• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    MNQ的一種衍生物對(duì)LPS體外誘導(dǎo)的牛卵巢卵泡顆粒細(xì)胞炎性損傷的保護(hù)作用

    2024-09-19 00:00:00楊小峰秦小偉呂麗華
    畜牧獸醫(yī)學(xué)報(bào) 2024年5期
    關(guān)鍵詞:炎癥反應(yīng)

    摘 要: 旨在采用鳳仙花(Impatiens balsamina L)提取物2-甲氧基-1,4-萘醌(MNQ)的衍生物D19消除體外暴露于脂多糖(LPS)中牛卵巢卵泡顆粒細(xì)胞(GCs)的炎癥反應(yīng),并緩減由LPS引起的卵泡GCs功能性紊亂。本研究采集性成熟且健康的荷斯坦牛卵巢組織,分離并培養(yǎng)卵泡GCs。MTT法測(cè)定D19和LPS對(duì)卵泡GCs存活率的影響。試驗(yàn)分為3組:對(duì)照組、LPS處理組和D19聯(lián)合LPS處理組,每組3個(gè)重復(fù)。qRT-PCR測(cè)定炎性因子和類固醇合成相關(guān)基因的相對(duì)表達(dá)量。TEM觀察D19對(duì)細(xì)胞炎性損傷的保護(hù)作用。ELISA檢測(cè)培養(yǎng)液上清中雌二醇(E2)和孕酮(P4)的含量。結(jié)果表明,D19對(duì)卵泡GCs作用12 h的最大安全濃度為64 μmol·L-1。LPS濃度為10 μg·mL-1時(shí),作用12 h對(duì)卵泡GCs存活率影響不大,但炎性因子IL-6、IL-1β和TNF-α的相對(duì)表達(dá)量顯著升高(Plt;0.01),D19+L組與LPS組比較炎性因子的相對(duì)表達(dá)量卻顯著降低(Plt;0.01)。通過(guò)TEM觀察表明D19對(duì)LPS引起的細(xì)胞器結(jié)構(gòu)性損傷具有保護(hù)作用。ELISA結(jié)果表明,LPS處理后培養(yǎng)液中E2和P4的含量顯著降低(Plt;0.01),qRT-PCR檢測(cè)到類固醇合成相關(guān)基因CYP19A1、CYP11A1、3β-HSD和STAR的相對(duì)表達(dá)量也顯著降低(Plt;0.01)。但D19聯(lián)合LPS處理后,E2和P4分泌量以及類固醇合成相關(guān)基因的相對(duì)表達(dá)量均比LPS組顯著升高(Plt;0.01)。本研究證明MNQ的衍生物D19具有消除LPS體外誘導(dǎo)卵泡GCs炎癥反應(yīng)的功能,并能一定程度緩減LPS導(dǎo)致的卵泡GCs功能性紊亂。

    關(guān)鍵詞: MNQ;D19;LPS;GCs;炎癥反應(yīng)

    中圖分類號(hào):S823.3

    文獻(xiàn)標(biāo)志碼:A""" 文章編號(hào):0366-6964(2024)05-2032-10

    收稿日期:2023-11-02

    基金項(xiàng)目:山西省基礎(chǔ)研究計(jì)劃項(xiàng)目(20210302123393);“1331工程”提質(zhì)增效建設(shè)計(jì)劃項(xiàng)目子課題資助(2023WTS03)

    作者簡(jiǎn)介:楊小峰(1981-),男,山西繁峙人,講師,博士,主要從事動(dòng)物生殖生理和繁殖生物技術(shù)研究,E-mail: xiaofengy2023@163.com

    *通信作者:呂麗華,主要從事動(dòng)物生殖生理和繁殖生物技術(shù)研究,E-mail: lihualyusxau@126.com

    Protective Effect of a Derivative of MNQ Against LPS-Induced Inflammatory Injury

    in Bovine Ovarian Follicular Granulosa Cells in Vitro

    YANG" Xiaofeng1,2, QIN" Xiaowei2, L Lihua2*

    (1.Department of Biology, Xinzhou Normal University, Xinzhou 034000," China;

    2.College of Animal Science, Shanxi Agricultural University, Taigu 030801," China)

    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 (Plt;0.01), whereas the relative expression of the inflammatory factors in the D19+L group was significantly lower compared with the LPS group (Plt;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 (Plt;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 (Plt;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 (Plt;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

    *Corresponding author: L Lihua,E-mail: lihualyusxau@126.com

    牛生殖系統(tǒng)炎癥是目前畜牧業(yè)面臨的問題之一。革蘭氏陰性菌細(xì)胞壁的內(nèi)毒素——脂多糖(lipopolysaccharide,LPS)是導(dǎo)致炎癥發(fā)生的主要原因[1]。之前研究表明,牛分娩后的子宮約有90%被細(xì)菌感染,其中40%發(fā)展為臨床疾病。生殖系統(tǒng)的細(xì)菌感染能破壞子宮和卵巢的功能,導(dǎo)致排卵延遲、生育能力下降甚至不孕,引發(fā)卵巢炎、盆腔炎、子宮內(nèi)膜炎等疾病[2-3]。卵巢中的卵泡顆粒細(xì)胞(granulosa cells,GCs)是組成卵泡的主要細(xì)胞群,在卵泡形成和發(fā)育過(guò)程中起著重要的作用,為卵母細(xì)胞的生長(zhǎng)和成熟提供必要的營(yíng)養(yǎng)[4]。在卵泡生長(zhǎng)發(fā)育過(guò)程中GCs進(jìn)行著增殖和分化,同時(shí)合成和分泌類固醇激素。類固醇生物合成信號(hào)通路對(duì)GCs的功能起著及其重要調(diào)控作用[5]。LPS對(duì)牛生理活動(dòng)產(chǎn)生嚴(yán)重的損害,如改變GCs基因的表達(dá),降低其產(chǎn)生雌二醇(E2)的量[6-7]。LPS可以引起免疫刺激的級(jí)聯(lián)反應(yīng)和機(jī)體的毒性病理生理活動(dòng),導(dǎo)致多種細(xì)胞因子和炎癥遞質(zhì)合成及釋放[8-9]。研究人員在患有子宮內(nèi)膜炎的牛卵巢卵泡液中測(cè)出高濃度的LPS[10]。由此推測(cè),患生殖系統(tǒng)炎癥的動(dòng)物,通過(guò)微環(huán)境中LPS的積累引發(fā)一系列炎性損傷并導(dǎo)致卵泡中GCs功能性紊亂。

    萘醌類化合物(NQs)已被證明具有抗炎活性,它們通過(guò)對(duì)各種細(xì)胞因子和調(diào)節(jié)蛋白的作用,影響細(xì)胞信號(hào)通路,保護(hù)細(xì)胞免受炎癥損傷[11]。Asiri等[12]從滇紫草提取物中分離得到了兩個(gè)呋喃型萘醌類化合物,它們能有效抑制NO的釋放,表明了其潛在的抗炎活性。Dong等[13]從onosma paniculatum甲醇提取物中分離到4種新的萘醌和6種已知的萘醌化合物,均對(duì)小鼠巨噬細(xì)胞(RAW 264.7)NO的產(chǎn)生具有抑制作用。Pinho等[14]評(píng)估了一系列單體和二聚物NQs的抗炎活性,結(jié)果表明一些二聚物能顯著降低NO的釋放量和促炎因子TNF-α和IL-6的表達(dá)量,并且沒有細(xì)胞毒性。2-甲氧基-1,4-萘醌(2-methoxy-1,4-naphthoquinone,MNQ)是存在于鳳仙花(Impatiens balsamina L)根、莖中的一種NQs,能作用于細(xì)胞壁、細(xì)胞膜及線粒體等多個(gè)靶點(diǎn),具有抗菌、消炎、抗過(guò)敏、抗瘙癢、抗腫瘤、抗氧化等活性[15]。

    目前,對(duì)MNQ的研究主要集中于細(xì)胞毒性和抗癌作用,對(duì)抗炎活性的研究較少。人工合成MNQ的衍生物,理論上增強(qiáng)了其生物學(xué)活性。課題組已經(jīng)證實(shí)MNQ及其一種衍生物D21具有消炎作用[16-17]。本試驗(yàn)將體外培養(yǎng)的牛卵泡GCs暴露于LPS中,形成炎癥損傷的細(xì)胞。本研究試圖用MNQ的另一種衍生物D19保護(hù)GCs免受炎癥損傷,并恢復(fù)其生理功能。

    1 材料與方法

    1.1 試驗(yàn)材料

    1.1.1 主要材料

    本試驗(yàn)從屠宰場(chǎng)采集健康的荷斯坦牛卵巢組織,將其置于含有1%的青鏈霉素混合液并滅菌的DPBS中,2 h內(nèi)冰上帶回實(shí)驗(yàn)室。

    從鳳仙花(Impatiens balsamina L)中提取MNQ,并合成MNQ的衍生物D19(圖1)。

    1.1.2 主要儀器

    細(xì)胞培養(yǎng)箱(ThermoFisher,美國(guó));熒光倒置顯微鏡(Leica,德國(guó));透射電子顯微鏡(JEOL,日本);酶標(biāo)儀(BIO-EKON,北京);超低溫冰箱(ThermoFisher,美國(guó));核酸/蛋白測(cè)定儀(NanoDrop,美國(guó))。

    1.1.3 主要試劑

    MNQ的衍生物D19(Luis實(shí)驗(yàn)室,英國(guó));青鏈霉素混合液(Gibco,美國(guó));DPBS(Solarbio,中國(guó));胎牛血清(Solarbio,中國(guó));DMEM/F12培養(yǎng)基(BOSTER,中國(guó));DMSO(Solarbio,北京);LPS(Solarbio,中國(guó));PBS(Solarbio,中國(guó));MTT(Solarbio,中國(guó));PrimeScriptTMRT reagent Kit with gDNA Eraser試劑盒(TaKaRa,北京);TB GreenTM Premix Ex TaqTMⅡ試劑盒(TaKaRa,北京);戊二醛(LILAI,成都);四氧化鋨(LILAI,成都);ELISA試劑盒(酶免,中國(guó))。

    1.2 方法

    1.2.1 卵泡GCs的分離和培養(yǎng)

    用提前滅菌的剪刀從牛卵巢組織中分離出卵泡。選擇直徑為4~8 mm的卵泡,從其內(nèi)壁輕輕刮取卵泡GCs。收集并洗滌GCs。用含有10%胎牛血清的培養(yǎng)基培養(yǎng)GCs,培養(yǎng)條件為37℃,5%的CO2[18]。

    1.2.2 MTT試驗(yàn)

    收集對(duì)數(shù)期的卵泡GCs接種于96孔板中培養(yǎng),培養(yǎng)條件為37℃,5% CO2。待細(xì)胞長(zhǎng)至20%~40%時(shí),去掉上清。

    加入含有D19的培養(yǎng)液(D19溶解于DMSO中,DMSO的終濃度小于0.1%),終濃度分別為1、2、4、8、16、32、64、128 μmol·L-1(n=6),設(shè)置對(duì)照組,分別培養(yǎng)12、24和48 h。

    加入含有LPS的培養(yǎng)液(LPS溶解于PBS),濃度分別為0.1、1、10 μg·mL-1(n=6),設(shè)置對(duì)照組,培養(yǎng)12 h。濃度梯度的設(shè)置參考生殖道炎癥卵泡液中LPS濃度和LPS用于免疫分析的濃度[19]。

    去掉上清,每孔加入90 μL培養(yǎng)液,再加10 μL MTT溶液,繼續(xù)培養(yǎng)4 h。棄去培養(yǎng)液,每孔加110 μL DMSO溶解結(jié)晶物。490 nm處測(cè)量OD值,計(jì)算細(xì)胞存活率。

    細(xì)胞存活率=(藥物組OD值-空白組OD值)/(對(duì)照組OD值-空白組OD值)×100%。

    1.2.3 實(shí)時(shí)熒光定量PCR(qRT-PCR)

    使用Trizol法提取總RNA,按照PrimeScriptTMRT reagent Kit with gDNA Eraser試劑盒說(shuō)明書進(jìn)行反轉(zhuǎn)錄[20]。根據(jù)NCBI上牛的預(yù)測(cè)序列,利用Primer 5.0設(shè)計(jì)目的基因引物,由上海生物工程股份有限公司合成。以荷斯坦牛β-actin作為內(nèi)參基因,引物序列及產(chǎn)物長(zhǎng)度見表1。依據(jù)TB GreenTM Premix Ex TaqTMⅡ試劑盒說(shuō)明書,以cDNA為模板,構(gòu)建10 μL反應(yīng)體系(TB Green Premix Ex TaqⅡ 5 μL、上游引物0.4 μL、下游引物0.4 μL、cDNA 2 μL、RNase Free dH2O 2.2 μL),進(jìn)行qRT-PCR反應(yīng)。反應(yīng)條件為95℃,30 s;95℃,5 s;60℃,30 s,共45個(gè)循環(huán)。擴(kuò)增后生成擴(kuò)增曲線、熔解曲線,計(jì)算CT值分析結(jié)果。

    1.2.4 試驗(yàn)分組

    試驗(yàn)分為3組:CK組、LPS組和D19+L組。CK組為對(duì)照組;LPS組,前12 h處理細(xì)胞同CK組,之后用濃度為10 μg·mL-1的LPS處理細(xì)胞12 h;D19+L組,前12 h用濃度為64 μmol·L-1的D19處理細(xì)胞,之后用濃度為10 μg·mL-1的LPS處理細(xì)胞12 h。每組設(shè)置3個(gè)重復(fù),作用時(shí)間共計(jì)24 h。

    1.2.5 TEM觀察D19對(duì)LPS損傷GCs的保護(hù)

    用60 mm的培養(yǎng)皿培養(yǎng)GCs,按照試驗(yàn)要求處理細(xì)胞。待細(xì)胞長(zhǎng)滿整個(gè)培養(yǎng)皿時(shí),胰酶消化并收集于EP管中。分別用3%戊二醛和1%四氧化鋨固定細(xì)胞。經(jīng)過(guò)丙酮逐級(jí)脫水,滲透和包埋后切片、染色,最后使用TEM采集圖像。

    1.2.6 酶聯(lián)免疫吸附試驗(yàn)(ELISA)

    收集細(xì)胞培養(yǎng)液,用ELISA試劑盒檢測(cè)培養(yǎng)液中E2和P4的濃度。在96孔板中加入100 μL的標(biāo)準(zhǔn)品和待測(cè)樣品,37℃下反應(yīng)60 min。洗滌后各孔中加入100 μL顯色劑,37℃避光反應(yīng)15~20 min。加入50 μL終止液,終止反應(yīng)。450 nm波長(zhǎng)下測(cè)定OD值。

    1.2.7 統(tǒng)計(jì)學(xué)分析

    試驗(yàn)數(shù)據(jù)利用SPSS 22.0進(jìn)行分析,并采用單因素方差分析和顯著性檢驗(yàn)的方法,P<0.05表示差異顯著,而P<0.01表示差異極顯著。使用GraphPad Prism 8.0軟件作圖。qRT-PCR相對(duì)表達(dá)量檢測(cè)結(jié)果采用2-ΔΔCt法計(jì)數(shù),各基因表達(dá)量均經(jīng)過(guò)內(nèi)參β-actin校正。E2和P4的濃度根據(jù)標(biāo)準(zhǔn)曲線確定。所有試驗(yàn)至少重復(fù)3次。

    2 結(jié) 果

    2.1 D19對(duì)GCs活性的影響

    在一定的濃度范圍內(nèi),D19對(duì)卵泡GCs的存活率影響較小,甚至?xí)龠M(jìn)其增殖。濃度在64 μmol·L-1以下,作用時(shí)間為12和24 h時(shí),GCs的存活率均在90%以上;濃度為128 μmol·L-1時(shí),作用時(shí)間為12 h時(shí),GCs的存活率小于90%,且與對(duì)照組相比存在顯著性差異(Plt;0.01)。因此D19對(duì)GCs作用12 h的最大安全濃度為64 μmol·L-1(最大安全濃度:MNTC,maximum no-cytotoxic concentration,是指能夠使90%以上細(xì)胞存活的化合物的最大濃度)。后期試驗(yàn)選擇濃度為64 μmol·L-1的D19處理細(xì)胞時(shí)間為12 h(圖2)。

    2.2 LPS誘導(dǎo)卵泡GCs產(chǎn)生炎癥反應(yīng)

    MTT試驗(yàn)檢測(cè)了不同濃度的LPS(0.1、1、10 μg·mL-1)處理卵泡GCs 12 h后對(duì)其存活率的影響。結(jié)果表明,試驗(yàn)濃度范圍之內(nèi)的LPS對(duì)卵泡GCs的存活率沒有顯著影響(圖3A)。

    不同濃度的LPS處理卵泡GCs 12 h,使用qRT-PCR檢測(cè)炎癥因子IL-6、IL-1β和TNF-α的相對(duì)表達(dá)量。結(jié)果表明,隨著LPS濃度的增加,炎"" 性因子的相對(duì)表達(dá)量逐漸升高,10 μg·mL-1的LPS處理后IL-6、IL-1β和TNF-α的相對(duì)表達(dá)量均顯著升高(Plt;0.01,圖3B)。后續(xù)選擇濃度為10 μg·mL-1的LPS誘導(dǎo)卵泡GCs產(chǎn)生炎癥反應(yīng)。

    2.3 衍生物D19的抗炎作用

    qRT-PCR檢測(cè)不同處理組IL-6、IL-1β和TNF-α的mRNA相對(duì)表達(dá)量,發(fā)現(xiàn)LPS組與CK組相比炎性因子mRNA的表達(dá)量顯著升高(Plt;0.01),但D19+L組與LPS組相比表達(dá)量卻顯著下降(Plt;0.01)(圖4)。結(jié)果表明濃度為64 μmol·L-1的D19能有效抑制由LPS引發(fā)的炎癥反應(yīng)。

    2.4 TEM觀察D19對(duì)LPS損傷GCs的保護(hù)

    使用TEM分別觀察了CK組、LPS組和D19+L組的亞顯微結(jié)構(gòu)圖。結(jié)果發(fā)現(xiàn)LPS組與CK組相比線粒體固縮、嵴減少變粗、膜密度增高、粗面內(nèi)質(zhì)網(wǎng)發(fā)生擴(kuò)張、胞漿中自噬溶酶體數(shù)量減少。D19+L組與LPS組相比線粒體形態(tài)結(jié)構(gòu)一定程度恢復(fù)正常,外膜平滑、內(nèi)膜向腔內(nèi)形成嵴、基質(zhì)電子密度均勻、粗面內(nèi)質(zhì)網(wǎng)扁囊輕度增大、高爾基體含量豐富、自噬溶酶體數(shù)量增多(圖5)。

    2.5 D19對(duì)LPS引起卵泡GCs功能性紊亂的保護(hù)作用

    用ELISA試劑盒檢測(cè)不同處理組培養(yǎng)液上清中E2和P4的濃度,并用qRT-PCR檢測(cè)類固醇合成相關(guān)基因的表達(dá)情況,結(jié)果見圖6。

    體外暴露在LPS中的卵泡GCs功能受到影響,表現(xiàn)為E2和P4的分泌量顯著降低(Plt;0.01),如圖6A、6B所示。同時(shí),檢測(cè)了CYP19A1(圖6C)、CYP11A1(圖6D)、3β-HSD(圖6E)和STAR(圖6F)的表達(dá)情況,結(jié)果表明與CK組相比LPS組均顯著下降(Plt;0.01)。但是,D19+L組的E2和P4分泌情況和類固醇合成相關(guān)基因表達(dá)情況,發(fā)現(xiàn)均比LPS組升高(Plt;0.01),因此表明D19能在一定程度恢復(fù)LPS對(duì)卵泡GCs造成的功能性損傷。

    3 討 論

    NQs是重要的細(xì)胞毒性化合物,通過(guò)不同的機(jī)制發(fā)揮作用,如氧化還原反應(yīng)、誘導(dǎo)DNA斷裂以及產(chǎn)生自由基、活性氧等[21]。MNQ屬于萘醌類化合物,它也表現(xiàn)出一定的細(xì)胞毒性。研究人員從海娜花(Impatiens balsamina)中分離得到MNQ,使用含有MNQ的培養(yǎng)基對(duì)肺癌人類肺泡基底上皮細(xì)胞(A549)進(jìn)行體外培養(yǎng),得到MNQ的IC50為7.5 μmol·L-1[22]。Chen等[23]研究表明,當(dāng)濃度為0.1和1 μmol·L-1時(shí),MNQ增強(qiáng)了細(xì)胞活力,在1 μmol·L-1時(shí)效果最明顯;但當(dāng)濃度達(dá)到10 μmol·L-1時(shí),細(xì)胞活力明顯下降,MNQ表現(xiàn)出細(xì)胞毒性。MNQ的萘醌環(huán)3號(hào)位氫原子被含有苯環(huán)的其他基團(tuán)取代,形成衍生物D19。通過(guò)試驗(yàn)表明D19表現(xiàn)出對(duì)卵泡GCs較小的毒性,因此推測(cè)MNQ及其衍生物的細(xì)胞毒性與其分子結(jié)構(gòu)密切相關(guān)。

    生命科學(xué)研究者經(jīng)常使用LPS誘導(dǎo)機(jī)體產(chǎn)生炎癥反應(yīng),建立體內(nèi)、外炎癥模型。以此模型為基礎(chǔ),驗(yàn)證某些提取物和藥物的抗炎作用[24-25]。研究人員在小鼠體內(nèi)注射LPS,使得血清中IL-6、IL-1β等炎性因子濃度顯著升高,表明成功誘導(dǎo)了小鼠炎癥模型[26-28]。此外,研究人員用LPS誘導(dǎo)巨噬細(xì)胞(RAW264.7)產(chǎn)生炎癥反應(yīng),并進(jìn)行了一系列相關(guān)的研究[29-30]。GCs位于卵巢卵泡的基底膜內(nèi),基底膜阻止了免疫細(xì)胞入內(nèi)。卵巢卵泡的基底膜可以攔截分子量為100~850 kDa的分子進(jìn)入,但是對(duì)LPS分子是高度通透的。Herath等[31]使用無(wú)菌針頭將產(chǎn)后患有不同程度(輕度、中度、重度)子宮內(nèi)膜炎奶牛的卵泡液吸出,檢測(cè)卵泡液中LPS的含量。結(jié)果發(fā)現(xiàn)隨著炎癥程度的增加,卵泡液中LPS的含量顯著增多。體外采集卵泡,檢測(cè)卵泡液中LPS的濃度,得到了同樣的結(jié)果。本試驗(yàn)將體外培養(yǎng)的卵泡GCs暴露在不同濃度的LPS中,發(fā)現(xiàn)隨著LPS濃度的增大,炎性因子IL-6、IL-1β和TNF-α的mRNA相對(duì)表達(dá)量逐漸增多,這和現(xiàn)有的研究結(jié)論基本一致。LPS可能通過(guò)炎癥相關(guān)信號(hào)通路誘導(dǎo)卵泡GCs的炎癥反應(yīng),如TNF/NF-κB信號(hào)通路、AMPK信號(hào)通路等。將體外培養(yǎng)的卵泡GCs暴露于LPS中,可以模擬患有生殖系統(tǒng)炎癥的卵泡GCs的狀態(tài)。

    之前研究表明LPS可以作用于卵泡GCs,并影響其生理功能。牛和鼠的卵泡GCs暴露于LPS中,細(xì)胞表達(dá)TLR4受體,受體與LPS結(jié)合導(dǎo)致炎癥因子的產(chǎn)生[32]。LPS通過(guò)AMPK和mTOR信號(hào)通路誘導(dǎo)牛卵泡GCs產(chǎn)生炎癥反應(yīng),炎性因子IL-1α、IL-1β、TNF-α、IL-6和IL-8表達(dá)量發(fā)生顯著改變[33-34]。Onnureddy等[35]用濃度為1 μg·mL-1的LPS成功誘導(dǎo)水牛(Bubalus bubalis)卵泡GCs促炎因子IL-6、IL-1β、TNF-α的表達(dá)升高。之后用IGF-1逆轉(zhuǎn)了LPS的作用,降低了促炎因子的表達(dá)。CYP19A1被稱為雌激素合成酶,是細(xì)胞色素P450超家族的成員,在卵泡GCs合成E2中起重要作用[36]。在哺乳動(dòng)物中,由膽固醇合成孕烯醇酮是甾醇類激素合成的第一個(gè)步驟。STAR將游離的膽固醇從細(xì)胞質(zhì)輸送到線粒體內(nèi)膜,在線粒體內(nèi)膜中CYP11A1(P450scc)催化膽固醇生成孕烯醇酮,然后將其運(yùn)輸?shù)絻?nèi)質(zhì)網(wǎng),通過(guò)3β-HSD轉(zhuǎn)化為P4[37]。研究表明將優(yōu)勢(shì)卵泡的GCs體外暴露于LPS中,產(chǎn)生的E2減少[31]。細(xì)菌產(chǎn)生的LPS通過(guò)TLR2和TLR4途徑作用于卵泡GCs引發(fā)炎癥并擾亂GCs內(nèi)分泌功能,使得E2和P4的分泌量減少[2]。Onnureddy等[35]發(fā)現(xiàn)LPS有效抑制了卵泡GCs中CYP19A1的表達(dá),進(jìn)而影響類固醇激素的合成。Shimizu等[38]研究表明LPS和PGN對(duì)顆粒細(xì)胞E2產(chǎn)生的抑制作用,導(dǎo)致了子宮炎奶牛的卵巢功能障礙。本研究也證實(shí)了LPS會(huì)影響GCs的正常生理功能,導(dǎo)致其E2和P4的合成和分泌量減少。同時(shí)我們檢測(cè)了類固醇激素合成相關(guān)基因的表達(dá)情況,推測(cè)LPS是通過(guò)改變類固醇激素合成相關(guān)基因的表達(dá)進(jìn)而影響了類固醇激素的合成。

    MNQ廣泛存在于鳳仙花等植物體內(nèi),其衍生物的合成簡(jiǎn)單易行,且有效增強(qiáng)了抗炎效應(yīng)。植物體內(nèi)活性物質(zhì)的藥用功能避免了傳統(tǒng)消炎藥物可能引起機(jī)體的免疫失調(diào)、伴有一系列不良反應(yīng)以及微生物對(duì)藥物的耐藥性等缺點(diǎn),為有效治療奶牛生殖系統(tǒng)炎癥,改善奶牛的生殖健康提供了理論依據(jù)。

    4 結(jié) 論

    試驗(yàn)確定了MNQ的衍生物D19對(duì)奶牛卵巢卵泡GCs作用12 h的最大無(wú)毒濃度為64 μmol·L-1。證明了D19在最大無(wú)毒濃度范圍之內(nèi)對(duì)LPS體外誘導(dǎo)的卵泡GCs炎癥損傷具有一定的保護(hù)作用,并能一定程度緩減LPS導(dǎo)致的GCs功能性紊亂。

    參考文獻(xiàn)(References):

    [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,VALENTO 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,F(xiàn)RANCHI 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,F(xiàn)ERREIRA 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.

    (編輯 郭云雁)

    猜你喜歡
    炎癥反應(yīng)
    多巴胺聯(lián)合多巴酚丁胺聯(lián)合治療小兒重癥肺炎的效果和對(duì)炎癥反應(yīng)的影響
    右美托咪定結(jié)合超聲技術(shù)對(duì)患者下肢手術(shù)中止血帶所致氧化應(yīng)激及炎癥反應(yīng)的影響
    兩種皮瓣修復(fù)術(shù)治療手外傷軟組織缺損的臨床對(duì)比研究
    右美托咪定對(duì)單肺通氣患者血漿IL—1β及肺組織AQP4、AQP5表達(dá)水平的影響
    血必凈注射液對(duì)改善嚴(yán)重多發(fā)傷患者預(yù)后作用的研究
    瑞舒伐他汀冠狀動(dòng)脈造影術(shù)后腎功能損害的保護(hù)作用及其機(jī)制
    中藥方劑對(duì)缺血性卒中患者血小板活化、內(nèi)皮功能、炎癥反應(yīng)的影響研究
    益氣扶正法在膿毒癥患者中的治療及對(duì)血清核因子—κB活性變化的影響研究
    右美托咪定對(duì)膿毒癥患者圍術(shù)期血漿中細(xì)胞因子的影響
    中西醫(yī)結(jié)合治療重度燒傷膿毒癥的效果研究
    新丰县| 武定县| 全椒县| 玉树县| 惠来县| 衡山县| 黑龙江省| 崇仁县| 姚安县| 和龙市| 阿勒泰市| 岫岩| 靖边县| 新乡县| 岗巴县| 唐海县| 集安市| 通化市| 云林县| 巴林左旗| 合川市| 扎赉特旗| 张家界市| 涞源县| 苍溪县| 浠水县| 唐海县| 永仁县| 长沙市| 噶尔县| 花莲县| 蕲春县| 晋中市| 沙洋县| 调兵山市| 吉木萨尔县| 凤山市| 盐池县| 迁西县| 泊头市| 永宁县|