[摘" "要]" "目的:比較不同給氧方案對脂多糖(lipopolysaccharide, LPS)誘導(dǎo)的急性腎損傷(acute kidney injury, AKI)模型小鼠腎組織炎癥性損傷及外周血炎癥因子的影響。方法:采用ICR雌性小鼠,腹腔注射LPS(5 mg/kg)建立小鼠AKI模型,隨機分成:(1)空白組(Control組),注射同等量的生理鹽水;(2)模型組(LPS組);(3)純氧治療組(LPS+100%O2組);(4)2.0 絕對大氣壓(atmosphere absolute, ATA)高壓氧(hyperbaric oxygen, HBO)治療組(LPS+2.0 ATA HBO組);(5)3.0 ATA HBO治療組(LPS+3.0 ATA HBO組),治療后24 h,通過流式微球技術(shù)及實時熒光定量PCR(real-time PCR, RT-PCR)實驗分別檢測腎組織和外周血中促炎因子IL-1β、IL-6和TNF-α,以及抑炎因子IL-10水平。此外,通過HE染色法觀察腎組織炎癥性損傷的組織形態(tài)變化。結(jié)果:IL-1β、IL-6和TNF-α在LPS誘導(dǎo)24 h后均顯著增加;100%O2和2.0 ATA HBO治療均可抑制促炎因子IL-1β、IL-6和TNF-α的釋放,且2.0 ATA HBO抑制作用更顯著(Plt;0.05或Plt;0.01);此外,LPS+2.0 ATA HBO組抑炎因子IL-10水平顯著增高(Plt;0.01)。HE染色結(jié)果表明,與LPS組相比,100%O2和2.0 ATA HBO治療后,腎組織炎癥性損傷均有所減輕,且LPS+2.0 ATA HBO組改善效果最好(Plt;0.05或Plt;0.01),而LPS+3.0 ATA HBO組腎組織損傷未改善。結(jié)論:100%O2和2.0 ATA HBO治療均可明顯改善AKI小鼠炎癥性損傷,且2.0 ATA HBO改善作用更好。
[關(guān)鍵詞]" "急性腎損傷;氧療;高壓氧;常壓氧;炎癥;細胞因子;小鼠
[中圖分類號]" "R692" " " " " " " "[文獻標志碼]" "A" " " " " " " "[文章編號]" "1674-7887(2024)02-0154-05
Comparison of the protective effects of different oxygen therapy regimens against
acute kidney injury in mice*
XU Feng1**, XIA Jing2, SHAO Yanan2, LI Dan2, WU Xinhe2***" " " " (1Department of Nephrology, 2Department of Rehabilitation Medicine, the Affiliated Rehabilitation Hospital of Nantong University, Jiangsu 226002)
[Abstract]" "Objective: Comparison of the effects of different oxygen therapy regimens on renal tissue inflammatory injury and peripheral blood inflammatory factors in a mouse model of lipopolysaccharide(LPS)-induced acute kidney injury(AKI). Methods: ICR female mice were injected intraperitoneally with 5 mg/kg of LPS to establish a model of AKI. They were randomly divided into the following groups: (1)Control group(injected with the same amount of physiological saline); (2)Model group(LPS group); (3)Pure oxygen treatment group(injected with LPS and 100%O2); (4)2.0 atmosphere absolute(ATA) hyperbaric oxygen(HBO) treatment group(LPS+2.0 ATA HBO group); (5)3.0 ATA HBO treatment group LPS+3.0 ATA HBO group). The pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and the anti-inflammatory cytokine IL-10 in peripheral blood and kidney tissue were detected by flow cytometric bead array and real-time PCR(RT-PCR) assays. The histomorphological changes of kidney tissue were observed by HE staining. Results: IL-1β, IL-6, and TNF-α levels were significantly increased after 24 hours of LPS induction. 100%O2 and 2.0 ATA HBO could inhibit the release of IL-1β, IL-6, and TNF-α, and 2.0 ATA HBO had a more pronounced impact, with a significantly increased IL-10 level in this treatment group. HE staining showed that compared with LPS, 100%O2 and 2.0 ATA HBO could alleviate the inflammatory injury of kidney, and 2.0 ATA HBO showed a better effect. Nevertheless, 3.0 ATA HBO did not reverse the damage to the kidney tissue. Conclusion:" The above results suggested that both 100%O2 and 2.0 ATA HBO treatments significantly improved inflammatory injury in AKI mice. Moreover, 2.0 ATA HBO had a superior effect on inhibiting tissue inflammation.
[Key words]" "acute kidney injury; oxygen therapy; hyperbaric oxygen; normobaric oxygen; inflammation; cytokines; mouse
急性腎損傷(acute kidney injury, AKI)是臨床上常見的危急重癥之一,具有高發(fā)病率和高死亡率的特點;其發(fā)病機制與炎癥反應(yīng)有關(guān),即促炎因子與抑炎因子的不平衡引起組織損傷,最終導(dǎo)致病情惡化及死亡率升高[1-3]。在脂多糖(lipopolysaccharide, LPS)誘導(dǎo)的AKI模型中,觀察到多種促炎因子浸潤導(dǎo)致腎臟組織損傷;因此,抑制機體炎癥過度反應(yīng)對LPS誘導(dǎo)的AKI的保護作用極為重要。
越來越多的研究[4]表明,氧療可調(diào)控炎癥反應(yīng),對組織炎癥性損傷具有保護作用。高壓氧(hyperbaric oxygen, HBO)治療是一種基于在增強大氣壓下暴露于純氧的治療方法,通常作為感染等不同疾病的主要或輔助療法,特別是深部或頑固性感染,如壞死性筋膜炎、骨髓炎和慢性軟組織感染以及感染性心內(nèi)膜炎等[5-7]。實驗動物研究[8]表明,2.0 絕對大氣壓(atmosphere absolute, ATA)HBO治療可減輕砷誘導(dǎo)的肝臟和腎臟組織損傷;2.5 ATA HBO治療可有效改善LPS誘導(dǎo)的小鼠急性肺損傷[9];100%O2和3.0 ATA HBO預(yù)處理均可減輕膿毒血癥模型大鼠急性肝組織損傷[10]。臨床研究[11]表明,2.0 ATA HBO治療減少AKI患者的住院時間,改善了腎血流動力學;2.5 ATA HBO治療可加速傷口愈合,改善糖尿病足潰瘍并提高患者的生存率[12];3.0 ATA HBO可有效治療頸部壞死性感染[13];但當壓力>3.0 ATA時,通常報道更多的不利影響[5]。然而AKI臨床常需依靠醫(yī)師經(jīng)驗選擇治療方案,關(guān)于腎組織損傷對于不同氧療方案的應(yīng)答反應(yīng)是否有差異也缺乏研究。因此,需要探究腎臟器官組織對不同氧療應(yīng)答反應(yīng),為輔助腎臟器官組織損傷治療摸索最佳氧療方案提供實驗依據(jù)。
目前臨床使用的氧療方法逐漸多樣化,如面罩給氧、機械通氣給氧和高壓氧等[14-16]。因此,采用LPS誘導(dǎo)AKI小鼠模型,模擬臨床上不同的氧療方法進行干預(yù),通過分析外周血和腎臟組織器官的炎癥因子表達以及器官損傷評分,從抑制炎癥角度比較不同吸氧方案對AKI模型小鼠腎組織損傷的治療效果,為腎組織器官炎癥損傷治療時氧療方案的選擇提供初步實驗依據(jù)。
1" "材料和方法
1.1" "動物來源與模型建立" "雌性ICR小鼠,體質(zhì)量22~26 g,購自南通大學實驗動物中心(機構(gòu)許可證:SYXK(SU)-2012-0030)。所有動物置于塑料籠中,室溫控制在(24±2)℃、日光燈照明(12 h/12 h明暗)、自由飲食,持續(xù)3 d。將LPS(貨號:L2630, Sigma Aldrich)粉末溶于生理鹽水制備成1 mg/mL的藥液避光保存,ICR小鼠按照5 mg/kg劑量腹腔注射形成膿毒血癥AKI模型,并立即對模型小鼠進行不同氧療方案治療。
1.2" "實驗分組與處理" "動物隨機分成5組:空白組(Control組)、模型組(LPS組)、純氧治療組(LPS+100%O2組)、2.0 ATA HBO治療組(LPS+2.0 ATA HBO組)、3.0 ATA HBO治療組(LPS+3.0 ATA HBO組),每組3~6只。Control組注射等量生理鹽水,與LPS組均未進行治療;LPS+100%O2組:將模型小鼠置于密封的帶有進(出)氣口的玻璃樹脂箱內(nèi),在常壓下接受純氧治療60 min;LPS+2.0 ATA HBO組與LPS+3.0 ATA HBO組:將模型小鼠置于100 L的艙內(nèi)(安徽省蕪湖潛水裝備廠),分別在2個絕對大氣壓和3個絕對大氣壓下接受純氧治療60 min,且升壓(減壓)時間各5 min。LPS誘導(dǎo)后24 h,對各組小鼠腹腔注射復(fù)合麻醉劑2.5%Avertin(2-甲基-2-丁醇,貨號152463;2, 2, 2-三溴乙醇,貨號T48402,Sigma),深度麻醉后處死并進行取材。本實驗方案獲南通大學實驗動物中心倫理審查批準(S20230315-003)。
1.3 " 實驗方法
1.3.1" "實時熒光定量PCR(real-time PCR, RT-PCR)" "按試劑盒說明書,使用TRIzol試劑(Invitrogen,CA,美國)從腎組織中分離總RNA,并逆轉(zhuǎn)錄成cDNA。使用SYBR qPCR Master Mix熒光定量PCR儀進行反應(yīng),β-actin(小鼠中的Actb)用于標準化目標基因的相對轉(zhuǎn)錄水平。引物序列如表1。
1.3.2" "HE染色及評分" "將10%甲醛固定的腎組織包埋在石蠟中,切片并行HE染色形態(tài)學檢查。腎組織的病理評分由專業(yè)病理科醫(yī)師進行評估。腎小管損傷程度,包括腎小管擴張、變平和空泡形成,分為5個等級(0,無;1,≤25%;2,>25%~50%;3,>50%~75%;4,gt;75%),基于光學顯微鏡下高倍視野中受影響的腎小管百分比;各分數(shù)相加,將總分的平均值作為腎損傷評分[17-18]。
1.3.3" "流式細胞微球技術(shù)(cytometric bead array, CBA)" "使用BDTM CBA Flex Set試劑盒和BDTM Mouse Enhance Sensitivity Master Buffer Kit試劑盒(貨號562246,BD Bioscience)測量血清中4種不同炎癥因子的濃度水平;IL-1β(貨號562278,BD Bioscience),IL-6(貨號562236,BD Bioscience),TNF-α(貨號562336,BD Bioscience),IL-10(貨號562263,BD Bioscience)。使用BriCyteE6(邁瑞,中國)流式細胞儀設(shè)置珠進行校準,按照試劑制造商的說明倍比稀釋制備標準品,每個樣本采集200個細胞,將不同目的蛋白捕獲微球在待測樣品中的檢測熒光強度代入各自的標準品曲線方程中,計算每組樣品中各種炎癥因子的濃度。
1.4" "統(tǒng)計學方法" "使用SPSS 25.0軟件進行統(tǒng)計分析,所有實驗數(shù)據(jù)均以均數(shù)±標準誤(mean±standard error, SEM)形式表示。通過單因素方差分析,差異有統(tǒng)計學意義后再行兩兩t檢驗,Plt;0.05表示差異有統(tǒng)計學意義。
2" "結(jié)" " " 果
2.1" "不同給氧方案對AKI模型小鼠腎臟組織病理損傷的減輕程度不同" "與Control組相比,腎組織的病理損傷有腎小球細胞增多,腎小管上皮細胞渾濁腫脹,間質(zhì)水腫充血,中性粒細胞浸潤。LPS+100%O2組治療后,腎小管上皮細胞渾濁腫脹減輕,中性粒細胞浸潤減少;LPS+2.0 ATA HBO組腎組織病理損傷均顯著改善,而LPS+3.0 ATA HBO組腎臟組織病理損傷無明顯改善(Plt;0.05或Plt;0.01,圖1)。結(jié)果表明,在一定的氧分壓范圍內(nèi),氧療對于組織病理損傷的改善效果隨著氧分壓的升高而升高,但超過一定閾值后,改善效果消失。
2.2" "不同給氧方案對LPS誘導(dǎo)的AKI模型小鼠外周血炎癥因子的影響" "通過CBA實驗檢測各組小鼠血清中炎癥因子水平。與Control組相比,LPS誘導(dǎo)24 h后IL-1β、IL-6和TNF-α(Plt;0.01,圖2)水平均顯著升高。與LPS組相比,LPS+100%O2組和LPS+2.0 ATA HBO組均抑制LPS誘導(dǎo)的IL-1β、IL-6和TNF-α升高,LPS+2.0 ATA HBO組作用更顯著(Plt;0.01或Plt;0.05, 圖2A~C),但LPS+3.0 ATA HBO組治療前后炎癥因子水平差異無統(tǒng)計學意義。LPS+2.0 ATA HBO組治療后血清IL-10水平顯著提高(Plt;0.01, 圖2D)。結(jié)果表明,LPS誘導(dǎo)的小鼠AKI模型中,100%O2和2.0 ATA HBO治療均能不同程度地抑制促炎因子的釋放,2.0 ATA HBO抑制炎癥因子的效果最佳。
2.3" "不同給氧方案對AKI模型小鼠腎臟組織炎癥因子的影響" "LPS誘導(dǎo)后,腎組織中促炎因子IL-1β、IL-6、TNF-α和IL-10的mRNA水平均顯著上調(diào)(Plt;0.01, 圖3A~D);與LPS組相比,LPS+100%O2組和LPS+2.0 ATA HBO組IL-1β、IL-6和TNF-α水平下降,LPS+2.0 ATA HBO組下降幅度更大(Plt;0.05或Plt;0.01, 圖3A~C);而LPS+3.0 ATA HBO組炎癥因子水平治療前后差異無統(tǒng)計學意義。與LPS組相比,LPS+2.0 ATA HBO組治療后IL-10 mRNA水平顯著升高(Plt;0.01, 圖3),其他組相比差異無統(tǒng)計學意義(P>0.05)。結(jié)果表明,不同的給氧方案對于腎臟組織炎癥因子的表達影響不同,2.0 ATA HBO治療既抑制了腎臟組織促炎因子的表達,也促進了腎臟組織抑炎因子的表達。
3" "討" " " 論
本研究探討了不同給氧方案對LPS誘導(dǎo)的AKI模型小鼠腎組織炎癥性損傷的保護作用。100%O2和2.0 ATA HBO治療均抑制了促炎因子IL-1β、IL-6和TNF-α的表達,減輕了AKI模型小鼠腎組織的損傷,僅2.0 ATA HBO治療上調(diào)抑炎因子IL-10的表達,且改善腎組織損傷的效果更好,為HBO治療AKI的臨床應(yīng)用提供了重要的實驗依據(jù)。
炎癥的過度反應(yīng)導(dǎo)致組織炎癥性缺氧[19],缺氧可激活炎癥細胞并釋放大量促炎因子,如TNF-α、IL-1β、IL-6和IL-8等,加劇組織損傷[20-21]。本研究中,100%O2和2.0 ATA HBO治療均能抑制促炎因子的表達,減輕腎組織的炎癥性損傷,這與J.L.HALBACH等[22-23]的研究結(jié)果相似,其中2.0 ATA HBO改善組織損傷效果最好,這可能與改善組織缺氧效率有關(guān)。眾所周知,隨著氧濃度以及氧分壓的增加,血液中的氧含量增加,而 HBO通過增加血漿中的溶解氧和氧氣的擴散距離更有效改善組織缺氧[5, 19],這種作用已在局灶性腦組織缺血中被證實[24]。此外,無論是在外周血還是在腎組織中,LPS+2.0 ATA HBO組的IL-10水平均明顯高于其他給氧治療組,說明不同的給氧方案對組織中炎癥因子水平的影響不同。已有文獻[25]表明,抑炎因子IL-10通過抑制IL-6的分泌降低膿毒血癥相關(guān)AKI模型小鼠的死亡率,而HBO可上調(diào)IL-10的表達。文獻[8-9, 11]報道,HBO通過抑制促炎因子的釋放和增加抑炎因子的分泌來減輕小鼠急性腎組織損傷,這與本研究結(jié)果一致。但是當壓力升到3.0 ATA時,促炎因子(IL-1β、IL-6和TNF-α)和抑炎因子IL-10的表達水平與LPS組均無差異,同時3.0 ATA HBO治療對LPS誘導(dǎo)的多器官組織損傷均無改善作用,這可能與氧毒性水平有關(guān)。越來越多的研究[26-27]表明,氧毒性會誘導(dǎo)細胞死亡和疾病發(fā)生。而HBO產(chǎn)生的氧毒性與暴露的壓力和持續(xù)時間有關(guān),O.SUKRU等[28]發(fā)現(xiàn),隨著暴露時間的延長,3.0 ATA HBO治療會導(dǎo)致大鼠肺和腦組織氧化應(yīng)激損傷。本研究表明氧療對腎組織炎癥性損傷具有保護作用,且2.0 ATA HBO的保護作用最好。
本研究結(jié)果表明,單次2.0 ATA HBO治療雖明顯改善了腎組織炎癥性損傷,但未觀察其遠期效果。因此,下一步計劃采用生化以及病理指標對小鼠腎損傷進行長期檢測,并比較不同療程的治療效果。此外,還可考慮在大動物模型上開展臨床前的療效觀察及機制探討。
總之,在AKI時,2.0 ATA HBO治療在調(diào)節(jié)機體炎癥反應(yīng)和減輕腎組織損傷方面效果最好。這不僅加深了對 HBO腎組織炎癥性損傷保護作用的認識和理解,也為拓展HBO的臨床適用范圍以及AKI的救治提供了重要的實驗依據(jù)。
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[收稿日期] 2023-12-27