Toll-like receptor-4 deficiency alleviates chronic intermittent hypoxia-induced renal injury,inflammation, and fibrosis

Background

Obstructive sleep apnea (OSA)-associated chronic kidney disease is mainly caused by chronic intermittent hypoxia (CIH) triggered renal damage. This study aims to investigate the role of toll-like receptor-4 (TLR4) in underlying mechanism involved chronic intermittent hypoxia (CIH)-induced renal damage.

Methods

C57BL/6J mice with normal TLR4 (TLR4 WT) or deficient TLR4 (TLR4 KO) were divided into four groups and exposed to normal air (NA) and CIH: TLR4 WT + NA, TLR4 KO + NA, TLR4 WT + CIH, and TLR4 KO + CIH. CIH lasted for 8 h/day and 7 days/week for 6 weeks. Renal injury and inflammation were evaluated by histology and ELISA. Renal tubular apoptosis, macrophages, and fibroblasts recruitment were determined by TUNEL assay, immunofluorescence, and western blot.

Results

In response to CIH, TLR4 deficiency alleviated renal histological injury, renal dysfunction, and fibrosis. TLR4 deficiency ameliorated renal dysfunction (serum BUN and creatinine) and tubular endothelial apoptosis determined by immunofluorescence staining of CD31 and TUNEL, and western blot of apoptotic protein (caspase-3, c-caspase-3, and Bax/Bcl-2 ratio). Furthermore, we also found TLR4 deficiency abrogated CIH-induced macrophages (CD68) and fibroblasts (α-SMA) recruitment, further reducing expression of extra-cellular matrix protein (collagen I and collagen IV) and inflammatory cytokines release (IL-6, TNF-α, and MCP-1). Finally, we used immunohistochemistry to demonstrate that TLR4 deficiency attenuated increased expression of MyD88 and NF-kB p65 after CIH treatment.

Conclusions

Our data suggest that TLR4 plays a vital role in CIH-induced renal injury, inflammation and fibrosis, and inhibition of TLR4 probably provides a therapeutic potential for CIH-induced kidney damage.

     

慢性间歇低氧对大鼠颏舌肌细胞线粒体功能的影响及脂联素的干预作用

目的 探讨慢性间歇低氧(CIH)对大鼠颏舌肌细胞线粒体功能的影响及脂联素干预作用.方法 健康雄性Wistar大鼠39只,采用随机数字表法分成健康对照(NC)组、CIH组、CIH脂联素干预组(CIH+Ad组),每组13只.NC组大鼠呼吸正常空气,CIH组与CIH+Ad组均接受CIH环境(CIH 8 h/d,共5周),CIH+Ad组加用经静脉脂联素注射10 μg/次,2次/周,共5周.于实验终止(第35天)时测定并比较各组大鼠血清脂联素浓度、颏舌肌线粒体膜电位、线粒体复合物Ⅰ活性、线粒体复合物Ⅳ活性.结果 CIH组血清脂联素浓度明显低于NC组[(1108±112)ng/ml,(2241±121)ng/ml,P＜0.01];CIH+Ad组高于CIH组[(1889±119)ng/ml]但低于NC组[(2241±121)ng/ml,均P＜0.01].CIH组颏舌肌线粒体膜电位相对值(1.82±0.11)明显低于NC组(2.09±0.14,P＜0.01),CIH+Ad组(1.98±0.09)较CIH组略高但低于NC组,差异均有统计学差异(均P＜0.05).CIH组线粒体复合物Ⅰ、Ⅳ浓度[(35.68±1.73)μmol·min-1·mg-1,(2.37±0.11)nmol·min-1·mg-1]最低,CIH+Ad组[(37.18±1.95)μmol·min-1·mg-1,(2.49±0.09)nmol·min-1·mg-1]及NC组[(39.02±1.38)μmol·min-1·mg-1,(2.81±0.12)nmol·min-1·mg-1]依次增高.NC组与CIH组比较差异有统计学意义(P＜0.01),CIH+Ad组与CIH组和NC组比较差异有统计学意义(均P＜0.05).结论 CIH可致大鼠血清脂联素水平降低,并能显著损伤颏舌肌细胞线粒体功能,补充外源性脂联素能部分改善CIH对大鼠颏舌肌细胞线粒体功能的损伤,提示低脂联素血症可能参与CIH导致的颏舌肌能量代谢障碍.

Abstract：

Objective To investigate the effect of chronic intermittent hypoxia (CIH) on mitochondrial function in genioglossus cells of rats and intervention role of adiponectin (Ad). Methods Thirty-nine healthy male Wistar rats were randomly divided into 3 groups, normal control (NC) group, CIH group and CIH + Ad group with 13 rats in each. Rats in NC group were kept breathing normal air, while rats in both CIH and CIH + Ad groups experienced the same CIH environment ( CIH 8 h/day for successive 5 weeks). However, rats in CIH + Ad group was given intravenous Ad supplement at the dosage of 10 μg,twice a week for sucessive 5 weeks. At the end of experiment ( day 35 ), the levels of plasma adiponectin,mitochondrial membrane potential activities of respiratory chain complexes Ⅰ and Ⅳ in mitochondrion of genioglossus cells were compared among different groups. Results Serum Ad level was significantly lower in CIH group than that in NC group [(1108 ± 112) ng/ml vs (2241 ± 121) ng/ml, P＜0.01 ]. Serum Ad level in CIH + Ad group [ ( 1889 ± 119) ng/ml] was significantly higher than that in NC group but lower than that in CIH group ( all P ＜ 0. 01 ). Mitochondrial membrane potential was significantly lower in CIH group than that in NC group [ ( 1.82 ± 0. 11 ) vs (2. 09 ± 0. 14), P ＜ 0. 01 ]. Mitochondrial membrane potential in CIH + Ad group ( 1.98 ± 0. 09) was higher than that in CIH group but lower than that in NC group ( all P ＜ 0. 05 ). The concentrations of mitochondrial respiratory chain complexes Ⅰ and Ⅳ in CIH group ( 35.68 ± 1.73 ) μmol · min - 1 · mg- 1 and (2. 37 ± 0. 11 ) nmol · min - 1 ·mg - 1, respectively) were the lowest but became higher from CIH + Ad group [ (37. 18 ± 1.95) μ mol· min-1 · mg-1 and (2. 49 ±0.09) nmol · min-1 ·mg-1 ,respectively] to NC group (39.02 ± 1.38) μmol · min-1 · mg-1 and (2. 81±0. 12) nmol · min-1 ·mg-1 ,respectively), with a significant difference between NC and CIH groups ( P ＜ 0. 01 ), between CIH + Ad and CIH groups ( P ＜ 0. 05 ), as well as between CIH + Ad and NC groups (P ＜ 0. 05 ). Conclusion CIH could lead to hypoadiponectinemia and impaired mitochondrial function in genioglossus cells of rats. Since such changes could be partially improved by supplement of adiponectin, it was suggested that hypoadiponectinemia might be involved in CIH-induced impairment of genioglossus energy metabolism.     

Adiponectin protects rat heart from left ventricular remodeling induced by chronic intermittent hypoxia via inhibition of TGF-β/smad2/3 pathway

Objective

Obstructive sleep apnea syndrome (OSAS) is associated with many cardiovascular disorders. Chronic intermittent hypoxia (CIH) is the primary player in OSAS of the many associated factors. This study was in order to investigate the effects of the Adiponectin (Ad) on left ventricular remodeling induced by CIH.

Methods

Forty-five rats were randomly divided into three groups: normal control (NC) group, CIH group and CIH plus Ad supplemented (CIH + Ad) group. After 35 days’ CIH exposure, masson analysis was used to detect the left ventricular fibrosis and western blot was used to measure the protein expression of collagen I, collagen III and TGF-β/smad2/3 pathway. Gene analysis by RT-PCR was used to study the MMP2 and TIMP2.

Results

After CIH exposure, the fibrosis of left ventricular in CIH group was significantly remarkable than that in both NC and CIH + Ad groups (P<0.05), although statistical difference existed between NC and CIH + Ad groups (P<0.05). In addition, the protein expression of collagen I as well as collagen III and the ratio of mRNA levels of MMP2/TIMP2 were the highest in CIH group but the lowest in NC group, with CIH + Ad group in between. There was a significant difference among three groups (all P<0.05). The TGF-β/smad2/3 pathway was activated obviously in CIH group, but less noticeably in CIH + Ad group (P<0.05) with a significant difference in the two groups.

Conclusions

The present study showed that Ad could ameliorate the left ventricular remodeling induced by CIH via inhibition of the expression of TGF-β/smad2/3 pathway.     

The effect of chronic intermittent hypoxia on respiratory sensitivity to morphine in rats

Purpose

Chronic intermittent hypoxia (CIH) is a characteristic of obstructive sleep apnea syndromes (OSAs). Recurrent hypoxia during the developmental period increases respiratory sensitivity to subsequent administration of opioids. However, it is unknown whether CIH affects respiratory sensitivity to opioids in adults. Our study aimed to assess the changes in respiratory sensitivity to morphine (MOR) under CIH and to explore the possible mechanisms in an adult rat model.

Methods

We applied CIH in adult Sprague-Dawley rats to simulate the hypoxia condition caused by OSAs. An atmosphere with room air was applied as the control environment. After 4 weeks of CIH, MOR was administered. Tests of respiratory function, including measurement of tidal volume (Vt), minute ventilation (MV), tidal volume divided by inspiratory time (Vt/Ti), and respiratory frequency (RF), were then performed. HIF-1α, δ-OR, and μ-OR expressions in the medulla were measured.

Results

After MOR administration, Vt, MV, RF, and Vt/Ti decreased in both the CIH and control groups. MOR caused a more profound depression of MV, RF, Vt, and Vt/Ti in CIH + MOR group compared with C + MOR group. Administration of either μ-OR-specific antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, or δ-OR-specific antagonist, naltrindole, attenuated the depression of Vt, MV, RF, and Vt/Ti. Intermittent hypoxia markedly increased the expression of δ-OR and μ-OR in the medullas of rats. HIF-1α protein expression increased significantly, and HIF-1α mRNA levels remained unchanged.

Conclusions

CIH increases the respiratory sensitivity of rats to MOR by upregulating expression of μ-OR and δ-OR in the medulla, which might be associated with increased levels of HIF-1α.

     

Angiotensin II receptor blocker irbesartan attenuates sleep apnea–induced cardiac apoptosis and enhances cardiac survival and Sirtuin 1 upregulation

Background

The purpose of this study was to investigate whether or not angiotensin II type 1 receptor blocker irbesartan (ARB) with a partial agonist of PPAR-γ could protect against chronic nocturnal intermittent hypoxia (CIH)–induced cardiac Fas/FasL-mediated to mitochondria-mediated apoptosis.

Methods

Sprague–Dawley rats were in a normoxic control group (CON-G), or rats were in a chronic nocturnal intermittent hypoxia group (HP-G, from 3 to 7% oxygen versus 21% oxygen per forty seconds cycle, nocturnally 8 h per day for 1 month), or rats were in a chronic nocturnal intermittent hypoxia group pretreated with ARB (50 mg/kg/day, S.C.) (ARB-HP-G). Echocardiography, H&E staining, TUNEL staining, and Western blotting were measured in the left ventricle.

Results

Hypoxia-induced SIRT1 degradation, Fas receptors, FADD, active caspase-8 and caspase-3 (Fas/FasL apoptotic pathway) and Bax, tBid, active caspase-9 and -3 (mitochondrial apoptotic pathway) and TUNEL-positive apoptosis were reduced in ARB-HP-G when compared with HP-G. IGF-I, IGF1 receptor, p-PI3k, p-Akt, Bcl2, and Bcl-XL (IGF1/PI3K/AKT pro-survival pathway) were increased in ARB-HP-G compared to HP-G.

Conclusions

Our findings suggest that the ARB may prevent cardiac Fas/FasL to mitochondrial apoptotic pathways and enhance cardiac IGF1/PI3K/AKT pro-survival pathway in the sleep apnea model associated with JNK de-activation and SIRT1 upregulation. ARB prevents chronic sleep apnea–enhanced cardiac apoptosis via enhancing survival pathways.

     

脂联素对大鼠慢性间歇性低氧所致心肌重塑的影响

目的：探讨慢性间歇性低氧（chronic intermittent hypoxia,CIH）对心肌重塑的影响及脂联素（adiponectin, Ad）的干预作用。方法将45只Wistar大鼠随机分为3组：正常对照组（NC）,CIH组和CIH＋Ad组。CIH 35d后,使用马松染色分析方法检测左室纤维化程度及使用Western blot方法来衡量Ⅰ型胶原蛋白、Ⅲ型胶原蛋白和TGF-β/smad2/3通路蛋白的表达。通过RT-PCR方法来研究基质金属蛋白酶-2（MMP2）/基质金属蛋白酶的组织抑制剂-2（TIMP-2）的mRNA表达比值情况。结果慢性间歇性低氧处理后,CIH组左心室的纤维化程度显著高于NC组和CIH＋Ad组（P＜0.05）,但NC组和CIH＋Ad组之间差异具有统计学意义（P＜0.05）。CIH组Ⅰ型胶原蛋白和Ⅲ型胶原蛋白和MMP2/TIMP-2的mRNA比值表达最高,NC组表达最低,CIH＋Ad组居中,3组之间均差异具有统计学意义（P＜0.05）。TGF-β/smad通路蛋白在CIH组中表达显著高于NC组和CIH组（P＜0.05）,且NC组和CIH＋Ad组差异具有统计学意义（P＜0.05）。结论慢性间歇性低氧可引起左室重构,而Ad可能通过抑制TGF-β/smad2/3通路改善此损害。     

慢性间歇性低氧对大鼠左心功能的影响及作用机制研究

目的了解慢性间歇性低氧对大鼠左心室功能的影响,及心肌细胞凋亡在其中的作用。方法SD雄性大鼠16只,随机分为慢性间歇性低氧组（CIH组,间歇性低氧舱内饲养8h／d,共5周）和正常对照组（NC组,空气饲养）。实验结束时测量体重、血压、心率,心导管测定平均动脉压、左室舒张末压,TUNEL法检测心肌细胞凋亡,Westernblot检测心肌组织caspase3、caspase8、Fas、Bax、Bcl-2蛋白水平。结果实验结束时CIH组左心室占体重的比值高于NC组（3．04±0．24‰和2．53±0．16％o,P〈0．001）,CIH组血压较NC组明显升高（136．3±6．8mmHg和122．3±4．1mmHg,P〈0．001）,两组间心率无明显差异。心导管检查显示CIH组左室舒张末压较NC组升高（13．6±1．0mmHg和5．7±O．5mmHg,P〈0．001）。TUNEL结果显示CIH组凋亡指数明显高于NC组（17．5％和1．9％,P〈0．001）,左室舒张末压与心肌细胞凋亡指数呈正相关（r=0．807,P〈0．001）。Westernblot检测结果显示与NC组相比,CIH组caspase3、Fas、caspase8表达增加,Bcl-2表达降低（P〈0．001）,Bax无明显差异（P=0．213）。结论慢性间歇性低氧可引起大鼠平均动脉压增高,左心室收缩和舒张功能降低,可能与慢性间歇性低氧诱导大鼠心肌细胞凋亡增加相关。     

Metallothionein alleviates oxidative stress-induced endoplasmic reticulum stress and myocardial dysfunction

Oxidative stress and endoplasmic reticulum (ER) stress have been implicated in cardiovascular diseases although the interplay between the two is not clear. This study was designed to examine the influence of oxidative stress through glutathione depletion on myocardial ER stress and contractile function in the absence or presence of the heavy metal scavenger antioxidant metallothionein (MT). FVB and MT overexpression transgenic mice received the GSH synthase inhibitor buthionine sulfoximine (BSO, 30 mM) in drinking water for 2 weeks. Oxidative stress, ER stress, apoptosis, cardiac function and ultrastructure were assessed using GSH/GSSG assay, reactive oxygen species (ROS), immunoblotting, caspase-3 activity, Langendorff perfused heart function (LVDP and ± dP/dt), and transmission electron microscopy. BSO led to a robust decrease in the GSH/GSSG ratio and increased ROS production, consolidating oxidative stress. Cardiac function and ultrastructure were compromised following BSO treatment, the effect of which was obliterated by MT. BSO promoted overt ER stress as evidenced by upregulated BiP, calregulin, phospho-IRE1α and phospho-eIF2α without affecting total IRE1α and eIF2α. BSO treatment led to apoptosis manifested as elevated expression of CHOP/GADD153, caspase-12 and Bax as well as caspase-3 activity, reduced Bcl-2 expression and JNK phosphorylation, all of which was ablated by MT. Moreover, both antioxidant N-acetylcysteine and the ER stress inhibitor tauroursodeoxycholic acid reversed the oxidative stress inducer menadione-elicited depression in cardiomyocyte contractile function. Taken together, these data suggested that ER stress occurs likely downstream of oxidative stress en route to cardiac dysfunction.     

Metabolic syndrome enhances endoplasmic reticulum,oxidative stress and leukocyte–endothelium interactions in PCOS

ObjectivePolycystic ovary syndrome (PCOS) is associated with insulin resistance, which can lead to metabolic syndrome (MetS). Oxidative stress and leukocyte–endothelium interactions are related to PCOS. Our aim was to evaluate whether the presence of MetS in PCOS patients can influence endoplasmic reticulum (ER) and oxidative stress and leukocyte–endothelium interactions.Material and MethodsThis was a prospective controlled study conducted in an academic medical center. The study population consisted of 148 PCOS women (116 without/32 with MetS) and 112 control subjects (87 without / 25 with MetS). Metabolic parameters, reactive oxygen species (ROS) production, ER stress markers (GRP78, sXBP1, ATF6), leukocyte–endothelium interactions, adhesion molecules (VCAM-1, ICAM-1, E-Selectin), TNF-α and IL-6 were determined.ResultsTotal ROS, inflammatory parameters and adhesion molecules were enhanced in the presence of MetS (p < 0.05), and the PCOS + MetS group showed higher levels of IL-6 and ICAM-1 than controls (p < 0.05). Increased adhesion and leukocyte rolling flux were observed in PCOS and PCOS + MetS groups vs their respective controls (p < 0.05). GRP78 protein expression was higher in the PCOS groups (p < 0.05 vs controls) and sXBP1 was associated with the presence of MetS (p < 0.05 vs controls without MetS). Furthermore, PCOS + MetS patients exhibited higher GRP78 and ATF6 levels than controls and PCOS patients without MetS (p < 0.05). In PCOS women, HOMA-IR was positively correlated with ICAM-1 (r = 0.501; p < 0.01), ROS (r = 0.604; p < 0.01), rolling flux (r = 0.455;p < 0.05) and GRP78 (r = 0.574; p < 0.001).ConclusionOur findings support the hypothesis of an association between altered metabolic status, increased ROS production, ER stress and leukocyte–endothelium interactions in PCOS, all of which are related to vascular complications.     

慢性间歇低氧大鼠氧化应激相关途径及抗氧化剂干预效果分析

目的 研究探讨慢性间歇低氧(CIH)大鼠氧化应激与血压及肾素-血管紧张素系统(RAS)相关性及N-乙酰半胱氨酸(NAC)对CIH相关性高血压的防治作用及机制.方法 24只SD雄性大鼠按随机数字表法分为CIH1、CIH2、CIH3和对照组,每组6只.CIH组大鼠循环给予氮气和压缩空气(每循环180 s,舱内最低氧浓度达6%～8%,维持20～25 s,然后恢复至21%,7 h/d),对照组大鼠常规饲养.CIH1组给予NAC 800 ml·kg-1·d-1腹腔注射,CIH2组予生理盐水5 ml·kg-1·d-1腹腔注射.结果第6周时CIH2、CIH3组大鼠收缩压(SBP)显著高于CIH1、对照组(P<0.05)和实验前水平(P<0.01).CIH2、CIH3组分别与CIH1和对照组比较:血管紧张素转化酶(ACE)及ACE2水平差异有统计学意义(P<0.05);血管紧张素(Ang)Ⅱ及血清丙二醛(MDA)、氧化低密度脂蛋白(ox-LDL)显著增高(P<0.05),Ang-(1-7)及血清抑制羟自由基能力则显著降低(P<0.05).SBP 与血清及肾Ang Ⅱ水平正相关;与Ang-(1-7)水平负相关;MDA、ox-LDL分别与血压及AngⅡ正相关,与Ang-(1-7)负相关;抑制羟自由基能力与血压、Ang Ⅱ负相关,与Ang-(1-7)正相关;oxLDL与MDA和抑制羟自由基能力分别呈正相关和负相关.CIH1组与对照组比较除SBP和血清AngⅡ(P<0.05)外其他指标差异无统计学意义(P>0.05),CIH2组与CIH3组以上指标差异均无统计学意义(P>0.05).结论 CIH 可引起大鼠氧化应激及RAS失衡,二者密切相关,可能是CIH诱导高血压发生的重要机制之一.NAC可能通过抗氧化作用调节RAS平衡而干预CIH相关性高血压的发生.

Abstract：

Objective To study the relation of oxidative stress with systolic blood pressure(SBP) and renin-agiotensin system(RAS) in a rat model of chronic intermitten hypoxia(CIH),and to investigate the preventive effect and mechanism of N-acetylcysteine (NAC) in CIH-induced hvpertension.Methods Twenty-four male Sprague-Dawley (SD) rats were divided into 4 groups:CIH + NAC group (CIH1),CIH +normal saline(NS) group(CIH2),CIH control group(CIH3)and blank control group(UC).CIH rats were subjected to alternating cycles of hypoxia (6%-8% O2 in N2 for 20-25 s) and normoxia (21% O2 in N2 for 2 min) every 180 s for 7 h per day.Rats in the CIH1 group were treated with NAC(800 ml · kg-1 · d-1)by intraperitoneal injection,and those in the CIH2 group were treated with NS (5 ml · kg-1 · d-1).Results SBP in the CIH2 and CIN3 groups at the end of 6th week was significantly elevated compared with the baseline SBP(P<.001)and those in the CIH1 and the UC groups (P<.05).The expression of angiotensinconverting enzyme(ACE) and ACE2 in renal arterioles was significantly different (P <0.05),and the levels of angiotensin Ⅱ(Ang Ⅱ)in the serum and kidney tissues,oxidation of low density lipoprotein (ox-LDL) and malondialdehyde (MDA)in the serum were increased.Ang-(1-7)and the inhibitory capability for hydroxyl free radicals in the serum were decreased significantly in CIH2 and CIH3 groups compared with CIH1 and UC (P <0.05)groups at the end of 6th week.SBP showed a positive correlation with Ang Ⅱ in serum and kidney tissues,but showed a negative correlation with Ang-(1-7)in serum and kidney tissues.The levels of MDS and ox-LDL in serum showed a positive correlation with Ang Ⅱ in serum and kidney tissues respectively,but showed a negative correlation with Ang-(1-7) in serum and kidney tissues respectively.The inhibitory capability for hydroxyl free radicals in serum showed a positive correlation with Ang-(1-7),but a negative correlation with Ang Ⅱ.The level of ox-LDL showed a positive correlation with MDA,but a negative correlation with the inhibitory capability for hydroxyl free radicals.There were no significant difference between CIH1 and UC groups in parameters except of SBP and Ang Ⅱ(P <0.05).All the data were not different between CIH2 and CIH3 groups(P>.05).Conclusions CIH caused oxidative stress and RAS imbalance in rats.The imbalance of RAS in CIH rats was related with oxidative stress.The imbalance of RAS and oxidative stress may be one of the important mechanisms for CIH-induced hypertension.NAC can prevent CIH-induced hypertension through modulation of RAS by its anti-oxidative effect.     

不同频率间歇低氧下大鼠肝脏氧化应激损伤及Tempol的干预作用

目的 探讨不同频率间歇低氧(IH)对大鼠肝脏氧化应激损伤差异和Tempol的干预作用及可能机制.方法 应用慢性间歇低氧(CIH)大鼠模型,模拟OSAS周期性间歇低氧/再氧和病理生理过程.56只雄性Wistar大鼠随机分为不同频率IH组(IH1,IH2,IH3,IH4,频率依次为10、20、30、40次/h),30T组(...     

Effects of allopurinol on cardiac function and oxidant stress in chronic intermittent hypoxia

Rationale

Obstructive sleep apnea is associated with left ventricular (LV) dysfunction, oxidant stress, and chronic intermittent hypoxia (CIH). Allopurinol (ALLO) is a xanthine oxidase inhibitor that also scavenges free radicals.

Objectives

Using an animal model of CIH we hypothesized that ALLO decreases oxidant stress and cardiac injury.

Materials and methods

Rats were exposed to either CIH (nadir 4–6%, approximately once per minute) or room air (handled controls, HC) for 8 h a day for 10 days. Four treatment groups (six to ten animals per group) were studied: CIH/ALLO, CIH/placebo (PLAC), HC/ALLO, and HC/PLAC. Outcomes included myocardial lipid peroxides (LPO) for oxidant stress, fraction shortening of the LV cavity for cardiac function (LVFS) and an assay for myocyte apoptosis.

Results

LPO was lower in CIH/ALLO group compared to CIH/PLAC (179?±?102 vs. 589?±?68 mcg/mg protein, p?<?0.05). LVFS was greater in ALLO animals than PLAC in both CIH and HC (CIH/ALLO 48.6?±?2.3% vs. CIH/PLAC 38?±?1.4%; HC/ALLO 64.9?±?1.8% vs. HC/PLAC 51.5?±?1.5%; both p?<?0.05). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed fewer apoptotic nuclei in LV myocardium in CIH/ALLO compared to CIH/PLAC (38.0?±?1.4 vs. 48.6?±?2.3 positive nuclei per 2.5 mm² area, p?<?0.05).

Conclusion

ALLO is associated with improvement in CIH-associated oxidant stress, myocardial dysfunction, and apoptosis in rats.     

Fisiopatología de la miocardiopatía dilatada isquémica a través del microRNA-16-5p

Introduction and objectivesThe expression levels of microRNA-16-5p (miR-16) are upregulated in ischemic cardiomyopathy and in animal models of ischemic dilated cardiomyopathy (iDCM), inducing myocardial apoptosis. We investigated the role of miR-16 in the adaptive cellular response associated with endoplasmic reticulum (ER) stress and autophagy in the apoptotic iDCM environment.MethodsWe quantified the miR-16 plasma levels of 168 participants—76 controls, 60 iDCM patients, and 32 familial DCM patients with the pathogenic variant of BAG3—by quantitative real-time polymerase chain reaction and correlated the levels with patient variables. The effects of intracellular miR-16 overexpression were analyzed in a human cardiac cell line. Apoptosis and cell viability were measured, as well as the levels of markers associated with ER stress, cardiac injury, and autophagy.ResultsPlasma miR-16 levels were upregulated in iDCM patients (P = .039). A multivariate logistic regression model determined the association of miR-16 with iDCM clinical variables (P < .001). In vitro, miR-16 overexpression increased apoptosis (P = .02) and reduced cell viability (P = .008). Furthermore, it induced proapoptotic components of ER stress, based on upregulation of the PERK/CHOP pathway. However, we observed augmentation of autophagic flux (P < .001) without lysosomal blockade by miR-16 as a possible cytoprotective mechanism.ConclusionsMiR-16 is specifically associated with iDCM. In an ischemic setting, miR-16 activates ER stress and promotes inflammation followed by autophagy in human cardiac cells. Thus, autophagy may be an attempt to maintain cellular homeostasis in response to misfolded/aggregated proteins related to ER stress, prior to apoptosis.     

Additional benefit of combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cell against sepsis‐induced kidney injury

This study tested whether combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cells (A‐ADMSCs) offered additional benefit in ameliorating sepsis‐induced acute kidney injury. Adult male Sprague–Dawley rats (n = 65) were randomized equally into five groups: Sham controls (SC), sepsis induced by cecal‐ligation and puncture (CLP), CLP‐melatonin, CLP‐A‐ADMSC, and CLP‐melatonin‐A‐ADMSC. Circulating TNF‐α level at post‐CLP 6 hr was highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Immune reactivity as reflected in the number of splenic helper‐, cytoxic‐, and regulatory‐T cells at post‐CLP 72 hr exhibited the same pattern as that of circulating TNF‐α among all groups (P < 0.001). The histological scoring of kidney injury and the number of F4/80+ and CD14+ cells in kidney were highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups, and higher in CLP‐A‐ADMSC than in CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Changes in protein expressions of inflammatory (RANTES, TNF‐1α, NF‐κB, MMP‐9, MIP‐1, IL‐1β), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), fibrotic (Smad3, TGF‐β) markers, reactive‐oxygen‐species (NOX‐1, NOX‐2), and oxidative stress displayed a pattern identical to that of kidney injury score among the five groups (all P < 0.001). Expressions of antioxidants (GR+, GPx+, HO‐1, NQO‐1+) were lowest in SC group and highest in CLP‐melatonin‐A‐ADMSC group, lower in CLP than in CLP‐melatonin and CLP‐A‐ADMSC groups, and lower in CLP‐melatonin‐ than in CLP‐A‐ADMSC‐tretaed animals (all P < 0.001). In conclusion, combined treatment with melatonin and A‐ADMSC was superior to A‐ADMSC alone in protecting the kidneys from sepsis‐induced injury.     

Validity of endoscopic resection for clinically diagnosed T1a-MM/T1b-SM1 N0 M0 esophageal squamous cell carcinoma

Background

Previous guidelines have not described clear recommendations for performing endoscopic resection (ER) of T1a-muscularis mucosa (MM)/T1b-submucosal (SM1) cancers that have invaded?≤?200 μm because these are considered to have a non-negligible risk of metastasis based on previous analyses of pathologically diagnosed (p)MM/SM1 cancers. Considering that the indication for ER is determined based on a clinical diagnosis, the applicability of ER should be investigated in clinical (c)MM/SM1 but not pMM/SM1 cancers. This study aimed to evaluate validity of ER for cMM/SM1 cancers.

Methods

In total, 175 cMM/SM1 esophageal squamous cell carcinoma cases that were endoscopically or surgically resected between January 2008 and December 2018 were identified from a prospectively maintained database. We histologically evaluated resected specimens and divided them into low- (n?=?92) and high-risk (n?=?83) cancers for metastasis.

Results

Univariate analysis showed that longer tumor length and larger circumferential extent were significantly correlated with high-risk cancer (P?<?0.001). Multivariate analysis showed that tumor circumference was an independent predictor of high-risk cancer (P?=?0.036). The proportion of low-risk cancers among cases with?≤?3/4,?>?3/4 and?<?1, and whole circumferential extent were 59, 17, and 14%, respectively, and the post-ER stricture rates of these groups were 12, 33, and 100%, respectively.

Conclusion

ER is the first-line treatment for cMM/SM1 cancers with?≤?3/4 circumferential extent considering that 59% of cMM/SM1 cancers were low-risk cancers for which ER is mostly curative. ER is not recommended for whole circumferential cMM/SM1 cancers given the low proportion of low-risk cancers and the high risk of stricture after ER.

     

Aerobic training and L-arginine supplement attenuates myocardial infarction-induced kidney and liver injury in rats via reduced oxidative stress

IntroductionThe aim of the present study was to determine the effect of exercise training and l-arginine supplementation on kidney and liver injury in rats with myocardial infarction (MI).Material and methodsFour weeks after MI, 50 male wistar rats randomly divided into five followed groups: sham surgery without MI (Sham, n = 10), Sedentary-MI (Sed-MI, n = 10) 3: L-Arginine-MI (La-MI, n = 10) 4: Exercise training-MI (Ex-MI, n = 10) and 5: Exercise and L-arginine-MI (Ex + La-MI). Ex-MI and Ex + La-MI groups running on a treadmill for 10 weeks with moderate intensity. Rats in the L-arginine-treated groups drank water containing 4% L-arginine. Tissues oxidative stress and kidney and liver functional indices were measured after treatments.ResultUrea as a kidney function indexes, increased in Sed-MI group in compared to sham group and decreased significantly in Ex-MI and Ex + La-MI groups. The level of catalase (CAT) and glutathione stimulating hormone (GSH) of kidney were significantly lower in the MI-groups compared with the Sham group and kidney Malondialdehyde (MDA) levels increased after MI and significantly decreased in response to aerobic training and L-arginine. As well as, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as liver injury indices, increased in MI-groups and decreased by training and L-arginine. In this regards, liver MDA and CAT respectively increased and decreased in MI-groups, but aerobic training and L-arginine increased liver glutathione per-oxidase (GPx) and decreased liver MDA.ConclusionThese results demonstrated that kidney and liver function impaired 14 weeks after MI and aerobic training and L-arginine supplementation synergistically ameliorated kidneys and liver injury in myocardial infarction rats through oxidative stress reduction.     

Use of figure of eight suture for groin closure with no heparin reversal in patients undergoing cryoballoon ablation for atrial fibrillation

Purpose

We aimed to demonstrate the feasibility of figure of eight (FoE) suture for groin access closure in cryoballoon (CB) ablation and its impact on the lab workflow.

Methods

We retrospectively analyzed all patients who underwent CB ablation in our institution from June 2018. From June 2019, we have been consistently using FoE suture for hemostasis (FoE+ group), and before that conventional manual compression was utilized (FoE- group). Ablations were performed under uninterrupted oral anticoagulant strategy. Both femoral veins were punctured, and a single introducer was placed in each vein. In the FoE- group, after ablation, protamine was administered, and manual compression was applied. In the FoE+ group, the suture was placed in both groins without protamine administration and manual compression. All patients were in decubitus position until the next morning.

Results

A total of 190 consecutive patients (73.2% male, 59.7?±?11.0 years old) were evaluated, 90 being in the FoE+ group. There was no difference in the mean procedure duration between two groups (72.7?±?23.1 min vs 73.7?±?27.1 min, p?=?0.784). However, the total lab time was significantly longer in FoE- group (132.9?±?35.3 min vs 109.9?±?32.6 min, p?<?0.0001). There was no difference in complication rates. No major vascular complications were found in our cohort.

Conclusions

Utilization of FoE sutures for hemostasis seems to be a safe and effective after cryoballoon ablation. It abolishes the need for protamine administration which can cause serious adverse events. As manual compression is unrequired, EP lab workflow improves due to significantly shortened total lab time per patient.

     

Breast cancer patients with estrogen receptor-negative/progesterone receptor-positive tumors: being younger and getting less benefit from adjuvant tamoxifen treatment

Purpose  Most breast cancer patients with estrogen receptor-negative/progesterone receptor-positive (ER−/PgR+) tumors are premenopausal cases, with few alternatives of adjuvant endocrine therapy but tamoxifen (TAM). The efficacy of adjuvant TAM on ER−/PgR+ patients is still controversial. In this study, we evaluated the efficacy of adjuvant TAM on patients with ER−/PgR+ tumors. Methods  Among all 1,836 consecutive patients with operable primary breast cancer, 798 cases were with ER+/PgR+ tumors and 205 with ER−/PgR+ tumors. By sub-grouping the patients according to ER/PR phenotypes and whether the patients had been treated with adjuvant TAM therapy or not, we investigated the differences of survivals between groups. Results  Patients with ER−/PgR+ tumors were younger than those with ER+/PgR+ tumors (P = 0.021), and were mainly premenopausal (P = 0.013). ER−/PgR+ patients were related to more involved lymph nodes and later stage. In the absence of TAM treatment, ER+/PgR+ group had a similar survival to ER−/PgR+ group in terms of 5-year disease-free survival (DFS), as well as overall survival (OS). After TAM treatment, both groups had increased survival rates comparing with the baseline of non-TAM-treated groups. Moreover, significant survival differences were then observed between TAM-treated ER+/PgR+ group and TAM-treated ER−/PgR+ group either in DFS (P = 0.016) or OS (P = 0.007). Of the TAM-treated patients, by sub-dividing the chemotherapy-treated population into CMF (cyclophosphamide, methotrexate and 5-fluorouracil) group and CA(E)F (cyclophosphamide, doxorubicin/epirubicin and 5-fluorouracil) group, we found that ER−/PgR+ group got more benefits from CMF regimen than from CA(E)F. Subpopulation treatment effect pattern plot (STEPP) analysis showed that the ER−/PgR+ group had an obvious worse survival than ER+/PgR+ group in younger patients (<55 years). Axillary lymph nodes involvement was the only independent prognostic factor for ER−/PgR+ group. Conclusions  Our results indicate that patients with ER−/PgR+ tumors are mainly premenopausal and young. Although patients with ER−/PgR+ tumors are generally considered as candidates for endocrine therapy clinically, the ER−/PgR+ group gains less benefits from adjuvant TAM treatment than ER+/PgR+ group. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Yu Ke-da and Di Gen-hong have contributed equally to this work.