.
[8] ROSADINI C V,KAGAN J C.Early innate immune responses to bacterial LPS[J].Curr Opin Immunol,2017,44:14-19.
[9] RATHINAM V A K,ZHAO Y,SHAO F.Innate immunity to intracellular LPS[J].Nat Immunol,2019,20(5):527-533.
[10] WANG D J,WENG Y J,ZHANG Y L,et al.Exposure to hyperandrogen drives ovarian dysfunction and fibrosis by activating the NLRP3 inflammasome in mice[J].Sci Total Environ,2020,745(31):141049.
[11] RAUF A,Abu-IZNEID T,RASHID U,et al.Anti-inflammatory,antibacterial,toxicological profile,and in silico studies of dimeric naphthoquinones from Diospyros lotus[J].BioMed Res Int,2020,2020:7942549.
[12] ASIRI S M,SHAARI K,ABAS F,et al.Two new naphthoquinone derivatives from the stem bark of Callicarpa maingayi[J].Nat Prod Commun,2012,7(10):1333-1336.
[13] DONG M,LIU D,LI Y H,et al.Naphthoquinones from Onosma paniculatum with potential anti-inflammatory activity[J].Planta Med,2017,83(7):631-635.
[14] PINHO B R,SOUSA C,VALENTÃO P,et al.Is nitric oxide decrease observed with naphthoquinones in LPS stimulated RAW 264.7 macrophages a beneficial property?[J].PLoS One,2011,6(8):e24098.
[15] JIANG H F,ZHUANG Z H,HOU B W,et al.Adverse effects of hydroalcoholic extracts and the major components in the stems of Impatiens balsamina L. on Caenorhabditis elegans[J].Evid Based Complement Alternat Med,2017,2017:4245830.
[16] YANG X F,GUO T,DU Z S,et al.Protective effects of MNQ against Lipopolysaccharide-induced inflammatory damage in bovine ovarian follicular granulosa cells in vitro[J].J Steroid Biochem Mol Biol,2023,230:106274.
[17] YANG X F,QIN X W,WANG K,et al.MNQ derivative D₂₁ protects against LPS-induced inflammatory damage in bovine ovarian follicular GCs in vitro via the steroid biosynthesis signaling pathway[J].Theriogenology,2023,206:149-160.
[18] SANTOS P H,NUNES S G,FRANCHI F F,et al.Expression of bta-miR-222 and LHCGR in bovine cultured granulosa cells:impact of follicle deviation and regulation by FSH/insulin in vitro[J].Theriogenology,2022,182:71-77.
[19] TSATSANIS C,ANDROULIDAKI A,ALISSAFI T,et al.Corticotropin-releasing factor and the urocortins induce the expression of TLR4 in macrophages via activation of the transcription factors PU.1 and AP-1[J].J Immunol,2006,176(3):1869-1877.
[20] RIO D C,ARES JR M,HANNON G J,et al.Purification of RNA using TRIzol (TRI reagent)[J].Cold Spring Harb Protoc,2010,2010(6):pdb.prot5439.
[21] FUTURO D O,FERREIRA P G,NICOLETTI C D,et al.The antifungal activity of naphthoquinones:an integrative review[J].An Acad Bras Ciênc,2018,90(1 Suppl 2):1187-1214.
[22] AHMADI E S,TAJBAKHSH A,IRANSHAHY M,et al.Naphthoquinone derivatives isolated from plants:recent advances in biological activity[J].Mini Rev Med Chem,2020,20(19):2019-2035.
[23] CHEN M,VIAL M L,GEE L,et al.The plant natural product 2-methoxy-1,4-naphthoquinone stimulates therapeutic neural repair properties of olfactory ensheathing cells[J].Sci Rep,2020,10(1):951.
[24] WU H,WANG Y,ZHANG Y P,et al.Breaking the vicious loop between inflammation,oxidative stress and coagulation,a novel anti-thrombus insight of nattokinase by inhibiting LPS-induced inflammation and oxidative stress[J].Redox Biol,2020,32: 101500.
[25] LIU J,CHANG G J,HUANG J,et al.Sodium butyrate inhibits the inflammation of lipopolysaccharide-induced acute lung injury in mice by regulating the toll-like receptor 4/nuclear factor κB signaling pathway[J].J Agric Food Chem,2019,67(6):1674-1682.
[26] MASTINU A,BONINI S A,PREMOLI M,et al.Protective effects of Gynostemma pentaphyllum (var. Ginpent) against lipopolysaccharide-induced inflammation and motor alteration in mice[J].Molecules,2021,26(3):570.
[27] ROUSTA A M,MIRAHMADI S M S,SHAHMOHAMMADI A,et al.Protective effect of sesamin in lipopolysaccharide-induced mouse model of acute kidney injury via attenuation of oxidative stress,inflammation,and apoptosis[J].Immunopharm Immunot,2018,40(5):423-429.
[28] XIE W,LU Q C,WANG K L,et al.miR-34b-5p inhibition attenuates lung inflammation and apoptosis in an LPS-induced acute lung injury mouse model by targeting progranulin[J].J Cell Physiol,2018,233(9):6615-6631.
[29] TANG J,DIAO P,SHU X H,et al.Quercetin and quercitrin attenuates the inflammatory response and oxidative stress in LPS-induced RAW264.7 cells:in vitro assessment and a theoretical model[J].BioMed Res Int,2019,2019:7039802.
[30] CHEN L,LIN X J,XIAO J B,et al.Sonchus oleraceus Linn protects against LPS-induced sepsis and inhibits inflammatory responses in RAW264.7 cells[J].J Ethnopharmacol,2019,236:63-69.
[31] HERATH S,WILLIAMS E J,LILLY S T,et al.Ovarian follicular cells have innate immune capabilities that modulate their endocrine function[J].Reproduction,2007,134(5):683-693.
[32] SHIMADA M,HERNANDEZ-GONZALEZ I,GONZALEZ-ROBANYA I,et al.Induced expression of pattern recognition receptors in cumulus oocyte complexes:novel evidence for innate immune-like functions during ovulation[J].Mol Endocrinol,2006,20(12):3228-3239.
[33] VASHISHT M,RANI P,SUNITA,et al.Curcumin primed exosomes reverses LPS-induced pro-inflammatory gene expression in buffalo granulosa cells[J].J Cell Biochem,2017,119(2):1488-1500.
[34] HORLOCK A D,ORMSBY T J R,CLIFT M J D,et al.Manipulating bovine granulosa cell energy metabolism limits inflammation[J]. Reproduction,2021,161(5):499-512.
[35] ONNUREDDY K,RAVINDER,ONTERU S K,et al.IGF-1 attenuates LPS induced pro-inflammatory cytokines expression in buffalo (Bubalus bubalis) granulosa cells[J].Mol Immunol,2015,64(1):136-143.
[36] GIACOMINI E,MINETTO S,KLEEMAN F,et al.P-300 Evaluation of CYP19A1 gene expression in luteinized granulosa cells of women affected by endometriosis undergoing assisted reproductive technology (ART) treatments[J].Hum Reprod,2022, 37(Suppl 1): deac104.073.
[37] COLE T J,SHORT K L,HOOPER S B.The science of steroids[J].Semin Fetal Neonatal Med,2019,24(3):170-175.
[38] SHIMIZU T,MIYAUCHI K,SHIRASUNA K,et al.Effects of lipopolysaccharide (LPS) and peptidoglycan (PGN) on estradiol production in bovine granulosa cells from small and large follicles[J].Toxicol in Vitro,2012,26(7):1134-1142.