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

Curcumin Alleviates LPS-Induced Inflammation and Apoptosis in Vascular Smooth Muscle Cells Via Inhibition of the NF- κ B and JNK Signaling Pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Curcumin Alleviates LPS-Induced Inflammation and Apoptosis in Vascular Smooth Muscle Cells Via Inhibition of the NF- κ B and JNK Signaling Pathways Haohang Ruan, Qing Huang, Benting Wan, Ming Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-969399/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Background: Curcumin plays an important role in inflammation. 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 flow cytometry were used to monitor the effects of curcumin on LPS-induced cell viability and apoptosis. The expression and release of inflammatory 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 inflammatory injury by increasing VSMC's cell viability, reducing apoptosis and inhibiting the release of inflammatory 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 inflammatory 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. Immunology Clinical Pharmacology Curcumin Lipopolysaccharide vascular smooth muscle NF- κ B JNK atherosclerosis inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Atherosclerosis (AS) is the basic pathological basis of ischemic cardiovascular and cerebrovascular diseases [ 1 ]. Most scholars believe that AS is a chronic inflammatory reaction, and a variety of inflammatory factors and inflammatory 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 inflammatory 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 findings 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 anti-inflammatory, anti-tumor, anti-oxidation and immunomodulatory effects [ 5 ], which are related to its regulation of some molecular targets, including pro-inflammatory cytokines, growth factors, factors involved in proliferation and apoptosis, adhesion molecules and transcription factors [ 6 – 9 ]. Especially in vascular cells, curcumin can inhibit the over expression of pro-inflammatory cytokines induced by oxidized low density lipoprotein (ox LDL) in VSMCs [ 10 ]. However, the molecular mechanism of curcumin inhibiting LPS induced inflammation in VSMCs remains unclear. The purpose of this study was to investigate the therapeutic effect of curcumin on AS. The inflammatory 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. 2. Materials And Methods 2.1. Cell culture and treatment Rat cell line VSMCs was cultured in Dulbecco's modified Eagle medium ((DMEM; Gibco BRL,Gaithersburg, MD, USA) containing 10% (v / v) fetal bovine serum (FBS༌Gibco) in 37°C, 5%CO 2 atmosphere. Curcumin and LPS are purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Firstly, VSMCs cells were treated with 10 µ M LPS for 12 hours to establish the injury model. Then VSMCs cells were treated with different concentrations of curcumin (0, 50, 100, 150 µ g / mL) for 24 hours. 2.2. 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. 2.3. 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 flow cytometry (Beckman Coulter, Fullerton, CA, USA) within 1 hour, and the data were analyzed by Flow Jo software (tree star, Ashland, or, USA). 2.4. Enzyme-linked immunosorbent assay VSMCs were seeded in 24 well culture dish and treated with different methods for 24 hours. The culture supernatant was collected. The concentrations of inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α) were measured by ELISA kit (TaKaRa, Dalian). 2.5. 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 amplified as a control. The relative expression levels of IL-6, IL-1 β and TNF- α were calculated by the 2 −ΔΔCT method. 2.6. Western blot The total proteins of different groups of cells were extracted by a protein extraction kit. The protein concentration was determined by BCA (Pierce, Appleton, WI, USA). Total proteins were separated by SDS-PAGE electrophoresis with 50 µ g total proteins in each well. After electrophoresis for 2 hours, the membrane was transferred to the PVDF membrane (Millipore, Billerica, Ma, USA) by the wet method. The membrane was sealed with 5% skimmed milk powder for 1 hour, and the primary antibody anti-Bax (ab32503, 1:1000), anti-pro-Caspase3 (ab32150, 1:1000), anti cleaved-Caspase3 (ab32042, 1:1000), anti-IL-6 (ab233706, 1:1000), anti IL-1β (ab216995, 1:1000), anti-TNF-α (ab183218, 1:1000), anti-p65 (ab32536, 1:1000), anti-toll-like receptor 4 (anti-TLR4, ab22048, 1:500), anti-p-p65 (ab31624, 1:1000), anti-IκBα (ab109300, 1:1000), anti-JNK (ab176645, 1:1000), anti-p-JNK (ab176645, 1:1000), anti-c-Jun (ab40766, 1:1000) and anti-p-c-Jun (ab32385, 1:1000), anti-β-actin(ab8226, 1:2000), all purchased from Abcam (Cambridge, UK) was incubated at 4 ℃. Overnight, In the next morning, TBST was used to rinse the membrane, horseradish peroxidase-labeled second antibody was added and incubated at 37 ℃ for 1 h. ECL was used to develop and save the image. The optical density value was measured by quantity one. 2.7. 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 significant result. All experiments are repeated at least three times. 3. Results 3.1. Curcumin attenuated LPS induced cell injury in VSMCs To investigate the effect of curcumin on the inflammatory injury of VSMCs cells induced by lipopolysaccharide (LPS), the cell viability was observed by CCK- 8 assay, and the apoptosis rate was determined by flow 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 significantly 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 confirm our results, a western-blot was used to detect the protein expression levels of specific 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 significantly higher than those in the control group, but the promoting effect of LPS and curcumin combined treatment group was significantly 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. 3.2. Curcumin inhibited LPS-induced production of inflammatory cytokines in VSMCs cells QRT-PCR, western blot and ELISA assay were used to detect the effect of curcumin on the production of inflammatory 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 significantly 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 2 A&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 significantly 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 inflammatory cytokines decreased with the increase of curcumin concentration (Figure 2 C-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. 3.3. 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 significantly induced the expression of TLR4 in VSMCs cells, while curcumin significantly 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 ). 3.4 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). 4. Discussion AS is the basic pathological basis of coronary heart disease. At present, it is considered that lipid metabolism disorder, oxidative stress and inflammatory reaction are important factors in the occurrence and development of AS [ 13 – 15 ]. A clinical trial has been confirmed that anti-inflammatory intervention is more accurate and personalized in patients with coronary heart disease [ 16 ]. In this study, the LPS-induced VSMCs model was used to explore the effects of curcumin on apoptosis and inflammation of VSMCs cells. We found that curcumin could reduce the inflammatory injury induced by LPS by increasing the viability of VSMCs cells, reducing apoptosis and inhibiting the release of inflammatory cytokines. On the other hand, we found that curcumin increased the expression of toll-like receptor 4 (TLR4) in LPS-treated 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 scientific basis for the pharmacological effects of curcumin. A large number of studies have confirmed that curcumin has anti-inflammatory, 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-inflammatory and antioxidant effects [ 19 ]. Meng et al have shown that curcumin through ROS related TLR4-MAPK /NF- κB pathway inhibits LPS induced inflammation 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 inflammation, 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 inflammatory 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 significantly 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 inflammation [ 26 ]. Inhibition of this pathway can improve cell viability and reduce apoptosis and inflammatory injury. P65 and IκBα are key molecules of the NF-κB signaling pathway, and their phosphorylation usually increases apoptosis and inflammation [ 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 inflammatory 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 inflammation. 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 inflammatory injury by improving cell viability, reducing apoptosis and inhibiting the production of inflammatory 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. Declarations Disclosure Statement No potential conflict of interest was reported by the authors. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-969399","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":56628165,"identity":"c2cf2075-7edb-4c6a-9fe7-6ff25f6978ee","order_by":0,"name":"Haohang Ruan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYBACewYGNiB1AMa3SQBTCQW4tRg2oGpJSwDzEwxwazE4gKrlMEQLAz4ttw+wPfhRcUfenH/tM4mfO87n8ct3J354YMAgzy92ALuWcwnshj1nnhnunPHcTLL3zO1iyTbezRJAhxnOnJ2AXcsZBjYJ3rbDjBtuHAMxbiduOMa7AaQlweA2bi2Sf9sO24O0ABnnQFo2/yCkRRpoS+KG820gxgGQlm14bTHsYWyTljlzOHnDDTZma9m25MSZbbnbLBIMJHD6xZ6H+Zjkm4rDthvOH2O8+bbNLrGf+ezmmz8qbOT5pbFrYWBgbIDQEqgKJHAoRwb8B4hQNApGwSgYBSMSAABVBWPJyHKE8gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7809-5009","institution":"Jiangxi Provincial People's Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Haohang","middleName":"","lastName":"Ruan","suffix":""},{"id":56628166,"identity":"82b80683-8fe5-41b5-931d-7fdbf12a418a","order_by":1,"name":"Qing Huang","email":"","orcid":"","institution":"Jiangxi Provincial People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Huang","suffix":""},{"id":56628167,"identity":"3a002851-aa17-48d5-b130-98a6a9bd4e7a","order_by":2,"name":"Benting Wan","email":"","orcid":"","institution":"Jiangxi University of Finance and Economics","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Benting","middleName":"","lastName":"Wan","suffix":""},{"id":56628168,"identity":"c0d3922e-57f7-4e21-b451-13c386f655fa","order_by":3,"name":"Ming Yang","email":"","orcid":"","institution":"Jiangxi Provincial People's Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2021-10-14 18:27:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-969399/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-969399/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14632430,"identity":"9f25e5ab-fcbd-4193-bef5-d7c6300992f6","added_by":"auto","created_at":"2021-10-18 17:20:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3010668,"visible":true,"origin":"","legend":"Curcumin attenuated LPS induced cell injury in VSMCs. The effects of curcumin (0, 50, 100, 150μg/ml) on LPS-induced VSMCs. (A)MTT assay was applied to detect cell viability. (B)Flow cytometry was used to monitor cell apoptosis. (C)western blot was applied to detect apoptosis the levels of related protein cleaved caspase3 and cleaved caspase9. *P\u003c0.05; **P\u003c0.01; ***P\u003c0.001. LPS, liposaccharide.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-969399/v1/f9db1164d9352e385f907509.png"},{"id":14632429,"identity":"56ad9a6e-ba80-4574-b067-82bf609c1e02","added_by":"auto","created_at":"2021-10-18 17:20:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":789751,"visible":true,"origin":"","legend":"Curcumin inhibited LPS-induced production of inflammatory cytokines in VSMCs cells. The effects of curcumin (0, 50, 100, 150μg/ml) on LPS-induced VSMCs. (A)QRT-PCR was used to monitor the expressions of TNF-α，IL-1β and IL-6 mRNA. (B) western blot was applied to detect proteins level of TNF-α，IL-1β and IL-6. (C) ELISA was used to detect the content of TNF-α，IL-1β and IL-6 in VSMCs. **P\u003c0.01; ***P\u003c0.001. LPS, lipopolysaccharide.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-969399/v1/0e29509e6e64c6fd70db3dfe.png"},{"id":14632431,"identity":"0d800bf4-da8c-4e92-ab5d-308357563f38","added_by":"auto","created_at":"2021-10-18 17:20:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":818344,"visible":true,"origin":"","legend":"Curcumin increased TLR4 expression in VSMCs cells treated with LPS. The effects of curcumin (0, 50, 100, 150μg/ml) on LPS-induced VSMCs. Western blot was applied to detect the expression of TLR4. **P\u003c0.01; ***P\u003c0.001. LPS, lipopolysaccharide; TLR4, Toll-like receptor 4.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-969399/v1/a4b04f806e73fa8684eb7561.png"},{"id":14632432,"identity":"ac3dd8a8-e3a9-479f-89d2-5f7cb30f00b3","added_by":"auto","created_at":"2021-10-18 17:20:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3303021,"visible":true,"origin":"","legend":"Curcumin relieved LPS induced VSMCs cell injury by inhibiting NF-κB and JNK signaling pathways. The effects of curcumin(0, 50, 100, 150μg/ml) on LPS-induced VSMCs. (A, B) western blot was applied to detect the expressions of JNK / p-JNK, Jun / p-Jun, p65 / p-p65, IκBα / p-IκBα. *P\u003c0.05; **P\u003c0.01; ***P\u003c0.001. LPS, lipopolysaccharide.","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-969399/v1/7a8a3a8b1b68afe4e161e2bb.png"},{"id":14632433,"identity":"68395d6a-f65f-4e92-8c69-144a6d64a61f","added_by":"auto","created_at":"2021-10-18 17:20:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1504339,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-969399/v1/92f3edf9-076c-43cf-a8a9-7e366db07d99.pdf"},{"id":14632428,"identity":"a02c9a1c-efb1-4b51-857f-cad2816bb6a0","added_by":"auto","created_at":"2021-10-18 17:20:45","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54647,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-969399/v1/ca56f0a8076c87f211117e73.png"}],"financialInterests":"","formattedTitle":"\u003cp\u003eCurcumin Alleviates LPS-Induced Inflammation and Apoptosis in Vascular Smooth Muscle Cells Via Inhibition of the NF- κ B and JNK Signaling Pathways\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAtherosclerosis (AS) is the basic pathological basis of ischemic cardiovascular and cerebrovascular diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Most scholars believe that AS is a chronic inflammatory reaction, and a variety of inflammatory factors and inflammatory cells participate in its occurrence and development. Recent studies suggest that endothelial damage is an important initiating factor in the formation of atherosclerotic plaque [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Vascular endothelial cells and smooth muscle cells are damaged by various risk factors, which leads to an excessive chronic inflammatory proliferative reaction in the local blood vessels [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Recent studies have reported that several Chinese herbal medicines can improve the symptoms of AS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These findings have aroused people's interest in the treatment of AS with traditional Chinese medicine.\u003c/p\u003e \u003cp\u003eCurcumin is a plant polyphenol extracted from Curcuma longa and other medicinal plants. It has anti-inflammatory, anti-tumor, anti-oxidation and immunomodulatory effects [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which are related to its regulation of some molecular targets, including pro-inflammatory cytokines, growth factors, factors involved in proliferation and apoptosis, adhesion molecules and transcription factors [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Especially in vascular cells, curcumin can inhibit the over expression of pro-inflammatory cytokines induced by oxidized low density lipoprotein (ox LDL) in VSMCs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the molecular mechanism of curcumin inhibiting LPS induced inflammation in VSMCs remains unclear.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to investigate the therapeutic effect of curcumin on AS. The inflammatory 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.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell culture and treatment\u003c/h2\u003e \u003cp\u003eRat cell line VSMCs was cultured in Dulbecco's modified Eagle medium ((DMEM; Gibco BRL,Gaithersburg, MD, USA) containing 10% (v / v) fetal bovine serum (FBS༌Gibco) in 37\u0026deg;C, 5%CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Curcumin and LPS are purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Firstly, VSMCs cells were treated with 10 \u0026micro; M LPS for 12 hours to establish the injury model. Then VSMCs cells were treated with different concentrations of curcumin (0, 50, 100, 150 \u0026micro; g / mL) for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell viability assay\u003c/h2\u003e \u003cp\u003eAfter digestion, the cells in the logarithmic growth phase were inoculated into 96-well plates at the density of 5 \u0026times; 10 \u003csup\u003e3\u003c/sup\u003e/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 \u0026micro; 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Apoptosis assay\u003c/h2\u003e \u003cp\u003eCell 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 \u0026times; 10 \u003csup\u003e6\u003c/sup\u003e cells per well, cultured for 24 hours, the supernatant was discarded, pre-cold PBS was washed twice, 1 \u0026times; Binding buffer was used to suspend cells. The cell suspension was added with 5 \u0026micro; L annexin V-FITC and 5 \u0026micro; l PI, After mixing well, the cells were incubated at room temperature for 15 minutes, The apoptotic rate was detected by flow cytometry (Beckman Coulter, Fullerton, CA, USA) within 1 hour, and the data were analyzed by Flow Jo software (tree star, Ashland, or, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Enzyme-linked immunosorbent assay\u003c/h2\u003e \u003cp\u003eVSMCs were seeded in 24 well culture dish and treated with different methods for 24 hours. The culture supernatant was collected. The concentrations of inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α) were measured by ELISA kit (TaKaRa, Dalian).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Quantitative Real-time PCR\u003c/h2\u003e \u003cp\u003eAccording 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 amplified as a control. The relative expression levels of IL-6, IL-1 β and TNF- α were calculated by the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Western blot\u003c/h2\u003e \u003cp\u003eThe total proteins of different groups of cells were extracted by a protein extraction kit. The protein concentration was determined by BCA (Pierce, Appleton, WI, USA). Total proteins were separated by SDS-PAGE electrophoresis with 50 \u0026micro; g total proteins in each well. After electrophoresis for 2 hours, the membrane was transferred to the PVDF membrane (Millipore, Billerica, Ma, USA) by the wet method. The membrane was sealed with 5% skimmed milk powder for 1 hour, and the primary antibody anti-Bax (ab32503, 1:1000), anti-pro-Caspase3 (ab32150, 1:1000), anti cleaved-Caspase3 (ab32042, 1:1000), anti-IL-6 (ab233706, 1:1000), anti IL-1β (ab216995, 1:1000), anti-TNF-α (ab183218, 1:1000), anti-p65 (ab32536, 1:1000), anti-toll-like receptor 4 (anti-TLR4, ab22048, 1:500), anti-p-p65 (ab31624, 1:1000), anti-IκBα (ab109300, 1:1000), anti-JNK (ab176645, 1:1000), anti-p-JNK (ab176645, 1:1000), anti-c-Jun (ab40766, 1:1000) and anti-p-c-Jun (ab32385, 1:1000), anti-β-actin(ab8226, 1:2000), all purchased from Abcam (Cambridge, UK) was incubated at 4 ℃. Overnight, In the next morning, TBST was used to rinse the membrane, horseradish peroxidase-labeled second antibody was added and incubated at 37 ℃ for 1 h. ECL was used to develop and save the image. The optical density value was measured by quantity one.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe data of this study were expressed by mean \u0026plusmn;standard deviation (SD), and the statistical software SPSS 19.0 was used (IBM Analytics, New York, USA), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05was considered to be a statistically significant result. All experiments are repeated at least three times.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e3.1. Curcumin attenuated LPS induced cell injury in VSMCs\u003c/h2\u003e\n \u003cp\u003eTo investigate the effect of curcumin on the inflammatory injury of VSMCs cells induced by lipopolysaccharide (LPS), the cell viability was observed by CCK- 8 assay, and the apoptosis rate was determined by flow cytometry before and after curcumin treatment. Consistent with previous studies [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e], 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 \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(A)). Besides, the percentage of apoptotic cells in the LPS treatment group was significantly higher than that in the control group, but curcumin could inhibit the pro-apoptotic effect of LPS in a dose-dependent manner (Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(B)). In order to further confirm our results, a western-blot was used to detect the protein expression levels of specific 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 significantly higher than those in the control group, but the promoting effect of LPS and curcumin combined treatment group was significantly 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.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e3.2. Curcumin inhibited LPS-induced production of inflammatory cytokines in VSMCs cells\u003c/h2\u003e\n \u003cp\u003eQRT-PCR, western blot and ELISA assay were used to detect the effect of curcumin on the production of inflammatory cytokines TNF- \u0026alpha;, IL-1 \u0026beta; 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-\u0026alpha;, IL-1\u0026beta; and IL-6 in VSMCs cells treated with LPS were significantly higher than those in the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Whereas, the increased expression of TNF- \u0026alpha;, IL-1\u0026beta; and IL-6 induced by LPS decreased in a dose-dependent manner under curcumin treatment (Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026amp;B). The results of ELISA were consistent with the above results. Compared with the control group, the release of TNF-\u0026alpha;, IL-1\u0026beta; and IL-6 in the LPS treatment group was significantly increased, while the promoting effect of LPS and curcumin on the release of TNF- \u0026alpha;, IL-1 \u0026beta; and IL- 6 induced by LPS was decreased. The release of inflammatory cytokines decreased with the increase of curcumin concentration (Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). It was suggested that curcumin could inhibit the excessive secretion of TNF-\u0026alpha;, IL-1\u0026beta; and IL-6 in VSMCs cells induced by LPS in a dose-dependent manner.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003e3.3. Curcumin increased TLR4 expression in VSMCs cells treated with LPS\u003c/h2\u003e\n \u003cp\u003eTo 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 \u0026micro; g / ml) co-treated with LPS for 24 hours. The results showed that LPS significantly induced the expression of TLR4 in VSMCs cells, while curcumin significantly 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 \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003ch2\u003e3.4 Curcumin relieved LPS induced VSMCs cell injury by inhibiting NF- \u0026kappa; B and JNK signaling pathways\u003c/h2\u003e\n \u003cp\u003eNext, we discussed the effect of curcumin on the regulation of NF- \u0026kappa; B and the JNK signal pathway. Curcumin (50,100,150 \u0026micro; g / ml) was co-treated with LPS for 24 hours. The protein levels of NF- \u0026kappa; 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-l\u0026kappa;B\u0026alpha;, p-JNK and p-c-Jun, indicating that LPS could promote the signal transduction of NF- \u0026kappa;B and JNK pathways. Under the action of curcumin, the expressions of p-p65, p-l\u0026kappa;B\u0026alpha;, p-JNK and p-c-Jun were down-regulated in a dose-dependent manner, suggesting that curcumin could inhibit NF-\u0026kappa;B and JNK signal pathways. In a word, curcumin attenuated LPS induced VSMCs cell injury by inhibiting NF- \u0026kappa; B and JNK signal pathways (Figure A\u0026amp;B).\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAS is the basic pathological basis of coronary heart disease. At present, it is considered that lipid metabolism disorder, oxidative stress and inflammatory reaction are important factors in the occurrence and development of AS [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A clinical trial has been confirmed that anti-inflammatory intervention is more accurate and personalized in patients with coronary heart disease [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, the LPS-induced VSMCs model was used to explore the effects of curcumin on apoptosis and inflammation of VSMCs cells. We found that curcumin could reduce the inflammatory injury induced by LPS by increasing the viability of VSMCs cells, reducing apoptosis and inhibiting the release of inflammatory cytokines. On the other hand, we found that curcumin increased the expression of toll-like receptor 4 (TLR4) in LPS-treated 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.\u003c/p\u003e \u003cp\u003eCurcumin and its analogs have long been used to treat various diseases, and modern science has also provided a reliable scientific basis for the pharmacological effects of curcumin. A large number of studies have confirmed that curcumin has anti-inflammatory, antioxidant and other pharmacological effects by regulating various targeting factors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. 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-inflammatory and antioxidant effects [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Meng \u003cem\u003eet al\u003c/em\u003e have shown that curcumin through ROS related TLR4-MAPK /NF- κB pathway inhibits LPS induced inflammation in rat vascular smooth muscle cells in vitro [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. On the other hand, curcumin decreased the expression of NF- κBp65 and DNA binding activity of NF- κB in VSMCs exposed to LPS [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. AS is an important cause of coronary heart disease, cerebral infarction, peripheral vascular disease and other diseases, and is related to inflammation, 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 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Zhang \u003cem\u003eet al\u003c/em\u003e 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 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, more and more studies have shown that oxidative stress and inflammatory 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 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Wang \u003cem\u003eet al\u003c/em\u003e have shown that curcumin inhibiting the JNK pathway can prevent diabetic cardiomyopathy [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, we further discussed the effect of curcumin on the expression of TLR4. We found that curcumin could significantly promote the expression of TLR4 in LPS-treated VSMCs cells.\u003c/p\u003e \u003cp\u003eMoreover, 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 inflammation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Inhibition of this pathway can improve cell viability and reduce apoptosis and inflammatory injury. P65 and IκBα are key molecules of the NF-κB signaling pathway, and their phosphorylation usually increases apoptosis and inflammation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 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 inflammatory 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 inflammation. JNK and its downstream molecule c-Jun are activated after injury [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eTo sum up, curcumin can protect VSMCs cells from LPS-induced inflammatory injury by improving cell viability, reducing apoptosis and inhibiting the production of inflammatory 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWan 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 \u0026amp; D project of Jiangxi Provincial Department of science and technology, the second undertaker.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQi Kong, Xin Ma, Jun-Xuan Lyu, et al. Plasma RANTES level is correlated with cardio-cerebral atherosclerosis burden in patients with ischemic cerebrovascular disease. 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Toxicol Appl Pharmacol. 2019;371:3-11.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Curcumin, Lipopolysaccharide, vascular smooth muscle, NF- κ B, JNK, atherosclerosis, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-969399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-969399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Curcumin plays an important role in inflammation. This study was aimed to investigate the effect of curcumin on vascular smooth muscle cells (VSMCs) injury induced by lipopolysaccharide (LPS) and its mechanism. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eVSMCs cells were treated with different concentrations of curcumin (0, 50, 100 and 150 μ g / mL). CCK-8 assay and flow cytometry were used to monitor the effects of curcumin on LPS-induced cell viability and apoptosis. The expression and release of inflammatory 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. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCurcumin could reduce LPS induced inflammatory injury by increasing VSMC's cell viability, reducing apoptosis and inhibiting the release of inflammatory 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. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eCurcumin can protect VSMCs from LPS induced inflammatory 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.\u003c/p\u003e","manuscriptTitle":"Curcumin Alleviates LPS-Induced Inflammation and Apoptosis in Vascular Smooth Muscle Cells Via Inhibition of the NF- κ B and JNK Signaling Pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-10-18 17:20:43","doi":"10.21203/rs.3.rs-969399/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-10-21T23:13:20+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2021-10-13T13:15:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammopharmacology","date":"2021-10-11T15:53:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammopharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"iphm","sideBox":"Learn more about [Inflammopharmacology](https://www.springer.com/journal/10787)","snPcode":"10787","submissionUrl":"https://submission.nature.com/new-submission/10787/3","title":"Inflammopharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d52cfddf-6db2-4daa-bb4b-f26b2c0d0b5f","owner":[],"postedDate":"October 18th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":7918800,"name":"Immunology"},{"id":7918801,"name":"Clinical Pharmacology"}],"tags":[],"updatedAt":"2021-12-08T08:01:28+00:00","versionOfRecord":[],"versionCreatedAt":"2021-10-18 17:20:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-969399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-969399","identity":"rs-969399","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}