DOI: https://doi.org/10.21203/rs.3.rs-944266/v1
Obesity is a growing global health problem and chronic over-nutrition disease with lipid accumulation that results in low-grade chronic inflammation in the microenvironment of adipose tissue. Triptolide is a diterpene lactone compound extracted from the roots of the Chinese herb TWHF and possesses a therapeutic potential due to its immunosuppressive and anti-inflammatory properties. In this study, we built obesity-related inflammatory models of adipocytes using LPS, Ma-CM and raw264.7 macrophages, while the obesity-related inflammatory models of macrophages were built using LPS and Ad-CM system. We used these inflammatory models to investigate the anti-inflammatory property of triptolide. Treatment of triptolide (0.005, 0.010, 0.020 and 0.040 μ M) inhibited LPS-induced or macrophages conditioned medium-stimulated activation of AMPK/mTOR signaling pathway (p < 0.05). The results showed that triptolide reduced the release of chemokines MCP-1, RANTES, EOTAXIN and KC in LPS, Ma-CM or RAW264.7 macrophages-stimulated 3T3-L1 adipocytes. Triptolide also diminished MCP-1, RANTES, EOTAXIN, KC and TNF-α in Ad-CM stimulated RAW264.7 macrophages, while expression of MCP-1, RANTES, TNF-α, GM-CSF and IL-6 was decreased in LPS stimulated RAW264.7 macrophages (p < 0.05). These results demonstrate that triptolide is not only effective against inflammatory response of RAW264.7 macrophages or 3T3-L1 adipocytes, but also disrupts the crosstalk between macrophages and adipocytes, particularly by inhibiting secretion of pro-inflammatory mediators through inhibiting the activation of AMPK/mTOR signaling pathway. Triptolide might benefit to ameliorate obesity-induced inflammatory diseases.
Obesity is a widespread and growing global health problem. In 2016, more than 1.9 billion adults were overweight, and over 650 million were obese, and these numbers are expected to continue to rise over the next decade[1]. In obesity body metabolic abnormalities and long-term hyperglycemia or hyperlipidemia are likely to induce overactive and continuous inflammatory reactions, which may type 2 diabetes, hepatic steatosis, arteriosclerosis, hyperlipidemia, arthritis, Alzheimer’s disease, hypertension and cancer[2].
The adipose tissue in which fat is stored contained adipocytes, fibroblasts, immune and endothelial cells. Adipocytes distended by droplets of fat not only act as a reservoir for energy storage, but also secrete paracrine factors to regulate other metabolic tissues[3, 4]. In obesity, adipocytes may not expand properly to store the energy excess, then releases adipokines, cytokines and chemokines (e.g., regulated upon activation normal T cell expressed and secreted factor(RANTES), monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF-α) to elicit recruitment of immune cells, such as macrophages, B cells and T cells[5]. Macrophages are closely correlated with inflammatory responses under obesity stress and correspond to about 40% of total adipose cells, as compared to only 18% in lean controls[6]. s obesity progresses, the macrophages become the major producers of chemokines and proinflammatory cytokines[7, 8]. This mechanism leads to lipolysis in adipocytes, systemic insulin resistance and chronic low-grade systemic inflammation which is an increased risk of metabolic diseases, pulmonary diseases and cancer[9].
Tripterygium wilfordii Hook.F. (TWHF, Leigongteng Family Celastraceae), commonly known as thunder god vine in China, and its preparations are widely used to treat inflammatory and autoimmune diseases, including rheumatoid arthritis, systemic lupus, psoriatic arthritis and nephritis[10–12]. Triptolide is a diterpene lactone compound extracted from the roots of the Chinese herb TWHF[13], it possesses a broad-spectrum therapeutic potential due to its immunosuppressive, anti-inflammatory and anti-tumor properties[14–17], through modulating the relevant signaling pathways involved in the regulation of HSP70[18], MAPK, PI3K[19], Jak2, Bcl-2/Bax[20], JNK-1/2, MPK1, ERK-1/2, NF-κB[21], reactive oxygen species (ROS) and caspase 8[22].
Adenosine 5-monophoshate-activated protein kinase (AMPK), a cellular energy sensor, is an upstream regulator of key enzymes in oxide synthesis and fat metabolism[23, 24]. AMPK is a heterotrimeric kinase consisting of one catalytic subunit α and two regulatory subunits, β and γ. The α subunit, of which two isoforms exist (α1 and α2), contains the kinase domain and a critical reversible phosphorylation site αThr172, the phosphorylation of which is required for full kinase activity[25–27]. Once AMPKα activated, it switches adipocytes from anabolic to catabolic mode, regulate energy metabolism by promoting glycolysis and fatty acid oxidation, and prevent gluconeogenesis[28, 29]. AMPK also regulates genes of cytokines and chemokines transcription by modulating mammalian target of rapamycin (mTOR) which is a serine-threonine kinase[30]. AMPK/mTOR signaling pathway is a vital link between immune function and metabolism. In the present study, we set up models to explore the crosstalk between RAW264.7 macrophages and 3T3-L1 adipocytes, then investigated the anti-inflammatory property of triptolide through modulating AMPK/mTOR signaling pathway on 3T3-L1 adipocytes.
Cell Culture and reagents 3T3-L1 adipocytes and raw264.7 macrophages were purchased from the China Infrastructure of Cell Line Resource (Beijing, China) and maintained in DMEM (Gibco) supplied with 10% heat-inactivated calf serum or fetal bovine serum (Gibco), and 1% Penicillin and Streptomycin solution (Beyotime) at 5% CO2 and 37 ℃ humidified incubator with 5% CO2 atmosphere. When cell confluence reached 70–80%, cells were passaged by trypsin with 0.05% EDTA (Gibco). 3T3-L1 adipocytes were planted in 24 wells plates to induce differentiation(1×105cells/well). Confluent 3T3-L1 adipocytes were incubated with high-glucose DMEM(Gibco) which contained 10% fetal bovine serum, 1 µM of dexamethasone (DEX, Sigma), 0.5 mM of 3-isobutyl-1-methylxanthine (IBMX, Sigma), and 10 µg/mL of insulin (Beyotime) for three days. Then the medium was replaced with high-glucose DMEM containing 10% fetal bovine serum and 10 µg/mL of insulin and again every 2 days. After ten days, the cells that accumulated large lipid droplets were used as hypertrophied 3T3-L1 adipocytes. 3T3-L1 adipocytes or raw264.7 macrophages planted in 24 wells (1×105cells/well) were cultured by DMEM (Gibco) supplied with 2% heat-inactivated fetal bovine serum for 24 h, then the supernatant was collected and named as adipocytes-conditioned Medium (Ad-CM) or macrophages-conditioned Medium(Ma-CM).99.6% Triptolide was purchased from National Institutes for Food and Drug Control (Beijing, China) dissolved in dimethyl sulfoxide (DMSO, Sigma) as a stock solution, and then stored at − 20°C. The concentration of DMSO in the vehicle group was equal to the 3.9×10− 4 % DMSO in the highest (0.040µM) dose of triptolide.
Lipopolysaccharide (LPS)-stimulated inflammatory assay 3T3-L1 adipocytes or RAW 264.7 macrophages were seeded in 24-well plates at a density of 1 × 105 cells/well and stimulated with LPS (100 ng/mL) in the presence or absence of different concentrations of triptolide for 24 h. Then the medium supernatant and cells were collected and stored at − 80°C for Luminex assay or Western blotting experiments.
Conditioned Medium (CM)-stimulated inflammatory assay 3T3-L1 adipocytes or RAW 264.7 macrophages were seeded in 24-well plates at a density of 1 × 105 cells/well for 24 h. Then the supernatants were collected and centrifuged to remove cell debris. 3T3-L1 adipocytes or RAW 264.7 macrophages were treated in 50% Ma-CM (RM) or Ad-CM (AM) medium followed by triptolide stimulation for 24 h. Then the medium supernatant and cells were collected and stored at − 80°C.
Macrophages-stimulated inflammatory assay The 3T3-L1 adipocytes (1×105 cells/well) were seeded in 24-well plates and differentiated into adipocytes until day eleven. Then The RAW264.7 macrophages (2.5×104 cells/well) were plated in transwell inserts (0.4 µ m pore size, Corning) which would be put in plates with 3T3-L1 adipocytes. The cells treated with or without different concentrations of triptolide were cultured for 24 h and harvested for analysis. 3T3-L1 adipocytes without RAW264.7 macrophages cultured in fresh culture medium alone was as a control.
Cell proliferation assay The extent of triptolide’s cytotoxicity on 3T3-L1 adipocytes or RAW264.7 macrophages was examined by the Cell Counting Kit-8 (Dojindo) according to manufacturer’s instructions.3T3-L1 adipocytes or RAW264.7 macrophages were four repeats for one group, when were seeded in a 96-well plate with 1×104 cells/well, and treated with the following conditions: fresh culture medium alone (control), fresh culture medium with different concentrations (0.005,0.010,0.020 and 0.040 µM) of triptolide according to 24 h. Then the CCK-8 solution was added to the culture medium and incubated at 37°C for 2 h. The absorbance was read at 450 nm with a microplate reader (Bio-Tek). Each experiment was repeated three times.
Oil Red O Staining Differentiated 3T3-L1 adipocytes grown in 24-well plates were washed twice with PBS, fixed with 10% of formalin in PBS, for 1 h and washed twice with 60% isopropanol. The fixed cells were then stained using oil red O(Solarbio) solution for 30 min and washed with distilled water at room temperature. After drying, the fixed cells were imaged by an inverted microscope༈CKX53,Olympus༉.
Western blotting Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitor cocktail (Beyotime). Protein quantification was performed with the BCA Protein Assay Kit (Beyotime). Twenty micrograms of total protein extract were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and were transferred to polyvinylidene difluoride (PVDF,Millipore) membranes. The membranes were blocked with 5% BSA and were then incubated with specific antibodies overnight at 4°C. A horseradish peroxidase-labeled secondary antibody was used and detected using enhanced chemiluminescence (ECL) kits (Beyotime). The primary antibodies used were listed as follows: anti- AMPKα (Cell Signaling Technology, 5831S, 1:1000), anti-Phospho-AMPKα (Thr172) (D79.5E) (Cell Signaling Technology, 4188S, 1:1000), anti-mTOR (Cell Signaling Technology, 2983S, 1:1000), anti-Phospho-mTOR (Ser2448) (D9C2) (Cell Signaling Technology, 5536S, 1:1000), anti-4EBP1 (Cell Signaling Technology, 9452S, 1:1000), anti-Phospho-4EBP1 (Ser65) (Cell Signaling Technology, 9451S, 1:1000), anti-GAPDH (proteintech,10494-1-AP, 1:10000).
Luminex assay The 9-Plex Mouse Magnetic Luminex Assay (R&D Systems) was used to measure the cytokine levels of Supernatant with analyses performed in triplicate. The cytokines included MCP-1, RANTES, EOTAXIN, GM-CSF, IL-6, KC and TNFα. The concentration values were obtained from the mean fluorescent intensity (MFI) according to the manufacturer’s instructions and the results were analyzed by the Bio-Plex manager software (version 2.0). Standard curves were generated from the reference cytokines gradient concentrations; the concentrations of these cytokines in samples were calculated from the standard curves.
Statistical analysis All data were analyzed from three independent experiments, and data are presented as means ± standard deviation (SD). Differences between the groups were analyzed by one-way ANOVA, followed by the Dunnett T3 for multiple comparisons using SPSS software (version 16.0), A value of P < 0.05 was considered significant.
Triptolide did not affect 3T3-L1 adipocytes or raw264.7 macrophages viability. To evaluate the cytotoxic effects of triptolide on 3T3-L1 pre-adipocytes or raw264.7 macrophages, cells were incubated with various concentration (0.005, 0.010, 0.020 and 0.040 µM) for 24 h. As compared with control cells that received no treatment, cells were treated with triptolide at concentrations up to 0.040 µ M showed viability more than 90% (Fig. 1A), suggesting that triptolide is safe and non-toxic at doses up to 0.040 µ M in both of 3T3-L1 pre-adipocytes and RAW 264.7 macrophages. To investigate the effect of triptolide on hypertrophied 3T3-L1 adipocytes that accumulated large lipid droplets, cells were incubated with or without triptolide (0.040 µM) for 24 h, then they were fixed with formalin and stained using oil red O. There was not significant difference in Oil Red O staining between the two group cells (Fig. 1B), suggesting that triptolide did not affect 3T3-L1 adipocytes growth and fat accumulation storage.
Triptolide inhibited inflammatory chemokines and cytokines production in LPS stimulated 3T3-L1 adipocytes or RAW 264.7 macrophages
To evaluate the anti-inflammatory actions of triptolide on 3T3-L1 adipocytes, the effects on inflammatory mediators were analyzed. We examined the effect of triptolide on the production of inflammatory cytokines in 3T3-L1 adipocytes or RAW 264.7 macrophages in response to LPS, a stimulus that induces the production of MCP-1, RANTES, EOTAXIN, GM-CSF, IL-6, KC and TNFα in immune cells. Production of MCP-1, RANTES, IL-6, and TNFα in LPS stimulated raw 264.7 macrophages were reduced by triptolide at highest concentration (0.040 µ M), when secretion of GM-CSF exhibited a dose-dependent decrease with triptolide (0.005,0.010,0.020,0.040µM) (Fig. 2). Expression of EOTAXIN and KC was not repressed by triptolide at any concentration. In LPS stimulated 3T3-L1 adipocytes, triptolide showed a dose-dependent decrease of RANTES, MCP-1, KC and EOTAXIN, when the expression of GM-CSF, IL-6 and TNFα was low and undetectable in 3T3-L1 adipocytes (Fig. 3).
Triptolide inhibited inflammatory chemokines and cytokines production in conditioned Medium (CM) stimulated 3T3-L1 adipocytes or RAW 264.7 macrophages In order to evaluate the anti-inflammatory effect of triptolide in obesity-related environment, raw264.7 cells were cultured in 50% Ad-CM medium when 3T3-L1 cells were cultured in 50% Ma-CM medium. Secretion of RANTES, MCP-1 and KC was lower by triptolide (0.005,0.010,0.020,0.040µM) in Ad-CM stimulated raw 264.7 macrophages(Fig. 4). Triptolide treatments showed a dose-dependent decrease in TNFα secretion and secretion of EOTAXIN was diminished by triptolide at only two concentrations (0.005,0.040µM) (Fig. 4).
Expression of GM-CSF and IL-6 was unable to detected Significant differences between the groups in Ad-CM stimulated RAW 264.7 macrophages. Productions of RANTES, MCP-1, KC and EOTAXIN in Ma-CM stimulated 3T3-L1 adipocytes had a significant decrease at concentrations of (0.005,0.010, 0.020, 0.040µM) triptolide (Fig. 5).
Triptolide inhibited inflammatory chemokines and cytokines production in macrophages stimulated 3T3-L1 adipocytes Potential mediators of the interactions between 3T3-L1 adipocytes and RAW 264.7 macrophages were evaluated using a transwell co-culture system. When both cell types were present, secretion of MCP-1 and KC was obviously pared all concentrations of triptolide when production of RANTES and EOTAXIN was lessened at only two concentrations (0.005,0.040µM) (Fig. 6).
Triptolide suppresses the activity of AMPKα/mTOR pathway in Inflammatory adipocytes We next analyzed the relationship between triptolide and AMPK/mTOR signaling pathway in (Ma-CM) -treated or LPS-induced 3T3-L1 adipocytes. Activation of AMPKα, mTOR and 4EBP1 was assessed by measuring the levels of expression and phosphorylation of these proteins (Fig. 7 and Fig. 8). Compared with the control group, we found that treatment of Ma-CM for 24 h obviously increased pAMPKα (Thr172) and p4EBP1(Ser65) protein expression in 3T3-L1 adipocytes, when the expression of 4EBP1 and pmTOR (Ser2448) was reduced. We also observed that LPS enhanced AMPKα, pAMPKα (Thr172), p4EBP1(Ser65) and inhibited 4EBP1 and pmTOR (Ser2448) protein expression. 3T3-L1 adipocytes were
Incubated with triptolide (0.005,0.010,0.020,0.040µM) for 24 h. The western blot assay (Fig. 7) showed triptolide at highest concentration (0.040 µ M) increased expression level of 4EBP1, pmTOR (Ser2448) and p4EBP1 (Ser65), while triptolide at higher concentration (0.020 µ M) decreased expression level of pAMPKα (Thr172). The enhancement effects were found of triptolide at low concentration (0.005,0.010 µ M) on 4EBP1 and pmTOR (Ser2448) protein expression, while inhibition effect of triptolide (0.010 µ M) was discovered on pAMPKα (Thr172) protein expression (Fig. 7). Triptolide (0.040 µ M) treatment suppressed AMPKα, pAMPKα (Thr172), 4EBP1 and p4EBP1 (Ser65)
while raised pmTOR (Ser2448) protein expression in LPS-induced 3T3-L1 adipocytes (Fig. 8). The expression levels of pAMPKα (Thr172) protein were significantly diminished by triptolide (0.050,0.020 µ M) and 4EBP1 protein expression was increased by triptolide (0.050,0.010 µ M) (Fig. 8).
With consequent adverse economic impact estimated to be $2 trillion or 2.8% of annual global GDP, obesity is one of the top five risk factors for mortality globally, and reduced prevalence has been identified as the most likely strategy to prevent loss of life in modeling to 2040[31].Obesity is a chronic over-nutrition disease with lipid accumulation that results in low-grade chronic inflammation in the microenvironment of adipose tissue. The increasing intestinal permeability in obesity leads higher circulating levels of LPS from intestinal Gram-positive bacterial species[32, 33]. LPS may initiate an inflammatory cascade via activation of pattern recognition receptors (PRRs) such as TLR4 in adipose tissue, leading to significant inflammatory reactions and adipocytes dysfunction[34]. Recently, various Many treatments are focused on obesity-related inflammation[35–37]. Triptolide is a promising herb with anti-inflammatory and immunosuppressive activity. It is used to suppress immune rejection following organ transplantation, and treat autoimmune disorders, asthma and arthritis[38, 39]. In this study, we built obesity-related inflammatory models of adipocytes using LPS, Ma-CM and raw264.7 macrophages, while the obesity-related inflammatory models of macrophages were built using LPS and Ad-CM system. We used these inflammatory models to investigate whether triptolide works as a potential natural agent with anti-inflammatory property in both macrophages and adipocytes, and disrupts the crosstalk between the two cells.
AMPK is a widely expressed multi-substrate serine/threonine kinase and a well-known sensor of the intracellular energy state that responds to metabolic stresses and other regulatory signals[40]. MTOR is a serine/threonine-protein kinase which senses various environmental and intracellular changes including nutrient availability and energy status, and coordinates diverse cellular processes including cell growth, differentiation, autophagy, survival, and metabolism, it is a common catalytic subunit both in mTORC1 and mTORC2[41]. MTORC1 consists of mTOR, regulatory-associated protein of mTOR(Raptor), proline-rich Akt substrate (PRAS40), DEP domain-containing mTOR-interacting protein (DEPTOR), and G protein beta subunit like(mLST8)[42]. MTORC1 complex promotes protein synthesis largely through the phosphorylation of two key effectors, p70S6 Kinase 1 (S6K1) and eIF4E Binding Protein (4EBP)[43].AMPK kinase activation required that phosphorylation of AMPKα at Thr172 in the activation loop, activated AMPKα phosphorylates and suppresses the activity of mTOR and 4EBP through phosphorylation of Raptor at Ser722/Ser792, 4E-BP1 inactivation leads to the translation of mRNA of chemokines and cytokines.To clarify the mechanism underlying the anti-inflammatory effects of triptolide, we examined whether treatment with triptolide influenced the phosphorylation and expression of AMPKα,mTOR and 4E-BP1.Both LPS and Ma-CM increased the phosphorylation of AMPKα and 4E-BP1, when the phosphorylation of mTOR and expression of 4E-BP1 was decreased. Ma-CM increased the expression of AMPKα, but LPS did not. The result indicated that Ma-CM may be a inflammatory property similar to LPS, and stimulate inflammatory response in adipocytes through AMPKα/mTOR pathway. The phosphorylation of AMPKα and 4E-BP1 was suppressed by treatment with triptolide, when the phosphorylation of mTOR and expression of 4E-BP1 and AMPKα was stimulated. The result indicated that triptolide inhibited inflammatory response of adipocytes and may involve in inhibition of AMPKα/mTOR pathway.
In this study, the cytokines release of macrophages and adipocytes were evaluated in vitro to investigate the inhibitory effects of triptolide on obesity-related inflammatory responses. Chemokines are small (8–12kDa) chemotactic cytokines that regulate cell migration and positioning in development, homeostasis, and inflammation[44]. Chemokines are defined by their primary amino acid sequence and the arrangement of specific structurally important cysteine residues within the mature protein. These form disulfide bonds that maintain the structure of the chemokine monomer, which consists of a central three stranded β-sheet, an overlying C-terminal α-helix, and a short unstructured N terminus that plays a critical role in receptor activation[45]. On a structural basis, chemokines can be subclassified into four subfamilies: the XC, CC, CXC, and CX3C chemokines.MCP-1/CCL2, RANTES/CCL5 and EOTAXIN/CCL11 are members of CC chemokine family, while KC is a member of CXC chemokine family[46]. These chemokines are ubiquitously expressed in adipose tissue to recruit Eosinophils, neutrophils, T cells and monocytes/ macrophages[47]. Eosinophils are critical for maintaining adipose tissue M2 macrophages as a major contributor of IL-4, a key factor for M2 polarization[48]. Neutrophils, T cells and monocytes/ macrophages are the producers of chemokines and inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, which have been shown to trigger inflammatory pathways, culminating in decreased insulin signaling[49]. Insulin regulates uptake of blood glucose by metabolic tissues including liver, skeletal muscles, and fat tissues[50]. Insulin signaling decreasing means insulin resistance that cells of the body don’t respond properly to the hormone insulin. Insulin resistance manifests as impaired glucose disposal in muscle and enhanced triglyceride lipolysis in adipose tissue, and leads to type 2 diabetes, cardiovascular disease and hyperglycemia[51, 52]. The results showed that triptolide reduced the release of chemokines MCP-1, RANTES, EOTAXIN and KC in LPS, Ma-CM or RAW264.7 macrophages-stimulated 3T3-L1 adipocytes. Triptolide also diminished MCP-1, RANTES, EOTAXIN, KC and TNF-α in Ad-CM stimulated RAW264.7 macrophages, while expression of MCP-1, RANTES, TNF-α, GM-CSF and IL-6 was decreased in LPS stimulated RAW264.7 macrophages. The results indicated that triptolide might improve insulin sensitivity through inhibiting expression of cytokines and chemokines in both macrophages and adipocytes.
In summary, this study indicated that triptolide reduced inflammatory responses in adipocytes possibly through inhibiting AMPKα/mTOR pathway. We demonstrated that triptolide is not only effective against inflammatory response of RAW264.7 macrophages or 3T3-L1 adipocytes, but also disrupts the crosstalk between macrophages and adipocytes, particularly by inhibiting secretion of pro-inflammatory mediators, might benefit to ameliorate obesity-induced inflammatory diseases. Triptolide could be a candidate for treating adipose tissue inflammation and related metabolic disorders and warrants further investigation.
Ethics statement
Our study did not involve human subjects, human data or tissue, or animals.
Authors' Contributions
Zhao Liu：Conceptualization, Methodology and Writing-Original draft preparation. Xue-li Li：Investigation, Validation and Funding acquisition. All authors consulted for the study and proofread the paper.
Conflicts of Interest
The authors declare no conflict of interest.
Acknowledgments
This study was supported by grants from Experimental Research Center, China Academy of Chinese Medical Sciences (ZZ2018019) and the Fundamental Research Funds for the Central public welfare research institutes (ZZ13-YQ-081).