未分割ヘパリンはホスファチジルイノシトール-3キナーゼ/セリン/スレオニンキナーゼ/核因子κ-B経路を介して内皮バリア機能障害を減衰させる

Unfractionated heparin attenuates endothelial barrier dysfunction via the phosphatidylinositol-3 kinase/serine/threonine kinase/nuclear factor kappa-B pathway.

• Sheng-Tian Mu
• Jie Tang
• Jian-Qi Ma
• Yu Zhong
• Han-Zhe Liu
• Xiao-Chun Ma
• Zhen Zheng

抄録

背景:

血管内皮機能障害は急性肺損傷の発症に重要な病態生理的プロセスと考えられている。本研究では、未分割ヘパリン（UFH）がリポ多糖（LPS）誘発性の血管内皮カドヘリン（VE-カドヘリン）の変化に及ぼす影響とその潜在的な機序について検討した。

BACKGROUND: Vascular endothelial dysfunction is considered a key pathophysiologic process for the development of acute lung injury. In this study, we aimed at investigating the effects of unfractionated heparin (UFH) on the lipopolysaccharide (LPS)-induced changes of vascular endothelial-cadherin (VE-cadherin) and the potential underlying mechanisms.

方法:

雄のC57BL/6 Jマウスを、ビヒクル群、LPS群、LPS"Zs_200A"+"Zs_200A"UFH群の3群に無作為に割り付けた。敗血症を誘発するために30mg/kgのLPSを腹腔内注射した。LPS"Zs_200A"+"Zs_200A"UFH群のマウスは、LPS注射の0.5時間前に8UのUFHを皮下注射した。マウスの肺組織を採取し、肺湿潤/乾燥（W/D）重量比を測定し、組織学的変化を観察することにより、肺損傷の評価を行った。ヒト肺微小血管内皮細胞（ＨＰＭＥＣ）を培養し、ＵＦＨのＬＰＳまたは腫瘍壊死因子α（ＴＮＦ-α)誘導性血管過疎化、VE-カドヘリン、p120-カテニン、リン酸化ミオシン軽鎖(p-MLC)の膜発現、F-アクチンリモデリング、LPS誘導性ホスファチジルイノシトール-3キナーゼ(PI3K)/セリン/スレオニンキナーゼ(Akt)/核因子κB(NF-κB)シグナル伝達経路の活性化に対するUFHの影響を解析しました。

METHODS: Male C57BL/6 J mice were randomized into three groups: vehicle, LPS, and LPS + UFH groups. Intraperitoneal injection of 30 mg/kg LPS was used to induce sepsis. Mice in the LPS + UFH group received subcutaneous injection of 8 U UFH 0.5 h before LPS injection. The lung tissue of the mice was collected for assessing lung injury by measuring the lung wet/dry (W/D) weight ratio and observing histological changes. Human pulmonary microvascular endothelial cells (HPMECs) were cultured and used to analyze the effects of UFH on LPS- or tumor necrosis factor-alpha (TNF-α)-induced vascular hyperpermeability, membrane expression of VE-cadherin, p120-catenin, and phosphorylated myosin light chain (p-MLC), and F-actin remodeling, and on the LPS-induced activation of the phosphatidylinositol-3 kinase (PI3K)/serine/threonine kinase (Akt)/nuclear factor kappa-B (NF-κB) signaling pathway.

結果:

インビボでは、UFHの前処理は、LPSによって誘発された肺の病理組織学的変化（好中球浸潤と赤血球の滲出、肺胞の崩壊、および中隔の肥厚）を有意に減衰させ、肺のW/Dを減少させ、タンパク質濃度を増加させた（LPS vs. LPS; +x200A.LPS"Zs_200A"+"Zs_200A"UFH:0.57"Zs_200A"±"Zs_200A"0.04"Zs_200A"0.04"Zs_200A"mg/mL、P"Zs_200A"="Zs_200A"0.0092）、総細胞数（LPS対LPS"Zs_200A"+"Zs_200A"UFH:9。57"Zs_200A"±"Zs_200A"1.23 vs. 3.65"Zs_200A"±"Zs_200A"0.78"Zs_200A"×"Zs_200A"10/mL、P"Zs_200A"="Zs_200A"0．0155）、多形核好中球率（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH:88.05%"Zs_200A"±"Zs_200A"2.88%vs.22.20%"Zs_200A"±"Zs_200A"3.92%、P"Zs_200A"="Zs_200A"0.0002）、TNF-α（460.33"Zs_200A"±"Zs_200A"23．48対189.33"Zs_200A"±"Zs_200A"14.19"Zs_200A"pg/mL、P"Zs_200A"="Zs_200A"0.0006）を気管支肺胞洗浄液中で測定した。インビトロでは、UFHの前処理は、VE-カドヘリンの膜発現のLPS誘発性の減少を防止した（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH: 0。368"Zs_200A"±"Zs_200A"0.044 vs. 0.716"Zs_200A"±"Zs_200A"0.064、P"Zs_200A"="Zs_200A"0.0114）およびp120-カテニン（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH: 0.208"Zs_200A"±"Zs_200A"0.018 vs. 0.924"Zs_200A"±"Zs_200A"0.092、P"Zs_200A"="Zs_200A"0.0016）、LPSによるp-MLCの発現増加（LPS vs.LPS"Zs_200A"+"Zs_200A"UFH：0.972"Zs_200A"±"Zs_200A"0.092 vs. 0.293"Zs_200A"±"Zs_200A"0.025、P"Zs_200A"="Zs_200A"0.0021）。)さらに、UFHは、HPMECsのLPS-およびTNF-α誘導性過疎化を減衰させた（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH：8.90"Zs_200A"±"Zs_200A"0.66 vs. 15.84"Zs_200A"±"Zs_200A"1.09Ω・cm、P"Zs_200A"="Zs_200A"0.0056; TNF-α vs. TNF-α + UFH: 11.28"Zs_200A"±"Zs_200A"0.64 vs. 18.15"Zs_200A"±"Zs_200A"0。98Ω・cm、P"Zs_200A"="Zs_200A"0.0042）、F-アクチンリモデリング（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH：56.25"Zs_200A"±"Zs_200A"1.51 vs. 39.70"Zs_200A"±"Zs_200A"1.51。98、P"Zs_200A"="Zs_200A"0.0027; TNF-α vs. TNF-α+UFH: 55.42"Zs_200A"±"Zs_200A"1.42 vs. 36.51"Zs_200A"±"Zs_200A"1.20、P"Zs_200A"="Zs_200A"0.0005)をインビトロで測定した。さらに、UFHはAktのリン酸化を減少させた（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH: 0.977"Zs_200A"±"Zs_200A"0.081 vs. 0.466"Zs_200A"±"Zs_200A"0.035。035、P"Zs_200A"="Zs_200A"0.0045）とIκBキナーゼ（IKK）（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH：1.023"Zs_200A"±"Zs_200A"0.070 vs. 0．578"Zs_200A"±"Zs_200A"0.044、P"Zs_200A"="Zs_200A"0.0060）、NF-κBの核内転座（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH: 1.003"Zs_200A"±"Zs_200A"0.0060）、NF-κBの核内転座（LPS vs. LPS"Zs_200A"+"Zs_200A"UFH: 1.003"Zs_200A"±"Zs_200A"0．0.077 vs. 0.503"Zs_200A"±"Zs_200A"0.065, P"Zs_200A"="Zs_200A"0.0078)のHPMECsでの効果は、PI3K阻害剤であるワルトマニンの効果と同様であった。

RESULTS: In vivo, UFH pretreatment significantly attenuated LPS-induced pulmonary histopathological changes (neutrophil infiltration and erythrocyte effusion, alveolus pulmonis collapse, and thicker septum), decreased the lung W/D, and increased protein concentration (LPS vs. LPS + UFH: 0.57 ± 0.04 vs. 0.32 ± 0.04 mg/mL, P = 0.0092), total cell count (LPS vs. LPS + UFH: 9.57 ± 1.23 vs. 3.65 ± 0.78 × 10/mL, P = 0.0155), polymorphonuclear neutrophil percentage (LPS vs. LPS + UFH: 88.05% ± 2.88% vs. 22.20% ± 3.92%, P = 0.0002), and TNF-α (460.33 ± 23.48 vs. 189.33 ± 14.19 pg/mL, P = 0.0006) in the bronchoalveolar lavage fluid. In vitro, UFH pre-treatment prevented the LPS-induced decrease in the membrane expression of VE-cadherin (LPS vs. LPS + UFH: 0.368 ± 0.044 vs. 0.716 ± 0.064, P = 0.0114) and p120-catenin (LPS vs. LPS + UFH: 0.208 ± 0.018 vs. 0.924 ± 0.092, P = 0.0016), and the LPS-induced increase in the expression of p-MLC (LPS vs. LPS + UFH: 0.972 ± 0.092 vs. 0.293 ± 0.025, P = 0.0021). Furthermore, UFH attenuated LPS- and TNF-α-induced hyperpermeability of HPMECs (LPS vs. LPS + UFH: 8.90 ± 0.66 vs. 15.84 ± 1.09 Ω·cm, P = 0.0056; TNF-α vs. TNF-α + UFH: 11.28 ± 0.64 vs. 18.15 ± 0.98 Ω·cm, P = 0.0042) and F-actin remodeling (LPS vs. LPS + UFH: 56.25 ± 1.51 vs. 39.70 ± 1.98, P = 0.0027; TNF-α vs. TNF-α + UFH: 55.42 ± 1.42 vs. 36.51 ± 1.20, P = 0.0005) in vitro. Additionally, UFH decreased the phosphorylation of Akt (LPS vs. LPS + UFH: 0.977 ± 0.081 vs. 0.466 ± 0.035, P = 0.0045) and I kappa B Kinase (IKK) (LPS vs. LPS + UFH: 1.023 ± 0.070 vs. 0.578 ± 0.044, P = 0.0060), and the nuclear translocation of NF-κB (LPS vs. LPS + UFH: 1.003 ± 0.077 vs. 0.503 ± 0.065, P = 0.0078) in HPMECs, which was similar to the effect of the PI3K inhibitor, wortmannin.

結論:

LPS 誘発性肺内皮バリア機能障害に対する UFH の保護効果は、VE-カドヘリンの安定化と PI3K/Akt/NF-κB シグナルに関与している。

CONCLUSIONS: The protective effect of UFH against LPS-induced pulmonary endothelial barrier dysfunction involves VE-cadherin stabilization and PI3K/Akt/NF-κB signaling.