N-乙酰半胱氨酸对大鼠肝脏缺血再灌注损伤的保护作用

目的探讨大鼠肝脏缺血再灌注损伤（HIRI）的机制及N-乙酰半胱氨酸（NAC）对肝组织的保护作用。方法将大鼠分成假手术组、HIRI组和NAC干预组,于再灌注1、3、6、12 h分别采用全自动生化分析仪测定各组血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平;黄嘌呤氧化酶法测定肝组织中超氧化物岐化酶（SOD）活性;免疫组织化学技术检测肝组织中肿瘤坏死因子α（TNF-α）表达。结果HIRI组肝脏损伤较假手术组更加明显,其中以再灌注6 h损伤最严重;血清AST、ALT均较假手术组明显升高;肝组织SOD活性明显低于假手术组（P〈0.01）,于再灌注6 h最低;肝组织TNF-α表达明显高于假手术组（P〈0.01）,于再灌注6 h达高峰。结论NAC对大鼠HIRI有保护作用,其机制与NAC清除氧自由基、减少细胞因子和炎症介质表达有关。     

S100蛋白在兔肝脏缺血再灌注损伤过程中的表达及意义

目的探讨肝脏缺血再灌注（HIRI）过程中S100蛋白表达变化及其意义。方法将21只大白兔随机分为模型组、对照组和正常组各7只，模型组建立免HIRI模型，对照组手术操作同模型组，但不夹闭门静脉和肝动脉；正常组不手术。普通HE染色观察各组肝组织细胞学形态；免疫组织化学方法染色检测各组肝组织细胞S100蛋白表达情况，并用Sampl-PCI图像分析系统进行半定量分析。结果模型组肝组织细胞可见大量S100染色阳性细胞，平均光密度值显著高于对照组和正常组（P〈0．01）。结论S100蛋白参与了HIRI过程，其表达升高对HIRI可能起保护作用，机制尚需进一步探讨。     

大鼠肝脏缺血再灌注损伤对氧自由基、一氧化氮、内皮素及炎性细胞因子的影响

肝脏缺血再灌注损伤（HIRI）是肝脏外科疾病中常见的病理过程.导致肝脏HIRI的原因很多,与以下三个因素有关：①肝脏缺血期的低氧损伤;②再灌注后肝窦内皮细胞和肝细胞的损伤;③继发于内皮细胞损伤的微循环障碍.但确切的发病机制仍不十分清楚.本研究旨在通过测定HIRI大鼠模型血浆超氧化物歧化酶（SOD）、丙二醛（MDA）、一氧化氮（NO）、内皮素（ET）、肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-8及IL-10含量的变化,探讨大鼠肝脏HIRI对氧自由基、NO、ET及炎性细胞因子的影响.     

人骨髓间充质干细胞及其外泌体在肝移植术后肝缺血再灌注损伤中潜在治疗作用的研究进展

［摘要］　肝缺血再灌注损伤（HIRI）是传统肝移植术中不可避免的病理生理过程,其严重程度受到来自供体和受体的多方面因素影响,最严重者可导致术后早期移植肝无功能。人骨髓间充质干细胞（BMSCs）及其外泌体已在临床试验中被证实具有促进组织修复与免疫调节的能力,这一治疗作用在HIRI的防治中具有较好的应用前景。该文旨在综述BMSCs及其外泌体在移植肝HIRI防治中潜在作用机制的研究进展及未来研究方向。     

左旋精氨酸对缺血-再灌注损伤肝脏的保护作用

目的：探讨左旋精氨酸（L-arginine,L-Arg）对肝缺血－再灌注损伤（hepatic ischemia-reperfusion injury,HIRI）的防治作用及其机理。方法：选择HIRI家兔及肝手术患者,观察一氧化氮水平、丙二醛浓度、血栓素B2和6－酮基－前列腺素F1α含量、血栓素B2/6－酮基－前列腺素F1α比值、ALT活性、肝细胞形态学的变化及L-Arg对上述指标的影响。结果：HIRI期间,NO、6－酮基－前列腺素F1α显著下降,MDA、血栓素B2、血栓素B2/6-酮基-前列腺素F1α及ALT明显升高,肝细胞形态学发生异常变化;使用L-Arg后,上述指标的异常变化显著减轻,其差异有显著意义（Ｐ＜0.05和Ｐ＜0.01＝。 结论：L－Arg通过提高机体NO水平、降低MDA含量及纠正血栓烷 A2与PGI2的平衡,对HIRI有积极的防治作用。     

肝脏非实质细胞在肝脏缺血再灌注损伤中的作用

肝脏非实质细胞在肝脏中发挥着非常重要的作用。回顾近几年关于非实质肝细胞,如Kupffer细胞、肝窦内皮细胞、肝星状细胞、树突状细胞等在肝脏缺血再灌注损伤(HIRI)中的作用。分析表明肝脏非实质细胞在HIRI过程中扮演着非常重要的角色,能通过各种途径最终加重或者减轻HIRI。     

中医药成分防治肝缺血再灌注损伤的研究进展

肝缺血再灌注损伤(HIRI)是肝移植、肝脏切除及休克等疾病的一种极为常见的并发症。目前对于HIRI的研究较多,但临床仍缺乏根治性的药物。相关细胞、分子机制及通路、活性氧及氧化应激反应、一氧化氮及线粒体等多种因素介导了HIRI的发生和发展,这不仅导致患者生活质量降低,且危及患者生命安全。在HIRI发生机制的基础上结合相关文献,总结中医药成分防治HIRI的研究进展,旨在为HIRI的基础实验及临床研究提供理论支持。     

p38MAPK信号通路与肝缺血再灌注损伤的关系研究

p38MAPK通路参与诸如细胞氧化应激、细胞凋亡和炎症等多种生理和病理过程并在其中起着重要作用。肝缺血再灌注损伤(HIRI)是肝脏外科手术面临的一大难题,由于HIRI发病机制复杂,临床上尚未发现更好的应对HIRI的防治策略。本文就p38MAPK信号通路的主要功能及其在HIRI中的相关作用作一综述,以便为防治HIRI提供一个新的思路。     

法舒地尔对肝纤维化大鼠肝脏缺血再灌注损伤的有效剂量

目的探讨法舒地尔对肝纤维化大鼠肝脏缺血再灌注损伤(HIRI)的影响及其有效剂量。方法以SD大鼠构建肝纤维化模型,80只大鼠分为假手术组、HIRI组、法舒地尔1组和法舒地尔2组,每组20只。开腹显露肝蒂,假手术组和HIRI组经腹腔注射生理盐水5 ml/kg,法舒地尔1组和法舒地尔2组分别注射20μg/kg和80μg/kg法舒地尔;HIRI组、法舒地尔1组和法舒地尔2组30 min后阻断肝门20 min,假手术组不阻断肝门,解除阻断并关腹。术毕4 h取各组6只大鼠血液和肝组织标本,检测谷草转氨酶(AST)、谷丙转氨酶(ALT)、丙二醛(MDA)、超氧化物歧化酶(SOD)和肿瘤坏死因子(TNF)-α。肝组织苏木素-伊红(HE)染色行组织病理检查。记录各组余14只大鼠96 h生存情况,统计生存率。结果与假手术组相比,HIRI组、法舒地尔1组和法舒地尔2组血清AST和ALT水平显著上升(P0. 05);法舒地尔1组和法舒地尔2组血清AST和ALT水平显著低于HIRI组(P0. 05),且法舒地尔2组显著低于法舒地尔1组(P0. 05)。与假手术组相比,HIRI组、法舒地尔1组和法舒地尔2组肝组织MDA含量显著上升,SOD含量显著下降(P0. 05);法舒地尔1组和法舒地尔2组肝组织MDA含量显著低于HIRI组(P0. 05),SOD含量显著高于HIRI组(P0. 05),且法舒地尔2组和法舒地尔1组比较差异有统计学意义(P0. 05)。与假手术组相比,HIRI组、法舒地尔1组和法舒地尔2组血清TNF-α水平显著上升(P0. 05);法舒地尔1组和法舒地尔2组血清TNF-α水平均显著低于HIRI组(P0. 05),且法舒地尔2组显著低于法舒地尔1组(P0. 05)。假手术组、HIRI组、法舒地尔1组和法舒地尔2组96 h生存率分别为78. 57%、28. 57%、50. 00%和64. 29%。法舒地尔1组和法舒地尔2组与HIRI组总体存活时间差异有统计学意义(P0. 05)。结论法舒地尔对肝纤维化大鼠HIRI后肝脏具有保护作用,且呈剂量依赖形式,可能通过提高机体的抗氧化能力,降低炎症细胞因子水平和脂质过氧化水平而发挥作用。     

靶向自噬缓解肝缺血再灌注损伤的研究进展

肝缺血再灌注损伤(HIRI)是肝切除和肝移植术后常见的临床问题,是导致移植术后肝功能障碍和肝功能衰竭的主要原因。近年来,自噬介导的途径成为缓解HIRI的研究热点。自噬是指细胞通过将大量细胞质、受损细胞器等底物运输到溶酶体内进行消化降解以不断更新重塑再利用细胞的过程。本文从基因、蛋白、信号通路、炎症反应、氧化应激反应、线粒体及内质网应激等方面总结了靶向自噬途径缓解HIRI相关机制的研究进展,并围绕研究中存在的问题进行了讨论和展望,以期为今后通过靶向自噬途径缓解HIRI的研究提供理论支持。     

葛根素对再灌注损伤肝能量代谢的保护作用及机制

[目的]研究葛根素对兔缺血再灌注损伤中肝脏能量代谢的改善作用及其机制。[方法]将30只家兔随机均分为对照组(C组)、缺血/再灌注组(IR组)和葛根素组(Pur组)。测定血浆中谷丙转氨酶(ALT)浓度和肝组织内一磷酸腺苷(AMP)、二磷酸腺苷(ADP)、三磷酸腺苷(ATP)、总腺苷酸量(TAN)、肝脏的细胞能荷(EC)、丙二醛(MDA)、超氧化物歧化酶(SOD)、一氧化氮(NO)、内皮素(ET)、NO/ET、血栓素B2(TXB2)、前列腺素F1α(PGF1α)、TXB2/PGF1α水平,并观察肝细胞形态学的改变。[结果]与C组比较,IR组血浆ALT浓度、肝组织AMP、ADP、MDA、ET含量显著升高(P0.01),TXB2、TXB2/PGF1α亦明显升高(P0.05),肝组织ATP、EC、SOD、NO/ET显著下降(P0.01),NO、PGF1α亦明显降低(P0.05),TAN差异无统计学意义(P0.05),肝细胞形态学异常改变;与IR组比较,Pur组血浆ALT浓度、肝组织AMP、MDA、ET含量显著降低(P0.01),肝组织ADP、TXB2/PGF1α亦明显减少(P0.05),TXB2略低于IR组(P0.05),肝组织ATP、EC、SOD、NO/ET显著升高(P0.01),肝组织NO、PGF1α亦明显增高(P0.05),TAN差异无统计学意义(P0.05),肝细胞形态学异常改变较大程度减轻。[结论]葛根素(60mg/kg)预处理可通过拮抗氧化损伤、调节NO/ET和前列腺素I2/血栓素A2(PGI2/TXA2)失衡而改善肝细胞的能量代谢,有效防治兔肝再灌注损伤。     

New progress in understanding roles of nitric oxide during hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury (HIRI) is a major clinical cause of morbidity and mortality in liver surgery and transplantation. Many studies have found that nitric oxide (NO) plays an important role in the HIRI and its increase or decrease can affect the progression and outcome of HIRI. However, the role of NO in HIRI is controversial and complicated. NO derived by endothelial NO synthase (eNOS) shows a protective role in HIRI, while excessive NO derived by inducible NO synthase (iNOS) accelerates inflammation and increases oxidative stress, further aggravating HIRI. Nevertheless, the overexpression of eNOS may exacerbate HIRI and iNOS-derived NO in some cases reduces HIRI. Here we review the new progress in the understanding of the roles of NO during HIRI: (1) NO possesses different roles in HIRI by increasing NO bioavailability, down-regulating leukotriene C4 synthase, inhibiting the activation of the nuclear factorκB (NFκB) pathway, enhancing cell autophagy, and reducing inflammatory cytokines and reactive oxygen species (ROS). And NO has both protective and deleterious effects by regulating apoptotic factors; (2) eNOS promotes NO production and suppresses its own overexpression, exerting a hepatoprotective effect reversely. Its activation is regulated by the PI3K/Akt and KLF2/AMPK pathways; and (3) iNOS derived NO mainly has deteriorating effects on HIRI, while it may have a protective function under some conditions. Their expression should reach a balance to reduce the adverse side and make NO protective in the treatment of HIRI. Thus, it can be inferred that NO modulating drugs may be a new direction in the treatment of HIRI or may be used as an adjunct to mitigate HIRI for the purpose of protecting the liver.     

Calcium entry blockade prevents leakage of macromolecules induced by ischemia-reperfusion in skeletal muscle.

Calcium kinetics and its intracellular mobilization are important in all biological processes. We used verapamil to examine the effect of calcium entry blockade on microvascular transport of macromolecules in ischemia-reperfusion injury. The rat cremaster muscle was splayed, placed in a Lucite intravital chamber, and suffused with bicarbonate buffer. The clearance of fluorescein isothiocyanate-conjugated dextran (FITC-dextran 150) was measured as an index of microvascular transport. After determination of baseline data (clearance of FITC-dextran 150, 3.0 +/- 0.5 microliters/5 min/g), the muscle was made ischemic for 2 hours by clamping its vascular pedicle and subsequently was reperfused for 2 hours. Ischemia-reperfusion produced a marked increase in FITC-dextran clearance. After a peak of 12 +/- 2-fold increase observed in the first 15 minutes into reperfusion, FITC-dextran 150 clearance decreased in magnitude and stabilized at about sixfold above baseline. Verapamil did not change the baseline clearance values. Importantly, verapamil inhibited the ischemia-induced increase in clearance and maintained the values at or near the baseline levels. We simultaneously determined the rate of release of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TXB2) into the suffusate. Verapamil decreased the baseline values of 6-keto-PGF1 alpha and increased those of TXB2. Verapamil inhibited the ischemia-reperfusion-induced increase in 6-keto-PGF1 alpha but did not alter the effect of ischemia-reperfusion on TXB2. Our main results demonstrate the effectiveness of verapamil in preventing microvascular alterations leading to increased leakage of macromolecules.     

生地-山茱萸对糖尿病大鼠血管的保护作用

目的探讨生地-山茱萸药对干预糖尿病大鼠血管病变的机制。方法高脂饲料喂养,一次性腹腔注射STZ,筛选合格的糖尿病大鼠模型,灌胃给药12 w。12 w末,戊巴比妥钠麻醉后取血,制备血浆和血清。测定血清中一氧化氮(NO)、总-氧化氮合酶(T-NOS)和总超氧化物歧化酶(SOD)活力,放射免疫测定血清中血栓素(TXB2)和前列腺素(6-keto-PGF1α)及血浆中内皮素(ET)含量。结果糖尿病大鼠NO下降,灌胃生地-山茱萸药对组有上升趋势,ET和SOD与之相反。模型组大鼠血清中TXB2升高,用药后,药对组明显下降(P<0.01),6-keto-PGF1α与之相反。结论生地-山茱萸药对可恢复NO/ET和TXA2/PGI2平衡,SOD活力增加,表明清除自由基能力增强,从而保护血管内皮,抑制因糖尿病引起的包括心脏病变、主动脉病变和肾病变在内的多种血管并发症的发生与发展。     

Interaction of L-arginine-methyl ester and Sonic hedgehog in liver ischemia-reperfusion injury in the rats

AIM： To investigate the role of Sonic hedgehog （Shh） on the course of liver ischemia and reperfusion （I/R） in rats, and the interaction between treatment with nitric oxide donor L-Arginine-methyl ester （L-Arg） and up-regulation of Shh expression.
METHODS： A total of 30 male Sprague-Dawley rats weighing 220-240 g were used in this study. Shamcontrol group （G1, n = 10）： a sham operation was performed （except for liver I/R）. I/R-untreated group （G2, n = 10）： rats underwent liver ischemia for 1 h followed by reperfusion for 45 min. I/R-L-Arg group （G3, n = 10）： after performing the same surgical procedure as in group 2, animals were treated with L-Arg. Liver tissues were taken for determination of malondialdehyde （MDA） levels, and biochemical and histological evaluations were made.
RESULTS： Plasma alanine aminotransferase （ALT）, aspartate aminotransferase （AS-T）, lactate dehydrogenase （LDH） and T-glutamyltranspeptidase （GGT） activities were higher in group 2 than in group 3. MDA values and the hepatic injury score decreased in the L-Arg treated group compared to the I/R-untreated group. In group 2, the hepatoo/tes were swollen with marked vacuolization. Group 3 rats showed well-preserved liver parenchyma, with hepatocytes extending from the central vein. The morphology of the hepatocytes and the sinusoidal structures was normal, without any signs of congestion. Mild Shh positive immunostaining was detected in group 2 animals. The expression of immunoreactive cells was increased markedly in liver tissue from I/R-L-Arg rats.
CONCLUSION： Our findings suggest that Shh molecules are critical factors in the pathophysiology of inflammatory liver injury induced by I/R. In addition, NO plays an important role in the immunohistochemical expression of these molecules.     

Effects of Wy14643 on hepatic ischemia reperfusion injury in rats

AIM： To investigate the effects and possible mechanisms of Wy14643 on hepatic ischemiareperfusion （I/R） injury in rats. METHODS： Thirty male Sprague-Dawley rats weighing 220-280 g were randomly divided into five experimental groups： sham group （G1, n = 6）： a sham operation was performed （except for liver I/R）, I/R-untreated group （G2, n = 6）： rats underwent liver ischemia for 90 min followed by reperfusion for 4h; and I/R ＋ Wy14643 groups （G3, G4, G5; n = 6）： after the same surgical procedure as in group 2, animals were pretreated with Wy14643 at the dose of 1, 5 and 10 mg/kg 1 h before ischemia, respectively. Hepatic ischemia-reperfusion （I/R） was induced by clamping blood supply to the left lateral and median lobes of the liver for 90 min, and atraumatic clamp was removed for 4 h reperfusion. Blood samples and liver tissues were obtained at the end of reperfusion to assess serum and hepatic tissue homogenate aminotransferase （ALT）, aspartate aminotransferase （AST）, myeloperoxidase （MPO）, serum interleukin- 1β（IL-1β） and tumor necrosis factor alpha （TNF-α）, as well as activity of superoxide dismutase （SOD） and content of malondialdehyde （MDA） in the hepatic tissue homogenate. RESULTS： Hepatic I/R induced a significant increase in the serum levels of ALT, AST, TNF-α, IL-1β and MPO, as well as the levels of ALT, AST and MDA in the liver tissue homogenate, which were reduced by pretreatment with Wy14643 at the dose of 1, 5 and 10 mg/kg, respectively. The activity of SOD in the liver tissue homogenate was decreased after hepatic I/R, which was enhanced by Wy14643 pretreatment. In addition, serum and liver tissue homogenate ALT and AST in the Wy14643 10 mg/kg group were lower than in the Wy14643 1 mg/kg and 5 mg/kg groups, respectively. CONCLUSION： Wy14643 pretreatment exerts significant protection against hepatic I/R injury in rats. The protective effects are possibly associated with enhancement of anti-oxidant and inhibition inflammation res     

A study of the role of plasma thromboxane B2 and 6-keto-prostaglandin F1 alpha in epidemic hemorrhagic fever

Plasma concentrations of Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) were dynamically measured with radioimmunoassay in 30 patients of epidemic hemorrhagic fever (EHF). It was found that the levels plasma TXB2 significantly increased and 6-keto-PGF1 alpha decreased in EHF patients as compared with those in controls. The more severe the patient's condition, the higher the level of TXB2 and the lower the level of 6-keto-PGF1 alpha. The ratio of TXB2/6-keto-PGF1 alpha was parallel with the severity of the patient's condition. Plasma TXB and TXB2/6-keto-PGF1 alpha ratio increased significantly in the hemorrhagic and shock group, while 6-keto-PGF1 alpha decreased significantly in the shock group. The results showed that there is a distinct imbalance of TXA2-PGI2 mediated through the increase of TXA2 and decrease of PGI2 in EHF. The imbalance of TXA2-PGI2 participates in the pathogenesis and pathophysiology of hemorrhage, shock and renal dysfunction.     

中、老年男性糖尿病阳萎患者血小板功能、血栓素B_2和6-酮-前列腺素F_(1α)的变化

测定32例糖尿病阳萎者和11例无阳萎者血小板最大聚集率(MAR)、血栓素B_2(TXB_2)和6-酮-前列腺素F_(1α)(6-keto-PGF_(1α)),并与正常人对照。结果表明,糖尿病有无阳萎组MAR、TXB_2及TXB_2/6-keto-PGF_(1α)比值均高于正常组(P<0.05～0.01),而6-keto-PGF_(1α)水平低下(P<0.01);糖尿病阳萎组这些指标变化更为明显;与无阳萎组比较,阳萎组MAR及TXB_2/6-keto-PGF_(1α)比值升高(P<0.05～0.01),6-keto-PGF_(1α)水平低下(P<0.05),而TXB_2则无明显变化。提示中老年男性糖尿病阳萎者血小板聚集释放功能亢进并与血栓素和前列环素平衡失调有关,从而促进阴茎血液高凝和阳萎的发生。     

Production of thromboxane A2 and prostaglandin i2 affected by interaction of heat aggregated IgG,endothelial cells,and platelets in lupus nephritis

OBJECTIVE: To examine the role of immune complexes in the prostanoid metabolism of glomerular capillary endothelial cells (EC) and platelets in lupus nephritis. Heat aggregated IgG (HA-IgG), instead of immune complexes, was incubated using an in vitro coculture system with human umbilical vein EC, instead of glomerular capillary EC, and platelets. The effect of complement component C1q and a novel imidazole-type thromboxane A2 (TXA2) synthetase inhibitor, DP-1904, on this prostanoid metabolism change was also investigated. METHODS: EC monolayers (1.5x10(5) cells/well) were incubated with various concentrations of HA-IgG, monomeric IgG, or medium alone for 1 h at 37 degrees C, and then incubated with platelet suspensions (1x10(8) cells/ml) for various times. Concentrations of TXB2 and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable hydrolysis products of TXA2 and prostaglandin I2 (PGI2), respectively, released in the supernatants were measured by ELISA. RESULTS: HA-IgG bound to EC monolayers produced TXB2 and 6-keto-PGF(1alpha) in a concentration dependent manner and much more than monomeric IgG or medium alone did. However, the production of 6-keto-PGF(1alpha) stimulated with HA-IgG was much lower than that of TXB2, indicating a large imbalance between TXA2 and PGI2. Preincubation of HA-IgG with purified C1q partially suppressed the production of TXB2, but not that of 6-keto-PGF(1alpha). DP-1904 suppressed the production of TXB2 completely, but by sharp contrast, it dramatically increased the production of 6-keto-PGF(1alpha) from EC and platelets by HA-IgG. CONCLUSION: The large imbalance of TXA2 and PGI2 produced by the interaction of EC, immune complexes, and platelets may be associated with alterations in glomerular pathological findings and hemodynamics mediated by immune complexes in lupus nephritis. C1q and a TXA2 synthetase inhibitor may improve the abnormal prostanoid metabolism change of lupus nephritis.     

Thromboxane analyses in patients with chronic inflammatory bowel diseases

Prostanoids are important for the pathogenesis of chronic inflammatory bowel diseases as mediators of inflammatory, immune and allergic reactions. The levels of thromboxane B2(TXB2), the stable hydrolysis product of thromboxane A2(TXA2) were determined in blood plasma of patients with chronic inflammatory bowel diseases. The platelet malondialdehyde (MDA) formation was determined as an indicator of the TXA2 synthetase activity. The TXB2 concentrations were measured radioimmunologically. The platelet MDA formation induced by N-ethylmaleimide was investigated with the thiobarbituric acid reaction. The investigated patients (n = 10) suffering from ulcerative colitis had a significant increasing (p less than 0.02) of the platelet MDA formation (mean = 4.39 nmol/10(9) platelets) in comparison to the normal group (n = 20; mean = 2.87; nmol/10(9) platelets). The increasing of TXB2 levels was not significantly different than in normal control subjects. The plasma concentrations of 6-keto-PGF1 were situated on the limit of detection.