Curcumin Alleviates LPS-Induced Inammation and Apoptosis in Vascular Smooth Muscle Cells Via Inhibition of the NF- κ B and JNK Signaling Pathways

Background: Curcumin plays an important role in inammation. This study was aimed to investigate the effect of curcumin on vascular smooth muscle cells (VSMCs) injury induced by lipopolysaccharide (LPS) and its mechanism. Methods: VSMCs cells were treated with different concentrations of curcumin (0, 50, 100 and 150 μ g / mL). CCK-8 assay and ow cytometry were used to monitor the effects of curcumin on LPS-induced cell viability and apoptosis. The expression and release of inammatory cytokines in VSMCs cells were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The proteins expressions of NF- κB and JNK signaling pathways were analyzed by western blot. Results: Curcumin could reduce LPS induced inammatory injury by increasing VSMC's cell viability, reducing apoptosis and inhibiting the release of inammatory cytokines. In addition, curcumin increased the expression of Toll-like receptor 4 (TLR4) in LPS treated VSMCs. In terms of mechanism, we found that curcumin attenuated LPS-induced cell injury in VSMCs via inhibition of NF- κ B and the JNK signal pathway. Conclusion: Curcumin can protect VSMCs from LPS induced inammatory injury, which may be related to the blocking of NF- κ B and the JNK signaling pathway. Curcumin may be a potential drug for the treatment of atherosclerosis.


Introduction
Atherosclerosis (AS) is the basic pathological basis of ischemic cardiovascular and cerebrovascular diseases [1]. Most scholars believe that AS is a chronic in ammatory reaction, and a variety of in ammatory factors and in ammatory cells participate in its occurrence and development. Recent studies suggest that endothelial damage is an important initiating factor in the formation of atherosclerotic plaque [2]. Vascular endothelial cells and smooth muscle cells are damaged by various risk factors, which leads to an excessive chronic in ammatory proliferative reaction in the local blood vessels [3]. Recent studies have reported that several Chinese herbal medicines can improve the symptoms of AS [4]. These ndings have aroused people's interest in the treatment of AS with traditional Chinese medicine.
Curcumin is a plant polyphenol extracted from Curcuma longa and other medicinal plants. It has antiin ammatory, anti-tumor, anti-oxidation and immunomodulatory effects [5], which are related to its regulation of some molecular targets, including pro-in ammatory cytokines, growth factors, factors involved in proliferation and apoptosis, adhesion molecules and transcription factors [6][7][8][9]. Especially in vascular cells, curcumin can inhibit the over expression of pro-in ammatory cytokines induced by oxidized low density lipoprotein (ox LDL) in VSMCs [10]. However, the molecular mechanism of curcumin inhibiting LPS induced in ammation in VSMCs remains unclear.

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The purpose of this study was to investigate the therapeutic effect of curcumin on AS. The in ammatory injury model of VSMCs cells induced by LPS was established. We found that curcumin could reduce the damage of VSMCs cells induced by LPS. The protective effect of curcumin on VSMCs cells may be through the inhibition of NF -κB and JNK signal pathways. It is suggested that curcumin may be a potential drug for the treatment of AS.

Cell viability assay
After digestion, the cells in the logarithmic growth phase were inoculated into 96-well plates at the density of 5 × 10 3 /ml for 24 hours, and the old culture was discarded. Curcumin of different concentrations was added to each experimental group for 24 hours. Each experimental group had 4 compound holes, and each well was cultured with MTT solution Sigma-Aldrich (St.Louis, MO) (concentration was 5mg/ml) for 4 hours. The supernatant was carefully discarded, and 200 µ l DMSO, was added to each well to dissolve and dissolve precipitation. The absorbance value was read by the enzyme labeling instrument at the wavelength of 490 nm.

Apoptosis assay
Cell apoptosis was detected by annexin V-FITC double staining. 24 hours after transfection, the cells were digested by trypsin, collected and inoculated in a 6-well plate, the cell density was adjusted to 2 × 10 6 cells per well, cultured for 24 hours, the supernatant was discarded, pre-cold PBS was washed twice, 1 × Binding buffer was used to suspend cells. The cell suspension was added with 5 µ L annexin V-FITC and 5 µ l PI, After mixing well, the cells were incubated at room temperature for 15 minutes, The apoptotic rate was detected by ow cytometry (Beckman Coulter, Fullerton, CA, USA) within 1 hour, and the data were analyzed by Flow Jo software (tree star, Ashland, or, USA).

Quantitative Real-time PCR
According to the instructions of the manufacturer, total RNA was isolated from VSMCs cells using TRIzol reagent (Invitrogen). Reverse transcription was carried out through multiple transcription kits (Applied Biosystems, Foster, CA, USA). To analyze IL-6, IL-1 β and TNF-α, SYBR green PCR kit (TaKaRa) was used to quantify the messenger RNA (mRNA) levels of IL-6, IL-1 β and TNF-α. β-actin was ampli ed as a control. The relative expression levels of IL-6, IL-1 β and TNF-α were calculated by the 2 −ΔΔCT method.

Statistical analysis
The data of this study were expressed by mean ±standard deviation (SD), and the statistical software SPSS 19.0 was used (IBM Analytics, New York, USA), P < 0.05was considered to be a statistically signi cant result. All experiments are repeated at least three times.

Curcumin attenuated LPS induced cell injury in VSMCs
To investigate the effect of curcumin on the in ammatory injury of VSMCs cells induced by lipopolysaccharide (LPS), the cell viability was observed by CCK-8 assay, and the apoptosis rate was determined by ow cytometry before and after curcumin treatment. Consistent with previous studies [11,12], our results showed that LPS could inhibit the viability of VSMCs cells. Curcumin could increase the viability of VSMCs cells in a dose-dependent manner ( Figure 1(A)). Besides, the percentage of apoptotic cells in the LPS treatment group was signi cantly higher than that in the control group, but curcumin could inhibit the pro-apoptotic effect of LPS in a dose-dependent manner (Figure 1(B)). In order to further con rm our results, a western-blot was used to detect the protein expression levels of speci c markers of apoptosis (Bax, Caspase-3 and Caspase-9). As shown in (Figures. 1C and D), the levels of Bax, Pro-Caspase-3 and Pro-Caspase-9 proteins in the LPS treatment group were signi cantly higher than those in the control group, but the promoting effect of LPS and curcumin combined treatment group was signi cantly decreased. Taken together, these data suggested that curcumin attenuates LPS-induced VSMCs cell injury in a dose-dependent manner by increasing cell viability and reducing apoptosis.

Curcumin inhibited LPS-induced production of in ammatory cytokines in VSMCs cells
QRT-PCR, western blot and ELISA assay were used to detect the effect of curcumin on the production of in ammatory cytokines TNF-α, IL-1 β and IL-6 in VSMCs cells induced by LPS. The results of qRT-PCR and western blot showed that the mRNA and protein levels of TNF-α, IL-1β and IL-6 in VSMCs cells treated with LPS were signi cantly higher than those in the control group (P < 0.05). Whereas, the increased expression of TNF-α, IL-1β and IL-6 induced by LPS decreased in a dose-dependent manner under curcumin treatment ( Figure 2A&B). The results of ELISA were consistent with the above results. Compared with the control group, the release of TNF-α, IL-1β and IL-6 in the LPS treatment group was signi cantly increased, while the promoting effect of LPS and curcumin on the release of TNF-α, IL-1 β and IL-6 induced by LPS was decreased. The release of in ammatory cytokines decreased with the increase of curcumin concentration ( Figure 2C-E). It was suggested that curcumin could inhibit the excessive secretion of TNF-α, IL-1β and IL-6 in VSMCs cells induced by LPS in a dose-dependent manner.

Curcumin increased TLR4 expression in VSMCs cells treated with LPS
To investigate the effect of curcumin on the expression of TLR4 in VSMCs cells, the protein level of TLR4 was detected by using curcumin (50,100,150 µ g / ml) co-treated with LPS for 24 hours. The results showed that LPS signi cantly induced the expression of TLR4 in VSMCs cells, while curcumin signi cantly inhibited the expression of TLR4. These data suggested that curcumin could inhibit the expression of TLR4 in VSMCs cells, indicating that TLR4 might play an important role in the regulation of LPS-induced cell injury by curcumin (Figure 3).

Curcumin relieved LPS induced VSMCs cell injury by inhibiting NF-κ B and JNK signaling pathways
Next, we discussed the effect of curcumin on the regulation of NF-κ B and the JNK signal pathway. Curcumin (50,100,150 µ g / ml) was co-treated with LPS for 24 hours. The protein levels of NF-κ B and the JNK signal pathway were detected by western blot. Western blot results showed that LPS treatment increased the expression of p-p65, p-lκBα, p-JNK and p-c-Jun, indicating that LPS could promote the signal transduction of NF-κB and JNK pathways. Under the action of curcumin, the expressions of p-p65, p-lκBα, p-JNK and p-c-Jun were down-regulated in a dose-dependent manner, suggesting that curcumin could inhibit NF-κB and JNK signal pathways. In a word, curcumin attenuated LPS induced VSMCs cell injury by inhibiting NF-κ B and JNK signal pathways ( Figure A&B).

Discussion
AS is the basic pathological basis of coronary heart disease. At present, it is considered that lipid metabolism disorder, oxidative stress and in ammatory reaction are important factors in the occurrence and development of AS [13][14][15]. A clinical trial has been con rmed that anti-in ammatory intervention is more accurate and personalized in patients with coronary heart disease [16]. In this study, the LPSinduced VSMCs model was used to explore the effects of curcumin on apoptosis and in ammation of VSMCs cells. We found that curcumin could reduce the in ammatory injury induced by LPS by increasing the viability of VSMCs cells, reducing apoptosis and inhibiting the release of in ammatory cytokines. On the other hand, we found that curcumin increased the expression of toll-like receptor 4 (TLR4) in LPStreated VSMCs cells. In terms of mechanism, we found that curcumin attenuated LPS induced VSMCs cell injury by inhibiting NF-κ B and JNK signal pathways. Therefore, it is suggested that diosgenin can inhibit the progress of AS.
Curcumin and its analogs have long been used to treat various diseases, and modern science has also provided a reliable scienti c basis for the pharmacological effects of curcumin. A large number of studies have con rmed that curcumin has anti-in ammatory, antioxidant and other pharmacological effects by regulating various targeting factors [17,18]. There are more and more studies on curcumin in cardiovascular diseases. For instance, curcumin can be used to prevent neointimal hyperplasia after angioplasty and regulate the proliferation and migration of VSMCs through anti-in ammatory and antioxidant effects [19]. Meng et al have shown that curcumin through ROS related TLR4-MAPK /NF-κB pathway inhibits LPS induced in ammation in rat vascular smooth muscle cells in vitro [20]. On the other hand, curcumin decreased the expression of NF-κBp65 and DNA binding activity of NF-κB in VSMCs exposed to LPS [21]. AS is an important cause of coronary heart disease, cerebral infarction, peripheral vascular disease and other diseases, and is related to in ammation, oxidation and other factors. There are more and more studies on curcumin against AS. For example, curcumin may alleviate AS by inhibiting PI3K / AKT and JAK2 / STAT5 signal pathways [22]. Zhang et al have shown that curcumin inhibits the activation of NF-κB in macrophages and reduces the expression of TLR4 induced by LPS, thereby reducing the degree of AS lesion and inhibiting the development of AS [23]. In addition, more and more studies have shown that oxidative stress and in ammatory pathways are involved in the biological process of curcumin. For instance, curcumin inhibits osteogenic differentiation of human aortic valve interstitial cells by interfering with the activation of the NF-κB / AKT / ERK signaling pathway [24]. Wang et al have shown that curcumin inhibiting the JNK pathway can prevent diabetic cardiomyopathy [25]. Consistent with the above studies, we found that curcumin had a protective effect on VSMCs damage caused by LPS. In recent years, more and more evidence shows that curcumin can reduce the expression of TLR4, and TLR4 can recognize LPS and induce related signal pathways to play an important role in LPS [23]. Therefore, we further discussed the effect of curcumin on the expression of TLR4. We found that curcumin could signi cantly promote the expression of TLR4 in LPS-treated VSMCs cells.
Moreover, we also studied the relevant signal pathways and revealed their potential mechanisms. NF-κB is an important signal pathway associated with many physiological functions, including cell proliferation, malignant transformation and in ammation [26]. Inhibition of this pathway can improve cell viability and reduce apoptosis and in ammatory injury. P65 and IκBα are key molecules of the NF-κB signaling pathway, and their phosphorylation usually increases apoptosis and in ammation [27]. Here, we found that the upregulation of p-p65 and p-lκBα induced by LPS was inhibited by curcumin. Therefore, these data suggested that the alleviating effect of curcumin on in ammatory injury induced by LPS might be achieved by inhibiting the NF-κB signal pathway in VSMCs. Recent evidence shows that JNK signaling pathway is also related to cell proliferation, apoptosis and in ammation. JNK and its downstream molecule c-Jun are activated after injury [28]. In this study, western blot showed that the expression of p-JNK and p-c-Jun increased in LPS treated group, but decreased after curcumin treatment. These data suggested that curcumin attenuated LPS-induced VSMCs cell injury by inhibiting NF-κB and JNK signaling pathways in VSMCs cells.
To sum up, curcumin can protect VSMCs cells from LPS-induced in ammatory injury by improving cell viability, reducing apoptosis and inhibiting the production of in ammatory cytokines. Curcumin may play a protective role by inhibiting NF-κB and JNK pathways. These results provide a basis for curcumin to be a potential drug for the treatment of AS. Our study may provide some reference for the treatment of AS.

Disclosure Statement
No potential con ict of interest was reported by the authors.

Funding
Wan benting, Ruan haohang,etc.,research on health monitoring and chronic disease prevention service management system for urban and rural residents, 2016-7-2019, key R & D project of Jiangxi Provincial Department of science and technology, the second undertaker.

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