姜黄素抗肝窦毛细血管化作用及机制

doi:10.3969/j.issn.2095-4344.0144

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (8): 1247-1252.doi: 10.3969/j.issn.2095-4344.0144

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇下一篇

姜黄素抗肝窦毛细血管化作用及机制

段雪琳，彭岳，赵铁建，韦燕飞，黎桂玉

广西中医药大学基础医学院人体机能系，广西壮族自治区南宁市 530200

出版日期:2018-03-18 发布日期:2018-03-18
通讯作者: Zhao Tie-jian, Department of Medical Physiology and Biochemistry, School of Basic Medical Science, Guangxi University of Chinese Medicine, Nanning 530200, Guangxi Zhuang Autonomous Region, China
作者简介:段雪琳，女，1977年生，汉族，2010年广西中医药大学毕业，硕士，副教授。并列第一作者：彭岳，男，1981年生，汉族， 2016年成都中医药大学毕业，博士，副教授。
基金资助:
国家自然科学基金项目(81403189，81460628，81660705，81560690)；广西教育厅高教科研项目(YB2014182)

Effect of curcumin against capillarization of hepatic sinusoids and its mechanism

Duan Xue-lin, Peng Yue, Zhao Tie-jian, Wei Yan-fei, Li Gui-yu

Department of Medical Physiology and Biochemistry, School of Basic Medical Science, Guangxi University of Chinese Medicine, Nanning 530200, Guangxi Zhuang Autonomous Region, China

Online:2018-03-18 Published:2018-03-18
Contact: 赵铁建。广西中医药大学基础医学院人体机能系，广西壮族自治区南宁市 530200
About author:Duan Xue-lin, Master, Associate professor, Department of Medical Physiology and Biochemistry, School of Basic Medical Science, Guangxi University of Chinese Medicine, Nanning 530200, Guangxi Zhuang Autonomous Region, China Peng Yue, M.D., Associate professor, Department of Medical Physiology and Biochemistry, School of Basic Medical Science, Guangxi University of Chinese Medicine, Nanning 530200, Guangxi Zhuang Autonomous Region, China Duan Xue-lin and Peng Yue contributed equally to this work.
Supported by:
the National Natural Science Foundation of China, No. 81403189, 81460628, 81660705 and 81560690; Scientific Research Project of Universities of Education Department of Guangxi Zhuang Autonomous Region, No. YB2014182

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
肝窦内皮细胞：是构成肝窦最主要的细胞，其具有特有的、密集的窗孔结构，使物质能从血液进入肝实质并在两者间自由交换。肝窦内皮细胞与其他内皮细胞不同，它没有基底膜结构，这些独特的结构使得其在物质交换过程以及肝纤维化形成发展中发挥重要作用。
肝窦毛细血管化：是一种以肝窦内皮细胞窗孔数量减少及连续性基底膜形成，肝内微循环障碍为主要特征的病理过程，其长期发展可导致肝纤维化。

摘要
背景：姜黄素对肝纤维化及肝硬化过程中必然发生的、可导致门脉高压的特征性病变“肝窦毛细血管化”是否有干预能力，其作用机制如何，目前尚不明确。
目的：观察姜黄科植物药用活性成分姜黄素对肝窦内皮细胞微观结构及分泌功能的影响，及其干预肝窦毛细血管化及抗肝纤维化的药理作用机制和靶位点。
方法：培养大鼠肝窦内皮细胞，实验分7组：空白对照组无干预措施，瘦素激活组建立瘦素激活细胞模型；姜黄素高、中、低剂量组为瘦素激活细胞后分别以32，16，8 μmol/L姜黄素干预；最后2组为瘦素激活细胞后分别以秋水仙碱和丹参酚酸B作为干预的阳性对照药物组；各组均干预48 h。
结果与结论：①扫描电镜及透射电镜观察显示，瘦素激活程度越高，肝窦内皮细胞的窗孔数量越少，孔径越小。姜黄素能够干预被瘦素缩小、消失了的窗孔，使其发生明显增多和增大，并使细胞外基底膜变薄及范围减小，降低肝窦内皮毛细血管化的程度，并且姜黄素干预肝窦内皮毛细血管化能力有剂量依赖性；②实时定量PCR和ELISA法检测显示，瘦素激活后，肝窦内皮细胞的内皮素1和血管内皮生长因子的mRNA水平及蛋白表达水平较正常对照组相比均出现明显增高(P < 0.05)。而上述情况在姜黄素高、中、低剂量组的肝窦内皮细胞中均出现了明显降低(P < 0.05)；而且随着姜黄素给药浓度上升，各蛋白的表达水平均出现了梯度下降(P < 0.05)。而且高剂量的姜黄素对内皮素1和血管内皮生长因子的转录和表达的干预强度大于秋水仙碱和丹参酚酸B组(P < 0.05)；③结果证实，姜黄素能明显降低肝窦内皮毛细血管化程度，从而干预肝纤维化进程，其机制可能与姜黄素下调内皮素1和血管内皮生长因子的表达有关。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程
orcid: 0000-0002-2481-9363(Duan Xue-lin)

关键词: 姜黄素, 肝窦内皮细胞, 组织工程, 肝窦毛细血管化, 窗孔, 基底膜, 分泌功能, 瘦素, 内皮素1, 血管内皮生长因子, 抗纤维化

Abstract:

BACKGROUND: Capillarization of hepatic sinusoids is an inevitable part in liver fibrosis and cirrhosis, and is a characteristic lesion inducing portal hypertension. However, curcumin effects on the capillarization of hepatic sinusoids and the underlying mechanism remain unclear.
OBJECTIVE: To observe the effect of curcumin (a natural polyphenolic compound derived from the rhizome of Curcuma longa) on the microstructure and secretion of hepatic sinusoidal endothelial cells (HSECs), and to further explore its intervention on sinusoidal capillarization and pharmacological action mechanism of anti-liver fibrosis and target sites.
METHODS: The rat HSECs were cultured and divided into seven groups: blank control group received no intervention and cells in the other groups were activated by leptin, followed by treatment with nothing (model group), high-, medium- and low-dose of curcumin, colchicine and salvia miltiorrhiza phenolic acid B, respectively, for 48 hours.
RESULTS AND CONCLUSION: Under scanning and transmission electron microscopes, with the increasing activation of leptin, the number of fenestrae in HSECs was increased and the aperture was decreased. Curcumin could increase and enlarge narrowed or disappeared fenestrae caused by leptin, attenuated the thickness and scope of extracellular basement membrane, and reduced the degree of capillarization of hepatic sinusoids in a dose-dependent manner. Real-time PCR and ELISA results showed that after activation of leptin, mRNA and protein expression levels of endothelin-1 and vascular endothelial growth factor in HSECs were significantly increased compared with the blank control group (P < 0.05), while the expressions showed a significant decrease after treatment with curcumin in a dose-dependent manner (P < 0.05). There was also a gradient reduction in the protein expression of endothelin-1 and vascular endothelial growth factor in HSECs treated with curcumin. Moreover, all above mRNA and protein expression levels in the high-dose curcumin group were significantly lower than those in the colchicine and salvia miltiorrhiza phenolic acid B groups. In summary, curcumin can significantly alleviate the sinusoidal capillarization, and thus delay the development of liver fibrosis, probably by down-regulating the expression levels of endothelin-1 and vascular endothelial growth factor.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Tissue Engineering, Curcumin, Liver

中图分类号:

R318

段雪琳，彭岳，赵铁建，韦燕飞，黎桂玉. 姜黄素抗肝窦毛细血管化作用及机制[J]. 中国组织工程研究, 2018, 22(8): 1247-1252.

Duan Xue-lin, Peng Yue, Zhao Tie-jian, Wei Yan-fei, Li Gui-yu. Effect of curcumin against capillarization of hepatic sinusoids and its mechanism[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(8): 1247-1252.

图/表（结果） 2

Ultrastructural changes in the HSECs

The results of scanning electron microscope showed that the cell wall of HSECs in the blank control group was mesoporous, with abundant fenestrae of uneven size (the large diameter was about 1 μm and the small one was about 0.1 μm), especially the large fenestrae (Figure 1A). The cell wall of HSECs in the leptin group showed less or none fenestrae, and its shape changes from honeycomb to flat plate (Figure 1B). Compared with the leptin group, the number of fenestrae in HSECs in the high-dose curcumin group was significantly increased, and the sinus wall returned to mesoporous shape with more large fenestrae (the diameter > 1 μm) (Figure 1C). The number of fenestrae in the medium-dose curcumin group was more than that in the leptin group, but lower than those in the high-dose curcumin group (Figure 1D). There was no significant difference in the number of fenestrae between low-dose curcumin and leptin groups. Compared with the medium- and high-dose curcumin groups, in the low-dose curcumin group, the number of fenestrae was less and with smaller diameter (Figure 1E).The number of fenestrae in the HSEC wall in the colchicines and salvianolic acid B groups was obviously more than that in the blank control and low-dose curcumin group, and the size of the fenestrae was larger, but compared with the medium- and high-dose curcumin groups, the number and size of fenestrae were reduced. The number and size of fenestrae did not differ significantly between colchicines and salvianolic acid groups (Figure 1F, G).

Results of transmission electron microscope revealed that there were a large number of hollow light-colored areas in the HSECs of the blank control group, and the fenestrae vwere dense and large in area, the monolayer cell membrane was thin, without basement membrane formation (Figure 2A). In the leptin group, the light-translucent area in HSECs almost disappeared, the number and area of fenestrae were reduced, the thickened cell membrane showed two-to-three layers, and the light-colored basement membrane area was obvious and continuous (Figure 2B). In the high-dose curcumin group, compared with the leptin group, the light translucent area within the HSECs increased significantly, the number and area of visible fenestrae were also increased, hollow-intensive sinus wall was almost sieve-shaped; thin membrane was observed, and the light-colored basement membrane area was formed outside the wall (Figure 2C). In the medium-dose curcumin group, compared with the leptin group, the light translucent area within the HSECs was enlarged, with a large number of fenestrae; two layers of the cell membrane could be still observed, but the light-colored basement membrane outside the cell wall was unobvious. However, compared with the high-dose curcumin group, the intracellular light-colored fenestrae area was smaller, and the extracellular basement membrane area existed (Figure 2D). Compared with the leptin group, in the low-dose curcumin group, the light-colored translucent area in the HSECs increased, the number of fenestrae increased; two distinct layers of the cell membrane were visible, obvious light-colored basement membrane area appeared outside the cell wall. Compared with the medium- and high-dose curcumin groups, the light-colored fenestrae area in the cells of the low-dose curcumin group was obviously smaller, and the light-colored basement membrane area was clearly seen (Figure 2E).

Compared with the leptin group, in the colchicine and salvianolic acid B groups, the light translucent area in the HSECs was significantly expanded, with increased number and area of fenestrae; the cell membrane was thinned, and it was hard to detect the two layers, and the light-colored basement membrane area was nearly observed outside the cell wall. There were no significant differences in the number and area of fenestrae, the thickness of the cell membrane and the basement membrane outside the cell wall between two control drug groups. Compared with the medium-and high-dose curcumin groups, the number and area of fenestrae in the two drug control groups were lower, and the formation of extracellular basement membrane was more obvious. Compared with the low-dose curcumin group, the light-colored fenestrae area in the two drug control groups was larger with unobvious cell membrane thickening, and unclear basement membrane (Figure 2F, G).

Gray scale results of the HSECs transmission electron microscope images showed that the gray scale score of HSECs in the high-dose curcumin group was the highest (P < 0.05; Table 2).

mRNA and protein expression levels of endothelin-1 and VEGF in each group

RT-PCR and EILSA results are shown in Tables 3, 4.

Compared with the blank control group, mRNA and protein expression levels of endothelin-1 and VEGF in HSECs were significantly increased in the other groups (P < 0.05), especially in the leptin group (P < 0.01). mRNA and protein expression levels of endothelin-1 and VEGF in the colchicines and salvianolic acid B groups were significantly lower than those in the leptin group (P < 0.01), suggesting that colchicines and salvianolic acid B could significantly inhibit the expression of endothelin-1 and VEGF. Compared with leptin group, the mRNA and protein expression levels of endothelin-1 and VEGF in each curcumin group were significantly reduced in a dose-dependent manner (P < 0.05). Moreover, the mRNA and protein expression levels of endothelin-1 and VEGF in the high-dose curcumin group were significantly lower than those in the colchicines and salvianolic acid B groups (P < 0.05), indicating that the inhibitory effect of high-dose curcumin was superior to colchicines and salvianolic acid B.

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引言

Liver fibrosis is a chronic, progressive and diffused phenomenon but not an independent disease, which is a continuous course of disease, and almost occurs in all types of chronic liver diseases^[1]. Intervention or reverse of liver fibrosis is a crucial issue in the treatment of chronic liver diseases^[2]. In the development of liver fibrosis, the decreased activities of plasminogen activator and plasmin down-regulate the matrix metalloproteinase activity, causing the formation of a continuous basement membrane between hepatic sinusoidal endothelial cells (HSECs) and hepatocytes accompanied with defenestration and HSEC activation^[3]. This process is termed the capillarization of the hepatic sinusoid, which is inevitable in liver fibrosis and cirrhosis progression^[4]. The sinusoidal capillarization of the HSECs precedes obvious inflammation in the early stage and promotes liver fibrosis progression^[5]. Defenestration of HSECs has been reported to occur before liver fibrosis, which can be reversed after risk factors are avoided, but is irreversible after a continuous basement membrane forms^[6]. Pathological activation of HSECs will secret abundant endothelin-1 and vascular endothelial growth factor (VEGF)^[7]. Endothelin-1-induced contraction of microvessel wall and HSECs^[8], as well as VEGF-induced vascular endothelial cell proliferation, increase in vascular permeability, changes in the extracellular matrix components and extravascular matrix deposition^[9] are directly involved in the capillarization of the hepatic sinusoid and the development of portal hypertension.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Curcumin is mainly derived from the tuber of Curcuma aromatica, rhizomes of Curcuma longa and Zedoary, which teem in Guangxi area, China, and other Zingberaceae^[10].

Curcumin has been found to inhibit the inflammatory reaction of liver, and hold antioxidant and anti-rheumatoid effects^[11]. In addition to hangover, it also exhibits anti-inflammation, anti-pathogenic microorganisms, anti-oxidation, hypolipidemic effect, hepato-protection, cardiovascular and cerebrovascular protection, anti-atherosclerosis, anti-aging, inhibit tumor growth, and other pharmacological actions^[12]. Among these properties, its liver protection has been widely used in the clinical practice, and has achieved remarkable treatment outcomes^[13].

In this study, we firstly observed and explored the changes in the number and size of fenestrae in HSECs after activation of leptin, as well as the formation of extracellular basement membrane, and then curcumin was added to observe its effect on the leptin-reduced or -closed number and size of fenestrae thus determining its protection against the sinusoidal capillarization; further detected the transcription, translation and expression levels of endothelin-1, and VEGF before and after curcumin intervention in order to understand the underling pharmacological mechanisms.

材料方法

MATERIALS AND METHODS

Design

Cytological experiment.

Time and setting

This study was finished from April 2015 to March 2016, at the Comprehensive Laboratory of Basic Medical Innovation of Guangxi University of Chinese Medicine.

Materials

HSECs (Shanghai Biosai Biotech Co., Ltd., China); Curcumin powder (Shanghai Yuanye Biological Co., Ltd., China, No. 20141024); Leptin (Shanghai Guduo Biotech Co., Ltd., China, No. 141003).

Methods

Preparation of curcumin solution

98% curcumin powder was dissolved in 95% ethanol, then diluted with water, and finally 5 g/L curcumin solution obtained.

Grouping and interventions

HSECs were cultured in 37 ℃, 5% CO₂, and 100% humidity of the incubator until in good attachment and growth (in logarithmic phase, and grew up to 80%-90% of the bottom), digested using 0.25% trypsin, and centrifuged. After removal of the supernatant, the cells were resuspended with fresh medium and then inoculated into 6-well or 96-well plates. After incubation for 48 hours in a CO₂ incubator, the cells were completely adhered and assigned.

There were seven groups: such as for the 6-well plates, there were three sub-pores in each. Different solutions were added and incubated in a CO₂ incubator for 48 hours as follows: (1) in blank control group, HESCs were cultured according to standard methods, without any intervnetion; (2) in leptin group, HESCs were cultured in the medium containing 100 μg/L leptin; (3) curcumin group was subdivided into three groups (low, medium, and high dose), followed by cultured in the medium containing leptin and 32, 16 and 8 μmol/L curcumin, respectively^[14]; (4) in colchicines group, 6.25 mg/L colchicines was added after activated by leptin^[15]; (5) in salvianolic acid B group, 10^-⁶ mmol/L colchicines was added after activated by leptin^[15]. All assays were performed in triplicate.

Electron microscopy

Cellular surface morphological changes were observed by scanning and transmission electron microscopes. Samples were processed for transmission electron microscope: 3 specimens were selected from each group, and 10 fields were observed. The obtained image was used for grayscale resolution by aoi software (0-255 grayscale. 0 was black, the darkest; 255 white, the brightest). The average grayscale of the cells in each group was compared to determine the differences in the number and area of the fenestration (in the transmission electron micrograph of HSECs, the sieve plate was black, the fenestration was white) and calculated the grayscale score.

Quantitative real-time PCR

All samples were collected and total RNA was extracted. Quantitative real-time PCR was used to detect the mRNA levels of endothelin-1, and VEGF in HSECs. The gene sequences produced by Sangon Biotech (Shanghai, China) are as follows (Table 1):

Table 1 Gene sequences

Gene	Sequence (5＇-3＇)	Length (bp)
β-actin	F: CAC GAT GGA GGG GCC GGA CTC ATC R: TAA AGA CCT CTA TGC CAA CAC AGT	240
Endothelin-1	F: TCC CGT GAT CTT CTC TCT GC R: TGA CCC AGA TGA TGT CCA GG	212
VEGF	F: CGT CTA CCA GCG CAG CTA TTG R: TGA CCC AGA TGA TGT CCA GG	245

Note: VEGF: vascular endothelial growth factor.

cDNA was obtained after reverse transcription. The target genes were detected at 50 °C for 2 minutes, 95 °C for 10 minutes, 95 °C for 30 seconds and 60 °C for 30 seconds. All steps were performed for 40 cycles. The relative expression levels of mRNAs were quantified by the 2^-ΔΔCtmethod with logarithmic transformation. 2% agarose gel was added into the electrophoresis pool, and 5 μL of PCR amplified products were added into each well, electrophoresis at 100 V for 30 minutes. The amplified DNA fragments were analyzed by gel imager to measure the absorbance value.

ELISA assay

The levels of endothelin-1, and VEGF in HSECs were determined using ELISA kits in accordance with the manufacturer’s instructions. After HSECs were incubated for 24 hours, the corresponding drugs were added, and the cells were then collected at 48 hours and centrifuged at 2 000 r/min for 20 minutes, and the supernatant was collected.

First, the ELISA plate was coated with anti-mouse endothelin-1, and VEGF antibodies. After overnight incubation at 4 °C, the plates were washed three times with sterile PBS, and the uncoated sites were blocked with 200 μL of PBS containing 10% FCS for 2 hours at 37 °C. After subsequent washing, 100 μL of the supernatant obtained from the culture sample was added, and the plates were incubated for 2 hours at 37 °C. The plates were subsequently washed and the biotinylated anti-mouse antibody solution was added to each well and incubated for 1 hour at 37 °C. After incubation and washing, the horseradish peroxidase (HRP)-streptavid in conjugated secondary antibody was added to each well and incubated for 30 minutes at 37 °C. After washing, 3,3’,5,5’- tetramethybenzidine (TMB) solution was added and incubated at room temperature, followed by the addition of stop solution to each well. The absorbance value at 450 nm was measured using a microplate reader. The cytokine level was determined using a standard curve.

Main outcome measures

Fenestration and basement membrane of HSECs in each group; mRNA and protein expression levels of endothelin-1 and VEGF.

Statistical analysis

Statistical analysis was performed using PEMS 3.1 software. Data are expressed as the mean±SD. Measurement data between groups were compared using one-way analysis of variance and chi-squaretest. P < 0.05 was considered statistically significant.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

讨论

Hepatic fibrosis is a major complication in chronic liver diseases. In China, especially in Guangxi Zhuang Autonomous Region, the incidence of liver fibrosis is on a rise, which has become a common disease endangering people’s health^[17]. Researchers have found that liver fibrosis in early- and mid-stage is reversible. Liver fibrosis is a characteristic lesion of liver cirrhosis, and its main pathological features include defenestration of HSECs, phenotypic changes and the formation of subepidermal basement membrane ^[18]. These lesions do harm to the hepatic microcirculation function, thereby promoting the occurrence of fibrosis, and are closely related to portal hypertension^[19]. Among which, the microstructural pathological changes of hepatic sinusoidal wall and the sinusoidal blood flow disorders are the main causes of portal hypertension. Previous studies found that the number and size of HSEC fenestrae are regulated by both neurological and humoral factors, and defenestration of HSECs occurs prior to liver fibrosis after liver injury, which is reversible^[20]. At present, studies on the structure and function of HSECs is an issue of concern in the prevention and treatment of liver fibrosis^[21].

Kojkind et al.^[22] reported that colchicine was able to inhibit liver fibrosis, so many researchers have performed a series of basical and clinical researches on the anti-fibrosis of colchicines. Notably, colchicine has been proved inhibit liver fibrosis probably by inhibiting HSEC proliferation, reducing HSEC autocrine and paracrine, and decreasing the synthesis and secretion of extracellular matrix, which has been the most commonly used drugs for liver fibrosis^[23-24]. Salvianolic acid B has been shown to hold good anti-liver fibrosis in patients with chronic hepatitis B similar with interferon, and is a novel anti-fibrotic drug^[25-27]. Yu et al.^[28]reported that high-dose salvianolic acid B could effectively inhibit the inflammatory response and fibrosis of pancreas, which may be via inhibiting the lipid peroxidation and reducing malondialdehyde production and the activation of hepatic stellate cells, thereby reducing the deposition of extracellular matrix. Thereafter, we chose these two drugs as controls.

Curcumin is mainly from the root tuber of Curcuma aromatic, rhizomes of Curcuma longa (3%-6%) and Zedoary. Curcumin has been found to inhibit the proliferation of various tumor cells, induce its apoptosis through cutting off the nutrient-rich blood supply to tumors, and do no damage to surrounding normal cells^[29]. In addition to anti-inflammation, curcumin also possesses anti-pathogenic microorganisms, anti-oxidation, hypolipidemic effect, hepato-protection, and cardiovascular and cerebrovascular protection, which has been extensively applied in clinic^[31-32]. Zhong et al.^[32] found that curcumin has the effect of protecting liver from carbon tetrachloride-induced liver fibrosis based on anti-lipid peroxidation; curcumin directly inhibits the proliferation of hepatic stellate cells, induce apoptosis of hepatic stellate cells, and reduce the synthesis and secretion of collage type I and III collagen as well as the deposition of extracellular matrix, further protecting against fibrosis. Liu et al.^[33]confirmed that curcumin exerts anti-fibrosis by inhibiting the collagen secretion of HSECs, regulating the expression of MMPS and accelerating the degradation of collagen. Therefore, curcumin plays a positive role in the prevention and treatment of liver fibrosis.

In the pathological process of liver fibrosis, the number of HSEC fenestrae is decreased, closed even disappears, and continuous basal membrane formation, which induce the “capillarization” of the hepatic sinusoid; basal membrane will combine with numerous cytokines, indirectly regulating the proliferation and activation of HSECs, which accelerates the progression of liver fibrosis^[34]^. Previous studies have found that the sinusoidal capillarization can be reversed after hepatotoxin avoided^[35]. In this study, the degree of liver fibrosis is negatively correlated with the number and area of fenestrae. Additionally, curcumin can reopen the closed or disappeared fenestrae, significantly enlarge its size, alleviate the basement membrane formation, and reduce sinusoidal capillarization, thereby significantly alleviates liver fibrosis. Furthermore, its pharmacological effect is in a dose-dependent manner, and that is to say the higher dose is, the stronger ability of protecting against sinusoidal capillarization.

HSECs are not only lined sinusoids, but also have a variety of secretory functions, which has been as endocrine cells. HSECs are damaged after hepatic fibrosis, leading to changes in secretory behavior and cell phenotype. On the one hand, the cells regulate the microcirculation of the liver through the change in the number and dameter of the fenestrae; on the other hand, it secretes a variety of active substances and other products, such as endothelin-1, VEGF, and connective tissue growth factors regulate intrahepatic microcirculation, especially the endothelin-1 induced vasoconstriction is closely related to portal hypertension^[36].

Endothelin-1, a potent vasoconstrictor in humans, is one of three isoforms of human endothelins. It is the only substance that can contract 50-nm capillaries, such as portal vein and hepatic sinusoid, decrease hepatic blood flow, induce hepatocellular degeneration, thus involved in the occurrence and development of liver diseases^[37]. HSECs partially secret endothelin-1 that regulates hepatic sinusoids, thereby affecting blood flow^[38]. However, HSECs will produce abundant endothelin-1 after liver injury, which has been shown to the cause of dysfunction and injury of HSECs^[39]. Pre-mRNA content of HSEC-derived endothelin-1 in animal model of live injury is increased by 60%, and while HSEC-derived endothelin-1 can significantly promote α-SMA protein expression and collagen deposition. Additionally, HSEC-derived endothelin-1 is involved in the regulation of sinusoidal blood flow and activation of HSECs^[38]. Endothelin-1 has also been recognized as the most stable and strongest factor for HSC contraction^[40]. Due to the longer cytoplasmic processes of HSC surrounding the hepatic sinusoidal and HSECs, and smooth muscle-like structures, the contracted HSCs can further constrict hepatic sinusoids, further aggravating the disorder of sinusoidal microcirculation.

In this study, the number of sinusoids and fenestrae in the HSECs were significantly reduced after leptin activation, which was involved in the sinusoidal capillarization. Molecular biology results found that HSECs-secreted endothelin-1 were deposited in the cell membrane and cytoplasm; mRNA and protein expression levels of endothelin-1 were significantly increased. After the intervention of curcumin, all above phenomena were significantly reversed in a dose-dependent manner. In summary, curcumin can inhibit HSECs synthesis and secretion of endothelin-1.

VEGF is a secreted glycoprotein that is mainly synthesized and secreted by hepatocytes, HSECs, and fat-storing cells. VEGF can induce the proliferation of endothelial cells, increase vascular permeability and change the compositions of the extracellular matrix^[41]. VEGF has been found to be related to the density of vessels and number of neovascularization, and is a specific mitogen of endothelial cells that can significantly promote the proliferation of vascular endothelial cells^[42]. Moreover, VEGF increases vascular permeability and causes extravasation of plasma protein, which is activated fibrinogen, being a part of extravascular matrix^[43]. In liver fibrosis, and liver cirrhosis, the significant increase in expression level of VEGF aggravates the development of liver fibrosis^[44]. VEGF can induce the formation of newborn capillaries after liver injury, resulting in the formation of continuous basement membrane and changes of intrahepatic microcirculation, further leading to ischemia and hypoxia of hepatocytes, which in turn induces the synthesis and secretion of VEGF. VEGF increases vascular permeability, causing a significant decrease in the blood flow, aggravate liver ischemia and hypoxia, and forming a vicious cycle^[45]. In this study, HSECs secreting VEGF were deposited in the cell membrane and cytoplasm; mRNA and protein expression levels of VEGF were significantly increased. After treated with curcumin, all above phenomena were significantly reversed in a dose-dependent manner. Therefore, curcumin can inhibit the HSECs synthesizing and secreting endothelin-1.

In summary, curcumin can regulate the capillarization of the hepatic sinusoid in liver fibrosis and reverse HSEC defenestration and endothelial basement membrane formation, which probably via inhibiting the expression of endothelin-1 and VEGF^[46], reducing endothelin-1-induced capillary wall contraction, HSEC contraction, thereby reopening the sinusoidal fenestrae; inhibiting VEGF-induced increased vascular permeability, exudation of extracellular matrix components and deposition of extravascular matrix, and reducing the formation of HSEC extracellular basement membrane. Our results show that all above pharmacological effects of curcumin are related to the capillarization of the hepatic sinusoid and portal hypertension, especially the high-dose curcumin, which provide a new idea for the treatment of liver fibrosis and lay a foundation for the development and utilization of curcumin in clinic.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

姜黄素抗肝窦毛细血管化作用及机制

Effect of curcumin against capillarization of hepatic sinusoids and its mechanism

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