Sulodexide   中文名称: 舒洛地特

Antithrombotic; antithrombin III (AT III); heparin cofactor II (HC II)

舒洛地特降低肝素酶III诱导的MLMECs中SDC1脱落[1]
SDC1是主要表达于细胞表面的跨膜蛋白聚糖小家族成员，也是内皮糖萼降解的主要标志物。为减少LPS刺激对内皮细胞炎症反应的影响，我们使用肝素酶III作为糖萼特异性水解剂来探究糖萼脱落对内皮细胞的作用。虽然根据肝素酶III在硫酸乙酰肝素分子上的预期作用位点推测其不会引起SDC1脱落，但通过添加细菌肝素酶III减少硫酸乙酰肝素含量可显著增加SDC1脱落。舒洛地特是由具有抗炎特性的硫酸乙酰肝素和硫酸皮肤素组成的糖胺聚糖，能减少巨噬细胞释放LPS刺激的炎症介质。作为舒洛地特主要成分的肝素及其衍生物，被认为可结合急性期蛋白、补体蛋白、细胞因子和生长因子。此外，舒洛地特通过调节丝氨酸蛋白酶、金属蛋白酶以及参与糖萼脱落的基质金属蛋白酶发挥多种抗蛋白水解作用。

为测试舒洛地特对内皮糖萼层的影响，我们在肝素酶III存在条件下培养MLMECs和HUVECs。如预期所示，肝素酶III去除了内皮表面的硫酸乙酰肝素（图S2A,B）。值得注意的是，肝素酶III给药还诱导了MLMECs膜上SDC1的脱落，而补充舒洛地特在不同时间点均呈现显著挽救效果（图2A-C）。在HUVECs中也发现相同结果（图S2C,D）。随后我们评估舒洛地特是否通过上调SDC1基因表达来增加MLMECs中SDC1水平。因此在这些条件下测量了SDC1 mRNA表达，但发现不同处理组间SDC1 mRNA表达无差异（图S2E）。这些数据表明舒洛地特能改善肝素酶III诱导的SDC1表达，但不是通过增加SDC1基因表达实现。

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舒洛地特通过增强ZO-1表达防止内皮通透性增加[1]
使用Transwell系统评估舒洛地特对内皮细胞（EC）屏障功能的影响（图3A）。发现SDX可降低肝素酶III诱导的内皮通透性（图3B）。舒洛地特同样能预防肝素酶诱导的HUVECs内皮通透性增加（图S3A）。值得注意的是，与对照组相比SDX组的通透性有所改善。VE-cadherin和ZO-1是维持EC屏障功能的重要组分（24,25）。我们旨在确定SDC1脱落导致的内皮屏障功能障碍是否与ZO-1和VE-cadherin相关。Western blot分析显示，肝素酶III处理2h或4h后VE-cadherin和ZO-1水平显著降低（图3C,D）。有趣的是，补充舒洛地特给药上调了肝素酶III诱导的ZO-1水平而非VE-cadherin水平（图3C,D；S3B,C）。这些数据共同表明舒洛地特可通过增加ZO-1表达来预防糖萼脱落诱导的内皮通透性增加。

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舒洛地特通过阻断NF-κB信号激活改善通透性[1]
在不同病理生理条件下，NF-κB作为内皮细胞中的转录因子在炎症表型变化中起重要作用。糖萼破坏后，剪切应力导致ICAM-1蛋白表达上调和NF-κB激活增强。但尚不清楚肝素酶III降解糖萼时ZO-1的表达是否由NF-κB通路介导。为此我们评估了NF-κB/p-p65和总NF-κB/p65水平。在MLMECs/HUVECs中，磷酸化p65水平在肝素酶处理15/30分钟内显著增加，而添加舒洛地特后降低（图S4A,B）。舒洛地特给药使MLMECs/HUVECs中肝素酶处理后的磷酸化p65减少（图4A,B）。同样，NF-κB抑制剂Bay 11-7082给药显著减弱肝素酶III诱导的磷酸化p65增加，并提升ZO-1表达（图4C-F）。这些结果共同揭示：当肝素酶III降解MLMECs/HUVECs中糖萼时，NF-κB信号激活参与调控ZO-1表达。值得注意的是，舒洛地特可通过阻止NF-κB/ZO-1信号过度激活来促进糖萼重塑并改善通透性（图4G）。

舒洛地特抑制SDC1脱落改善脓毒症小鼠肺损伤并防止死亡[2]
我们采用CLP和LPS建立脓毒症模型（图5A）。首先检测了脓毒症小鼠血浆中SDC1水平，发现SDC1在脓毒症小鼠中表达上调。使用舒洛地特后，SDC1水平下调且脓毒症小鼠存活率提高（图5B-E）。但舒洛地特治疗未降低脓毒症期间IL-6水平（图5B,D）。这些数据表明舒洛地特能恢复存活率并降低脓毒症小鼠血浆中SDC1表达。
我们评估了小鼠肺部组织学特征。值得注意的是，LPS/CLP组的肺组织出现显著损伤，与对照组差异明显。相比LPS/CLP组，LPS+SDX/CLP+SDX组的形态学特征有显著改善（图5F,S5A）。通过测量肺组织湿干重比（W/D）评估肺血管通透性。结果显示舒洛地特显著抑制了LPS/CLP引起的肺组织W/D比升高（图5G,S5B）。最后通过肺组织荧光标记检测SDC1表达来评估糖萼损伤。脓毒症模型中SDC1表达低于对照组，而舒洛地特帮助维持了脓毒症模型肺组织中SDC1表达（图5H,I,S5C）。这些数据共同表明舒洛地特能预防小鼠肺损伤和脓毒症诱导的内皮糖萼脱落。

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DN小鼠随时间进展出现进行性蛋白尿和肾功能恶化，伴随系膜扩张、PKC与ERK激活、肾脏TGF-β1/纤连蛋白/I/III/IV型胶原表达增加，但glomerular perlecan表达降低。舒洛地特治疗显著减少蛋白尿、改善肾功能、增加glomerular perlecan表达并降低I/IV型胶原表达及ERK激活。肾小球内PKC-α激活不受舒洛地特影响，而纤连蛋白和III型胶原表达增加。30mM D-葡萄糖刺激的MMC显示PKC和ERK介导的纤连蛋白/III型胶原合成增加。单独使用舒洛地特可剂量依赖性显著增加MMC中纤连蛋白/III型胶原合成，且在30mM D-葡萄糖存在时增强此效应。舒洛地特对30mM D-葡萄糖诱导的PKC-βII和ERK磷酸化呈剂量依赖性抑制，但不影响PKC-α或PKC-βI磷酸化。
结论：数据显示虽然舒洛地特治疗能减少蛋白尿并改善肾功能，但对DN模型C57BL/6小鼠肾脏信号通路和基质蛋白合成产生差异调节[3]。

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舒洛地特是由肝素和硫酸皮肤素组成的混合糖胺聚糖。本研究采用氧诱导视网膜病变（OIR）小鼠模型评估其抗血管生成效应。假注射OIR小鼠（P17）视网膜存在特征性中央无灌注区，而舒洛地特注射组该区域显著减小。通过SWIFT_NV测量的新生血管簇数量和平均新生血管管腔数在舒洛地特组显著降低。高压氧暴露导致VEGF、MMP-2和MMP-9水平升高，而舒洛地特治疗呈剂量依赖性降低这些因子水平。结果明确证实舒洛地特的抗血管生成作用，提示其可作为涉及新生血管形成的眼部病变辅助治疗候选药物[4]。

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舒洛地特是具有广泛药理活性的类肝素化合物，但其对肝纤维化的作用尚未见报道。本研究旨在评估舒洛地特对肝纤维化小鼠模型的治疗潜力并探索其抗纤维化机制。发现舒洛地特显著减轻TAA和DDC诱导的小鼠肝纤维化。转录组分析显示舒洛地特下调纤维化相关基因和肝窦内皮细胞（LSECs）毛细血管化相关基因。免疫组化证实纤维化肝组织中LSEC毛细血管化相关基因（CD34/CD31/Laminin）的表达升高被舒洛地特降低。扫描电镜显示舒洛地特能维持LSECs窗孔结构。qPCR和免疫荧光显示间充质标志物表达被舒洛地特下调，提示其抑制肝纤维化中LSECs的内皮-间质转化。体外实验证实舒洛地特保护原代LSECs免受内皮功能障碍。综上，舒洛地特通过恢复LSECs分化表型减轻小鼠肝纤维化，表明其可能成为肝纤维化患者的潜在疗法[5]。

细胞处理条件[2]
MLMECs被分为四组：（i）对照组；用无血清培养基处理细胞2小时；（ii）肝素酶III组；细胞用15mU/mL肝素酶III处理2小时、4小时或8小时，（III）SDX/Sulodexide组；细胞用30LSU/mL SDX处理2小时和（iv）肝素酶III+SDX组；用30 LSU/mL SDX预处理细胞2小时，然后用15 mU/mL肝素酶III预处理2小时、4小时或8小时。

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内皮屏障通透性评价[2]
使用Transwell系统（0.4μm孔径的聚酯膜插入物）培养MLMECs。通过测量内皮上的FD40来评估内皮屏障的通透性。MLMEC用或不用肝素酶III（15 mU/mL）或SDX/Sulodexide（30 LSU/mL）处理。我们向上部插入物中加入0.1 mg/mL的FD40，并向Transwell系统的下部隔室中加入等量的无血清培养基60分钟。分别在490和520 nm的激发和发射波长下测量上部插入物的荧光。

Endotoxemia model [2]
The mice were randomly allocated to four groups (n = 5/experiment): LPS+SDX, LPS, Sulodexide/SDX, and control. Within the groups, mice were injected intraperitoneally (ip.) with LPS (30 mg/kg body weight/mouse) and/or treated intragastrically (ig.) with Sulodexide (40 mg/kg/mouse). Equal amounts of saline or Sulodexide were injected into the control mice or mice in the SDX group. In the survival experiment, we recorded the mortality in each group three times a day for 120 h after LPS injection. For general anesthesia, 1% tribromoethanol was administered, and the mice were sacrificed 12 h later. Blood and lung samples were collected from the surviving mice. CLP-induced polymicrobial sepsis model [2]
We randomly grouped the mice into four (n = 5/experiment): control, CLP, SDX/Sulodexide, and CLP+SDX. The CLP-induced sepsis model was developed based on previous literature. Briefly, after anesthesia with 1% tribromoethanol, laparotomy was performed. Cecum was ligated with 4-0 silk to 1 cm and punctured with a 22-gauge needle. Subsequently, a small mound of feces was squeezed from the hole after removing the needle. The peritoneum was sutured with a 6-0 silk suture, and the skin was intermittently sutured with a 4-0 silk suture. The same operation was performed on the control mice without ligation and perforation. The CLP mice were and/or intragastric (ig.) sulodexide (40 mg/kg). An equivalent volume of normal saline (NS) was injected into the control mice. After surgery, all the mice were subcutaneously resuscitated in 40 mL/kg saline. All the mice were sacrificed 24 h later. Retro-orbital blood and lung samples were collected from the surviving mice.

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Male C57BL/6 mice were rendered diabetic with streptozotocin. After the onset of proteinuria, mice were randomized to receive Sulodexide (1 mg/kg/day) or saline for up to 12 weeks and renal function, histology and fibrosis were examined. The effect of Sulodexide on fibrogenesis in murine mesangial cells (MMC) was also investigated.
Male C57BL/6 mice at 6–8 weeks of age were fasted for 6 h prior to intra-peritoneal injection of streptozotocin (STZ, 50 mg/kg) in 10 mM citrate buffer, pH 4.5, administered on five consecutive days. Diabetes mellitus was confirmed by tail vein blood sampling of glucose concentration, measured with Accu-Chek Advantage II Glucostix test strips. Spot urine was tested weekly for albuminuria with QuantiChrom albumin assay kit until sacrifice. Mice with elevated blood glucose levels (>10 mM) and albuminuria (>100 mg/dl) on two separate occasions two days apart (defined as ‘baseline’ in the animal studies) were randomized to receive treatment with either saline (vehicle control) or sulodexide (1 mg/kg/day) by oral gavage for 2, 4, 8 or 12 weeks (6 mice per time-point for each group). After 2, 4, 8 and 12 weeks of treatment, mice were sacrificed, blood samples were obtained by cardiac puncture and the kidneys harvested, decapsulated and weighed. The left kidney was cut perpendicular to the long-axis and one half of the kidney was snap frozen in OCT followed by immersion in liquid nitrogen, while the second half was fixed in 10% neutral-buffered formalin followed by paraffin embedding. Renal cortical tissue from the right kidney was separated from the medulla and frozen at −80°C until mRNA isolation. Six diabetic mice that had just developed proteinuria were also sacrificed to obtain baseline values for clinical, histological and morphometrical parameters. Negative control groups included non-diabetic male C57BL/6 mice treated with either saline or sulodexide for 12 weeks. Serum creatinine and urea levels were measured using QuantiChrom creatinine and urea assay kits respectively. [3]

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Oxygen-induced retinopathy in mice [4]
ICR pups were randomly divided into three groups: a normoxia group (control group), an oxygen-exposed group (OIR group), and a Sulodexide group; each group had one nursing mother and 5-7 pups. Oxygen-induced retinopathy was induced in ICR pups, as described previously. For the OIR model, the newborn pups were transferred at post-natal day (P) 7 along with their mother to a chamber supplied with 75 ± 2% oxygen, under continual monitoring with a ProOx 110 oxygen controller for 120 h. On P12, the mice were returned to the room air and given daily intraperitoneal (IP) injections of vehicle (saline) or 5-15 mg/kg of sulodexide dissolved in the vehicle. The mice in the normoxia group were maintained in room air from birth until P17.

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Mouse models of liver fibrosis [5]
Liver fibrosis was induced by thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet. For the TAA model, mice were supplied with 400 mg/L TAA in drinking water for 16 weeks to establish the liver fibrosis mouse model. Sulodexide, known as Vessel Due F, was administrated to the mice by gastric gavage. Mice were randomly assigned to each of three groups (with six mice per group): (a) the control group, (b) the TAA + vehicle group, and (c) the TAA + sulodexide (SDX) group. All animals received food and water ad libitum. For the DDC model, mice were fed a diet containing 0.12 % w/w DDC for four weeks to induce liver fibrosis with cholangitis.

[1]. Andreozzi GM. Sulodexide in the treatment of chronic venous disease. Am J Cardiovasc Drugs. 2012 Apr 1;12(2):73-81.

[2]. Sulodexide improves vascular permeability via glycocalyx remodelling in endothelial cells during sepsis. Frontiers in immunology, 2023, 14: 1172892.

[3]. Sulodexide decreases albuminuria and regulates matrix protein accumulation in C57BL/6 mice with streptozotocin-induced type I diabetic nephropathy. PLoS One. 2013;8(1):e54501.

[4]. Sulodexide inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy. BMB reports, 2014, 47(11): 637.

[5]. Sulodexide attenuates liver fibrosis in mice by restoration of differentiated liver sinusoidal endothelial cell. Biomedicine & Pharmacotherapy, 2023, 160: 114396.

Background: Degradation of the endothelial glycocalyx is critical for sepsis-associated lung injury and pulmonary vascular permeability. We investigated whether Sulodexide, a precursor for the synthesis of glycosaminoglycans, plays a biological role in glycocalyx remodeling and improves endothelial barrier dysfunction in sepsis. Methods: The number of children with septic shock that were admitted to the PICU at Children's Hospital of Fudan University who enrolled in the study was 28. On days one and three after enrollment, venous blood samples were collected, and heparan sulfate, and syndecan-1 (SDC1) were assayed in the plasma. We established a cell model of glycocalyx shedding by heparinase III and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Sulodexide was administrated to prevent endothelial glycocalyx damage. Endothelial barrier function and expression of endothelial-related proteins were determined using permeability, western blot and immunofluorescent staining. The survival rate, histopathology evaluation of lungs and wet-to-dry lung weight ratio were also evaluated.
In summary, this study demonstrated a previously unknown role of SDC1 in prognosis for children with septic shock and promoting vascular permeability by inducing ZO-1 disruption mediated by NF-κB dependent signaling in ECs. Sulodexide administration may thus serve as a helpful treatment in sepsis by attenuating glycocalyx shedding and downstream EC signaling that promotes vascular leakage. [2]

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SSulodexide is a mixture of glycosaminoglycans that may reduce proteinuria in diabetic nephropathy (DN), but its mechanism of action and effect on renal histology is not known. We investigated the effect of sulodexide on disease manifestations in a murine model of type I DN.
In conclusion, we have demonstrated that Sulodexide treatment reduced albuminuria, improved serum levels of urea, restored perlecan expression and ameliorated selective renal histopathologic changes in male C57BL/6 DN mice that included reduced collagen type I and IV deposition, and ERK and PKC-βII activation. In contrast, sulodexide had no effect on PKC-α or PKC-βI activation, but increased glomerular but not tubulo-interstitial deposition of fibronectin and collagen type III. It is possible that an increase in glomerular expression of these matrix proteins and an inability to suppress PKC-α or PKC-βI activation during progressive disease may explain at least in part, why sulodexide showed no efficacy in recent clinical studies although further studies are warranted to confirm this. Whether sulodexide can provide renoprotection in sub-populations of DN patients with specific histopathology remains to be determined. [3]

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In conclusion, Sulodexide was shown to be effective, at least in part, in inhibiting retinal neovascularization in a mouse model of retinopathy. Sulodexide acts by inhibiting pro-angiogenic proteins including VEGF, MMP-1, and MMP-9. Because pathologic angiogenesis following retinal ischemia is one of the leading causes of blindness, drugs with low toxicity and potent anti-angiogenic activity are urgently needed. Our results show that sulodexide fits these criteria and can be suggested as a supplementary compound in the treatment of ocular pathologic angiogenesis. Detailed information on the pharmacokinetics of this substance in the eye will require further investigation. [4]

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In summary, we provided evidence for the efficacy of Sulodexide in treating two mouse models of liver fibrosis; sulodexide treatment ameliorated liver fibrosis in both TAA- and DDC-induced mouse models. Mechanistic studies revealed that sulodexide downregulated fibrosis-related signaling pathways and LSEC capillarization-related genes. Furthermore, sulodexide treatment preserved LSEC fenestrae and inhibited capillarization and EndMT in fibrotic livers. Consistent with these findings in vivo, sulodexide administration minimized LSEC capillarization and EndMT progression in primary LSECs in vitro. This suggested that the anti-fibrotic activity of sulodexide occurred through restoration of differentiated LSECs. Our findings provide the first insights into a potential mechanism of action for orally-administrated sulodexide in liver fibrosis. [5]

57821-29-1

Colorless to light yellow Solid-Liquid Mixture

114.55

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。