Effect of Curcumol on the Fenestrae of Liver Sinusoidal Endothelial Cells Based on NF-κB Signaling Pathway

Objective To study the effect of curcumol on liver sinusoidal endothelial cells (LSECs) and to analyze the mechanism of antihepatic fibrosis. Methods The effects of drug intervention on cell proliferation rates were detected by MTT assay. The expression of NF-κB was detected by RT-PCR and WB. The NF-κB expression and entry into the nucleus were detected by immunofluorescence; scanning electron microscopy was used to observe the changes of LSECs fenestrae. Results MTT results showed that the interference of cell proliferation in each group was small. RT-PCR showed that the expression of NF-κB in the curcumol intervention group was significantly lower than that in the positive control group (P < 0.05). The WB detection found that, in the curcumol intervention group, the expression of pNF-κB in the NF-κB signaling pathway was significantly lower than that in the positive control group (P < 0.05). Scanning electron microscopy showed that the LSEC fenestrae were significantly improved compared with the positive control group. Conclusion Curcumol may be one of the mechanisms of antihepatic fibrosis by inhibiting the activity of the NF-κB signaling pathway and increasing the fenestrae of LSECs.


Introduction
Hepatic fibrosis is a common result of chronic liver disease and is mainly characterized by extensive deposition of extracellular matrix (ECM) [1]. Inflammation, chronic viral hepatitis, and liver damage are considered to be the leading causes of liver fibrosis. Liver injury leads to the loss of function of hepatic sinusoidal fenestrae, and hepatic sinusoidal thrombosis may promote liver fibrosis [2]. Liver sinusoidal endothelial cells (LSECs) play an essential role in the development of liver fibrosis [3]. LSECs are the primary boundary component of the hepatic sinusoidal wall, which are with fenestrae and the loose connection between cells, but without the basement membrane under endothelium. e structure of the hepatic sinusoidal wall is beneficial to regulate the material exchange between hepatocytes and hepatic sinusoidal blood. LSECs also have an active endocytic function and play an important role in regulating liver microcirculation and secreting extracellular matrix. In the early stage of liver fibrosis, LSECs show loss of fenestrae and subendothelial basement membrane formation.
is phenomenon is named the capillarization of hepatic sinusoidal, which is an essential pathological change in the formation of liver fibrosis [4][5][6].
NF-κB is a crucial inflammatory transcription factor that triggers the massive release of inflammatory cytokines, such as IL-6, TNF-α, and IL-8 [7]. NF-κB enhances the inflammatory response of the liver to form an "inflammatory waterfall" and eventually leads to the formation of liver fibrosis. Studies have found that hepatic stellate cell (HSC) collagen expression correlates with NF-κB, inhibits NF-κB expression and activity, and can significantly impede collagen expression [8]. Reportedly, NF-κB has antiapoptotic effects, and some apoptosis stimuli activate NF-κB. Activated NF-κB blocks TNF-α mediated apoptosis [9]. As a fibrotic factor, NF-κB enhances liver inflammation, participates in the regulation of proliferation and apoptosis of LSECs, and promotes collagen synthesis and secretion. e eNOS-NO-cGMP signaling pathway is an important pathway regulating the formation of fenestrae in LSECs [10,11]. When the NF-κB signaling pathway is activated, the IκB kinase IKK is activated for phosphorylating and ubiquitinating the NF-κB inhibitory protein IκBα, resulting in a decrease in the cytoplasmic IκBα content. us, the NF-κB p65 subunit enters the nucleus from the inhibitory state to the activated state, activates the expression of multiple inflammatory factors, and regulates the transcription of the iNOS gene at the same time, leading to the disorder of the liver microcirculation [12][13][14][15].
In our previous study, we have found curcumol that suppresses HSC activation and contributes to the activation of HSC apoptosis during liver fibrosis [16]. Curcumol, a Chinese herbal medicine, promotes blood circulation for the removal of blood stasis. However, the effects of curcumol on LSECs remain undetermined.
We speculate that curcumol could reverse hepatic fibrosis by inhibiting the activity of the NF-κB signaling pathway and increasing the fenestrae of LSECs. We aimed to elucidate that curcumol changes the structure of LSECs by inhibiting the activity of the NF-κB signaling pathway, which might effectively reduce the pathogenesis of hepatic fibrosis.

Animals.
e experiment was conducted with 8-weekold male Sprague Dawley (SD) rats from the Medical Laboratory of Guangxi Medical University. e animal certificate number was SCXK (Gui) 2016-0002, and the animal license number was SYXK (X) 2015-0001. e weight of the rats is 150-180 g. e animal research is carried out at the Animal Experimental Center of Guangxi University of Traditional Chinese Medicine and approved by the Animal Ethics Committee of Guangxi Traditional Chinese Medicine Institute (protocol number: 2016-12-02-1). All authors ensure that the guidelines and regulations for conducting all experiments were approved. All animals were tested under standard conditions (25°C and 12 hr light/dark cycles).

Cell Isolation and
Culture. Rats were anesthetized by intraperitoneal injection with 3% pentobarbital sodium at a dose of 0.15 mL/100 g. e anesthetic was treated with an anticoagulant. Separation of LSECs was described by Oie et al. [17] using hepatic collagenase perfusion, differential centrifugation and Percoll density gradient sedimentation, and cell-selective adherence. e cells were identified by scanning electron microscopy and cultured in DMEM medium containing 10% FBS. Suitable conditions include moderate room temperature and 5% CO 2 humidified air to maintain cells. After reaching 80∼90% confluence, cells were cultured on a 6-well plate with a density of 1 × 10 5 cells per well for further experiments for 48 hr. To maintain the isolated LSEC phenotype, the culture was carried out, on the one hand, in DMEM containing 10% FBS and 10 ng/mL endothelial cell growth factor (ECGF). On the other hand, primary cells were seeded on collagen-coated coverslips for experiments and rapidly fixed by glutaraldehyde using an electron microscope.

Experimental Grouping and
Intervention. e cells were divided into five groups. Group 1 cells were treated with 2% DMEM as the control group. Group 2 cells were treated with LPS (activate the NF-κB signaling pathway) at a concentration of 5 μg/mL for 3 hr as the positive group. Group 3 cells were treated with curcumol at a concentration of 45 μg/ mL for 48 hr as the curcumol group. Group 4 cells were pretreated with curcumol at a concentration of 45 μg/mL for 48 hr and then treated with LPS 5 μg/mL for 3 hr as the curcumol intervention group. Group 5 cells were pretreated with 25 μg/mL PDTC (NF-κB inhibitor) for 30 minutes and then treated with LPS at concentration 5 μg/mL for 3 hr as the negative control group.

Cell Proliferation Experiment.
e cell density of the isolated rat LSECs was adjusted to 5 × 10 4 cells per mL with endothelial cell complete medium. Cells (100 μL per well) in a 96-well plate (blank well was set at 37°C) were incubated overnight (100 μL of sterile PBS was added to the wells around the wells). Each component was composed of three duplicate wells, cultured at 37°C, 5% CO 2 saturated humidity for 48 hr. MTT (10 μL) was added to each well and then incubated at 37°C for 4 hr. After the medium was aspirated, 150 μL of DMSO was added for 10 min. e absorbance value OD568 of each well was determined by an enzyme labeling instrument. e cell proliferation rate of each group was calculated from the OD value.

Western Blots.
e total protein was extracted and separated by SDS-PAGE and then transferred the PVDF membrane for incubation with the primary antibody at 4°C overnight. After being washed with PBS, the membranes were incubated with the corresponding HRP-labeled secondary antibody at 37°C for 2 hr and then were illuminated with ECL reagent. e gray value of the film was analyzed using BandScan.
e ABI system was used for data analysis.

Immunofluorescence Observation.
To investigate the expression and entry into nucleus of NF-κB in LSECs, cells were washed 3 times with PBS for 3 minutes and then fixed with 4% paraformaldehyde solution for 15 min. e cells were then incubated with 0.5% Triton X-100 containing goat serum and incubated with fluorescently labeled goat antirabbit IgG antibody. An antifluorescence quenching seal and DAPI mounting media were mounted on glass slides and visualized using an fluorescent inverted microscope.

Scanning Electron Microscope Observation.
To investigate the changes in fenestration in LSECs, we used a scanning electron microscope (SEM). All samples were subjected to scanning electron microscopy for 24 hr after administration.
e formulation was then dried in 100% isoamyl acetate. Platinum (15 nm) was used as a coating for surface preparation samples. e sample was observed using a HITACHI scanning electron microscope (SU-8010).

Statistical
Analysis. Data were analyzed using SPSS statistic 22.0 software and presented as the mean ± SD. ANOVA with Bonferroni posttests was used to evaluate significant differences between groups. P values < 0.05 were considered significant differences between independent groups.

Isolation and Culture Results of LSECs
Percoll Density Gradient Centrifugation. e hepatocyte suspension is divided into density gradients of 4 different regions. e upper layer (between the top layer of the Percoll gradient and the 25% Percoll gradient layer) mainly contains debris, damaged cells, and a small amount of nonparenchymal cells. e boundary layer between 25% Percoll and 50% Percoll is rich in LSEC, with a small amount of unknown small white and red blood cells. e third layer (50% Percoll gradient zone) contains Kupffer cells (KC) and red blood cells. A large number of red blood cells were deposited in the bottom layer.
After Percoll density gradient centrifugation, about 32 × 10 6 cells of LSEC were obtained. After selective attachment, 22 × 10 6 cells were obtained. As determined by trypan blue staining, the viability of the isolated LSEC cells reached 95-98%. e separate and cultured LSECs were observed under an optical microscope. At 12 hr, the cells were round and then gradually grew into an oval structure (as indicated by the arrow in Figure 1(a)). After 36 hr, the oval cells slowly gathered into a paving stone-like shape, and the cells gradually became a spinning cone (as indicated by the arrow in Figure 1(b)). After 60 hr, the cell morphology basically changed to a spinning cone (as indicated by the arrow in Figure 1(c)), and the cells grew better.

MTT Assay Showed at the Proliferation Rate of Each Group Was Higher.
e OD value of the curcumol group was statistically significant compared with the blank control group (P < 0.05).
e OD value of the model group was statistically significant compared with the blank control group (P < 0.05). As shown in Figure 2. According to the cell proliferation rate formula (experimental group OD/blank control group OD), the proliferation rate of each group was greater than 60%, so the effect rate of each group of drugs on cell proliferation rate was relatively small, and the test results of subsequent experiments were reliable.

Curcumol Downregulated the Expression of NF-κB mRNA and Phosphorylated NF-κB.
e expression of NF-κB mRNA in the curcumol intervention group was significantly lower than that in the model group (P < 0.05).
e expression of NF-κB mRNA in the model group was significantly higher than that in the blank control group (P < 0.05), and the expression of NF-κB mRNA in the curcumol intervention group was significantly higher than that in the PDTC group (P < 0.05). e expression of NF-κB mRNA in the curcumol group was significantly lower than that in the blank control group, and the difference was statistically significant (P < 0.05). RT-PCR detection showed that curcumol could inhibit the expression of NF-κB Mrna, as shown in Figure 3. e expression of phosphorylated NF-κB in the curcumol intervention group was significantly lower than that in the model group, and the difference was statistically significant (P < 0.05), e expression of NF-κB in the model group was significantly higher than that in the blank control group (P < 0.05). e expression of NF-κB in the curcumol intervention group was significantly higher than that in the PDTC group (P < 0.05). e expression of NF-κB in the curcumol group was significantly lower than that in the blank control group, and the difference was statistically significant (P < 0.05). WB assay showed that curcumol could inhibit the expression of phosphorylated NF-Κb, as shown in Figure 3. Curcumol inhibited the expression of NF-κB mRNA and phosphorylated NF-κB, which may be one of the mechanisms of its antifibrosis.    Evidence-Based Complementary and Alternative Medicine

Curcumol Can Reduce NF-κB Translocation from the Cytosol to Nuclear Translocation .
To detect the expression of NF-κB in LSECs and its translocation from cytosol to nuclear translocation, the immunofluorescence method was used to identify the expression of NF-κB in LSECs. e expression of NF-κB in curcumol group was lower than that in the blank control group, and that in curcumol intervention group was lower than that in the positive control group. Curcumol inhibits NF-κB translocation from cytosol to nuclear translocation, as shown in Figure 5. e results showed that curcumol could inhibit the NF-κB translocation from cytosol to nuclear translocation and regulate the expression of a series of downstream target genes, thereby improving the fenestrae of LSECs.

Discussion
Liver fibrosis is an essential stage for the further development of chronic hepatitis, and it is a pathophysiological process for the massive deposition of ECM [18]. It is well known that liver fibrosis eventually leads to cirrhosis and liver failure. Liver fibrosis is a common disease that requires effective treatment. Numerous studies have shown that liver fibrosis can be reversed [19]. Our previous studies have

Evidence-Based Complementary and Alternative Medicine
shown that curcumol has antifibrotic effects [16]. Our current data suggest that curcumol can restore the capillarization of hepatic sinusoidal in vitro. Sinusoidal capillarization is mainly characterized by the formation of basement membranes and the loss of fenestrae openings of LSECs. LSECs constitute the blood vessel wall of the hepatic sinusoids and are the main population of nonparenchymal cells in the liver, accounting for approximately 50% of parenchymal cells. Under differentiated LSEC electron microscopy, its fenestrae structure can be seen, and there is no intact basement membrane under the fenestration endothelium. e fenestrae occupies 6% to 8% of the total surface area. is porous structure is found in liver parenchymal cells and blood substances. Exchange plays a key role. When sinusoidal capillarization occurs, the loss of LSECs leads to the reduced permeability of LSECs, and various metabolites do not easily enter the blood circulation, aggravating liver pathological damage. At the same time, liver cells and blood are weakened. e exchange of oxygen and nutrients eventually leads to hepatocyte damage and sinus collapse [20,21]. LPS can induce liver sinusoidal endothelial cell activation. In contrast, curcumol reversed this pathology in LPS stimulated activated LSECs. In this study, we found that LPS promotes the expression of NF-κB, while curcumol can downregulate the expression of NF-κB.
Combining the above data, we confirmed that curcumol reverses the pathological changes of LPS stimulated LSECs, such as sinusoidal capillarization, by downregulating the expression of NF-κB. NF-κB is a pleiotropic transcription factor. NF-κB in healthy cells is in an inactive state. It mainly binds to its inhibitor kappa B (I-κB) as a p50/p65 dimer and exists in the cytoplasm. Various stimuli such as reactive oxygen species and cytokines can induce phosphorylation of I-κB, dissociate it from p50/p65, activate NF-κB, and transfer it into the nucleus. Combined with apoptosis-related genes to promote gene transcription and produce cells, apoptosis causes damage to the body. Oxidative stress is an important factor in inflammatory diseases of many diseases, and the NF-κB inducible nitric oxide synthase (iNOS)-NO signaling pathway is an important oxidative stress pathway [22][23][24]. e activity of LSECs is also regulated by the NF-κB/NO signaling pathway. LSECs can synthesize and secrete NO and ET, thereby regulating intrahepatic vasodilation and contraction, and play an important role in regulating hepatic sinus blood flow. NO has a powerful vasodilator effect, which directly stimulates soluble guanylate cyclase in a paracrine form, which increases cyclic guanylate, which causes Ca 2+ reduction and vasodilation [25,26]. Under physiological conditions, LSECs can express endothelial nitric oxide synthase (eNOS) and synthesize NO through eNOS, but the level of NO is low. Because of the strong ability of NO to diffuse and the long-term biological effects of metabolites, it is sufficient to maintain its normal blood perfusion. LSECs are extremely susceptible to damage by certain toxicants or drugs. Some studies have found that when LSECs is damaged, eNOS posttranslationally modifies abnormally, leading to a decrease in NO synthesis, while an increase in the expression of ET-1 with vasoconstriction can lead to HSC contraction [27]. HSC expresses the ET-1 receptor. ET-1 binds to the receptor to increase the Ca 2+ content in the cell, causing contraction of myosin and the like. At the same time, it increases the expression of α-SMA and regulates HSC proliferation. Increased Ca 2+ content and cell contraction induced by ET-1 are seen at any stage after HSC activation. ET-1 can also directly affect the hepatic sinusoids, cause liver microcirculation disorders, lead to liver cell damage, portal hypertension, and aggravate the liver disease. LSEC injury and dysfunction aggravate the liver injury and promote liver fibrosis and cirrhosis [28].
is lesion can activate hepatic stellate cells, leading to liver fibrosis [29]. Traditional Chinese medicine believes that blood stasis is the most important cause of liver fibrosis. Studies have shown that the capillarization may be the root cause of "blood stasis syndrome" in liver fibrosis [30]. Studies have found that [31] Curcuma is the top ten Chinese medicines commonly used in the treatment of chronic liver diseases. Curcumol is the main active ingredient of Guangxi's native medicinal herb Curcuma, and its antifibrotic effect is closely related to its pharmacological effect of promoting blood circulation and removing blood stasis. Our results show that the isolated and cultured LSECs grow better, have distinctive morphology, and grow regularly. e cells grow into a paving stone-like morphology and change from an initial round structure to an oval and spindle shape. e experiments provided the basis. MTT experiments showed that the cell proliferation in each group was excellent, indicating that various biological detection indicators were reliable. PCR, WB, and immunofluorescence analyses showed that the curcumol intervention group could inhibit the NF-κB signaling pathway activated by LPS. Scanning electron microscopy results showed that curcumol could improve the fenestrae size and number of LSECs by inhibiting the activity of NF-κB signaling pathway. Compared with the blank control group, the curcumol group can inhibit the activity of the NF-κB signaling pathway, which indicates that when LSECs are at rest, curcumol can also inhibit the NF-κB signaling pathway. Scanning electron microscopy observations show the hepatic sinusoids in the curcumol group. e number of LSECs fenestration was increased compared with the blank control group, which may be closely related to the pharmacological effect of curcumol on blood circulation and blood stasis. According to our research, curcumol can be used not only as an effective antifibrosis drug but also as a preventive agent for chronic liver diseases. Curcumol can inhibit the activity of NF-κB signaling pathway of LSECs and improve the fenestrae of LSECs. It may be one of its molecular mechanisms for preventing and treating liver fibrosis. LSECs as targets for the prevention and treatment of chronic liver diseases will become an increasing concern.

Conclusion
Curcumol can regulate the structure of LSECs by inhibiting the activity of the NF-κB signaling pathway in vitro.

Data Availability
e data used to support the findings of this study are included within the article and are available from the corresponding author upon request.

Disclosure
Yang Zheng and Jiahui Wang are co-first authors.

Conflicts of Interest
e authors declare that there are no financial conflicts of interest.

Authors' Contributions
Yang Zheng and Jiahui Wang contributed equally to this study.