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ラット重症熱傷初期の心筋組織におけるmicroRNA-126の発現と心筋損傷との関係
[Expression of microRNA-126 in myocardial tissue of rats in the early stage of severe burn injury and its relation with myocardial damage].
PMID: 26714406
抄録
目的:
重症熱傷初期のラットの心筋組織における microRNA-126 の発現量と血清中の心筋トロポニンI（cTnI）の発現量の変化を解析しながら観察し、細胞レベルでの microRNA-126 と心筋損傷との関係を検証すること。
OBJECTIVE: To observe the changes in the expressions of microRNA-126 in myocardial tissue and cardiac troponin I (cTnI) in serum of rats in the early stage of severe burn injury with analysis of their relationship, and to validate the relationship between microRNA-126 and myocardial damage in cellular level.
方法:
(1) SDラット48匹を、乱数表に従って、偽傷害群（n=8匹、偽傷害後に体液療法を行わない）と熱傷傷害群（n=40匹、背中に30%TBSA全厚のやけどを負い、以下熱傷と称し、乳酸リンゲル液の腹腔内注射を受けた）に分け、それぞれのラットの左室組織を採取した。傷害後１時間目（ＰＩＨ）に偽傷害群のラットの腹部大動脈から採血し、この８匹のラットを犠牲にして左室組織を得た。火傷損傷群のラット８匹の腹部大動脈から、それぞれＰＩＨ３、６、１２、２４、４８の時点で採血した後、これら８匹を犠牲にして各時点での左心室組織を得た。心筋組織におけるmicroRNA-126の発現は、リアルタイム蛍光定量RT-PCRにより評価した。cTnIの血清レベルはELISAで評価した。(2) ラット心筋細胞株H9C2を、乱数表に従って、正常対照群（NC、日常培養）、刺激群（S）、ネガティブトランスフェクション＋刺激群（NT＋S）、トランスフェクション＋刺激群（T＋S）に分けた。Ｓ群の細胞は、実験（１）のＰＩＨ６で熱傷群のラットから得た１０％熱傷血清を含むＤＭＥＭで培養した後、２４時間低酸素処理を行った。ＮＴ＋Ｓ群およびＴ＋Ｓ群の細胞は、それぞれマイクロＲＮＡ模倣体およびマイクロＲＮＡ-１２６模倣体のネガティブコントロールを用いて２４時間トランスフェクトした後、Ｓ群と同様の処理を行った。心筋細胞におけるｍｉｃｒｏＲＮＡ-１２６の発現は、リアルタイム蛍光定量ＲＴ-ＰＣＲ（サンプル数３）により決定した。心筋細胞の元気度を調べるために、セルカウンティングキット８を用いた（サンプル数４、吸光度値で表す）。心筋細胞のアポトーシス率をフローサイトメーターで調べた（サンプル数３とする）。データは一方向分散分析およびLSD-t検定で処理した。心筋組織におけるmicroRNA-126の発現とラットのcTnIの血清レベルとの関係を線形相関分析で評価した。
METHODS: (1) Forty-eight SD rats were divided into sham injury group (n=8, without fluid therapy after sham injury) and burn injury group (n=40, inflicted with 30% TBSA full-thickness scald on the back, hereinafter referred to as burn, and received intraperitoneally injection of lactic acid Ringer's solution) according to the random number table. Blood was collected from abdominal aorta of rats in sham injury group at post injury hour (PIH) 1, and then these 8 rats were sacrificed for obtaining left ventricular tissue. Blood was respectively collected from abdominal aorta of 8 rats in burn injury group at PIH 3, 6, 12, 24, and 48, and then they were sacrificed and the left ventricular tissue was obtained at each time point. The expression of microRNA-126 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR. Serum level of cTnI was assessed by ELISA. (2) Rat myocardial cell line H9C2 was divided into normal control group (NC, routinely cultured), stimulation group (S), negative transfection+stimulation group (NT+S), and transfection+stimulation group (T+S) according to the random number table. Cells in S group were treated with hypoxia for 24 h after being cultured with DMEM containing 10% burn serum obtained from rats in burn injury group at PIH 6 in experiment (1). Cells in NT+S group and T+S group were respectively transfected with the negative control of microRNA mimics and microRNA-126 mimics for 24 h, and then were given the same treatment as that of S group. The expression of microRNA-126 in myocardial cells was determined by real-time fluorescent quantitative RT-PCR (with the sample number of 3). Cell counting kit 8 was used to examine the vitality of myocardial cell (with the sample number of 4, denoted as absorbance value). Apoptotic rate of myocardial cells was determined by flow cytometer (with the sample number of 3). Data were processed with one-way analysis of variance and LSD-t test. The relationship between microRNA-126 expression in myocardial tissue and serum level of cTnI of rats was assessed by linear correlation analysis.
結果:
(1)熱傷群ラットの心筋組織における microRNA-126 の発現量は、PIH1 における偽装群と比較して、PIH3、6、12、24、48 の各時点で有意に減少し（t 値 5.68～9.79、P 値 0.01 以下）、PIH24 で最下降（0.40 ± 0.08）しました。やけど損傷群のラットの血清中cTnI濃度は、PIH1時の偽装損傷群と比較して、PIH3、6、12、24、48時に有意に上昇し（t値は6.68～12.79、0.01以下のP値）、PIH12時にピークを迎えた（(1035±177) pg/mL）。熱傷群のラットでは、各時点で心筋組織中のmicroRNA-126の発現量とcTnIの血清中濃度との間に有意な負の相関が認められた（r=-0.797、P<0.001）。(2) NC群と比較して、S群およびT+S群の心筋細胞におけるmicroRNA-126発現量はそれぞれ減少および増加し（t値はそれぞれ4.57および5.73、P<0.05またはP<0.01）、細胞活力レベルは明らかに低下し（t値はそれぞれ14.88および6.48、P<0.01以下）、アポトーシス率は有意に増加した（t値はそれぞれ13.82および6.96、P<0.01以下）。NT+S群と比較して、T+S群の心筋細胞におけるmicroRNA-126発現量は有意に増加し（t=6.77、P<0.01）、細胞活力は明らかに増加し（t=8.23、P<0.001）、アポトーシス率は有意に減少した（t=6.14、P<0.001）。
RESULTS: (1) Compared with that of sham injury group at PIH 1, the expression levels of microRNA-126 in myocardial tissue of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly decreased (with t values from 5.68 to 9.79, P values below 0.01), reaching its nadir at PIH 24 (0.40 ± 0.08). Compared with that of sham injury group at PIH 1, the serum levels of cTnI of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly increased (with t values from 6.68 to 12.79, P values below 0.01), peaking at PIH 12 [(1 035 ± 177) pg/mL]. A significant negative correlation between the expression level of microRNA-126 in myocardial tissue and serum level of cTnI was observed in rats of burn injury group at each time point (r=-0.797, P<0.001). (2) Compared with those of NC group, the microRNA-126 expression levels in myocardial cells of S group and T+S group were respectively decreased and increased (with t values respectively 4.57 and 5.73, P<0.05 or P<0.01), the cell vitality levels were obviously decreased (with t values respectively 14.88 and 6.48, P values below 0.01), and the apoptotic rates were significantly increased (with t values respectively 13.82 and 6.96, P values below 0.01). Compared with that in NT+S group, the microRNA-126 expression level in myocardial cells of T+S group was significantly increased (t=6.77, P<0.01), the cell vitality level was obviously increased (t=8.23, P<0.001), and the apoptotic rate was significantly decreased (t=6.14, P<0.001).
結論:
ラットの心筋組織におけるmicroRNA-126の発現レベルは、重度の熱傷の初期段階で低下した。microRNA-126は心筋損傷の制御に関与し、心筋保護の役割を果たしている可能性がある。
CONCLUSIONS: Expression level of microRNA-126 in myocardial tissue of rat was decreased in the early stage of severe burn injury. It may participate in regulating myocardial damage and play a protective role.