MCP-1 Expression Research Articles

E076 To investigate the mechanisms of action of novel compounds ex vivo in cells obtained from the inflammatory synovium of patients with rheumatoid arthritis

Abstract Background/Aims To investigate the ex vivo effect of JAK1/2 inhibitor baricitinib on the expression of pro-inflammatory mediators in normal and RA diseased fibroblasts stimulated with oncostatin M (OSM) as well as in cells derived from RA inflamed synovial membrane cells (RA SMC). Methods RA SMC and fibroblast like synoviocytes (RA FLS) were isolated from synovial membranes of patients undergoing surgery using enzymatic digestion and cell propagation. Normal fibroblast-like synoviocytes (FLS) and normal human skin fibroblasts (HSF) were purchased from commercial sources. All fibroblasts were cultured in media supplemented foetal bovine serum and antibiotics in incubators using established laboratory procedures, and serum-starved prior to stimulation with OSM with or without baricitinib. RA SMC were cultured in the presence of baricitinib without OSM as they spontaneously release pro-inflammatory cytokines. Levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, CCL5 and IP-10) were determined by qPCR, ELISA and MSD. Western blot was used to measure levels of phospho-JAK specific components. Results qPCR analysis found that baricitinib inhibited OSM induced IP-10 and MCP-1 mRNA expression but not RANTES mRNA expression in normal human skin fibroblasts and RA FLS. In normal FLS however, baricitinib was found to inhibit RANTES and MCP-1 mRNA expression, but not IP-10 mRNA expression. Expression levels of IL-8 were too low for accurate conclusions to be drawn in all cell types apart from the RA SMCs, and in this case baricitinib did not inhibit IL-8 expression. ELISA results showed that baricitinib inhibited OSM induced MCP-1 expression in all cell types investigated. OSM induced IL-6 expression was inhibited in RA FLS but no other cell type. Conclusion Baricitinib inhibits OSM stimulated pro-inflammatory cytokine production through JAK1, 2. It has a varying inhibitory effect on the production of different cytokines: it is shown to particularly inhibit MCP-1. Baricitinib was found to have no effect on the production of spontaneously released pro-inflammatory cytokines in RA mixed cell populations. Disclosure M. Turner: None.

Roflumilast alleviates adenine-induced chronic kidney disease by regulating inflammatory biomarkers

The present investigation was carried out to explore the role of roflumilast, a PDE4 inhibitor, as a potential treatment option for chronic kidney disease. Forty-six male Wistar rats were divided into five groups: Control, Disease control (50 mg/kg Adenine p.o.), Adenine + Roflumilast (0.5, 1 and 1.5 mg/kg, p.o.). Various urinary and serum biomarkers, antioxidant status, histopathology, and protein expression of inflammatory markers were measured to investigate the effects of roflumilast on kidney functions. Adenine was found to elevate the levels of serum creatinine, urea, uric acid, sodium, potassium, chloride, magnesium, and phosphorus and reduce the level of serum calcium. Further, adenine significantly increased the serum TGF-β levels and reduced the anti-oxidant indices. Significant elevation was observed in protein expression of IL-1β, TNF-α, MCP-1, ICAM-1, and Fibronectin. Histopathologically, adenine caused thickening of the glomerular basement membrane, inflammatory cells infiltration, atrophy, and glomeruli deterioration. However, Roflumilast administration (1 mg/kg) remarkably decrease serum creatinine, urea, uric acid, sodium, potassium, chloride, magnesium, phosphorus by 61%, 40%, 44%, 41%, 49%, 58%, 59% and 42% respectively, and increase in calcium by 158%. Moreover, Roflumilast (1 mg/kg) significantly reduced serum TGF-β levels by 50% and elevated anti-oxidant indices by 257%, 112%, and 60%, respectively. The protein expression was significantly reduced by 5.5-fold, 7-fold, 5.7-fold, 6.2-fold, and 5.1-fold individually. Roflumilast noticeably improved the structure of glomeruli, tubules, and cellular functioning. The study confirmed that Roflumilast has the potential to ameliorate renal injury by reducing and regulating inflammatory responses.

Dihydrotestosterone, and Not Testosterone, Enhances the LPS-Induced Inflammatory Cytokine Gene Expression in Human Adipocytes

The development of obesity-related complications lies in the low-grade inflammatory state consequent to adipocyte dysfunction. The direct involvement of sex hormones in adipose tissue inflammation has been previously suggested, but the evidence is scarce. In this study, we evaluated the effects of sex steroids on the in-vitroexpression of inflammatory mediators in human-derived adipocytes before and after lipopolysaccharide (LPS) exposure. Human adipocytes were differentiated from the vascular stromal fraction of adipose tissue samples of subjects undergoing abdominoplasty. We evaluated MCP-1, IL-1β, IL-6, and TNF-α gene expression in the presence of the main sex steroids, testosterone (T), and 17β-estradiol (E). Furthermore, we analyzed the effects of adipocytes exposure to the non-aromatizable androgen dihydrotestosterone (DHT), together with the effects of adipocytes pre-incubation with the aromatase inhibitor anastrozole alone (A), and in combination with T (A/T) before incubation with LPS. DHT, but not T, significantly enhanced the LPSinduction of MCP-1, IL-1β, IL-6, and TNF-α. Intriguingly, the exposure of adipocytes with A/T dramatically increased the LPS-induced expression of all considered inflammatory cytokines, even more than a hundred-fold. DHT and A/T dramatically enhance LPS-induced inflammatory cytokine expression in human-derived adipocytes. These results confirm the involvement of sex hormones in adipose tissue inflammation, suggesting a specific role for non-aromatizable androgens as the amplificatory sex hormones of the inflammatory response.

Sex-specific association between adipose tissue inflammation and vascular and metabolic complications of obesity.

Adipose tissue (AT) inflammation predisposes to insulin resistance and metabolic syndrome in obesity. To investigate the association between adipocyte size, AT inflammation, systemic inflammation, and metabolic and atherosclerotic complications of obesity in a sex-specific manner. Cross-sectional cohort study. University hospital in the Netherlands. 302 adult subjects with a BMI ≥ 27 kg/m2. We obtained subcutaneous abdominal fat biopsies and systematically assessed, in a sex-specific manner, associations of several parameters of AT inflammation (including adipocyte size, macrophage content, crown like structures (CLS), and gene expression) to biomarkers of systemic inflammation, leukocyte number and function, and to the presence of metabolic syndrome, insulin resistance, and carotid atherosclerotic plaques, assessed with ultrasound. Adipocyte size was associated with metabolic syndrome and AT macrophage content with insulin resistance. In contrast, none of the AT parameters were associated with carotid atherosclerosis, although mRNA expression of the anti-inflammatory IL-37 was associated with a lower intima-media thickness. We revealed profound sex-specific differences, with an association between BMI and adipocyte size, and between adipocyte size and metabolic syndrome only in men. Also, only men showed an association between adipocyte size, AT expression of leptin and MCP-1, and AT macrophage numbers), and between AT inflammation (CLS number) and several circulating inflammatory proteins, including hsCRP, and IL-6. Inflammation in abdominal subcutaneous adipose tissue is more related to the metabolic than the atherosclerotic complications of obesity, and there are profound sex-specific differences in the association between BMI, adipocyte size, AT inflammation, and systemic inflammation, which are much stronger in men than women.

Bacterial protease alleviate chronic liver fibrosis induced by thioacetamide through suppression of hepatic stellate cells consequently decrease its proliferative index

In chronic liver diseases, liver fibrosis occurs due to excessive extracellular matrix (ECM) protein accumulation. Approximately 2 million deaths occur yearly due to liver disease, while cirrhosis is the 11th most common cause of death. Therefore, newer compounds or biomolecules must be synthesized to treat chronic liver diseases. In this aspect, the present study focuses on the assessment of the anti-inflammatory and antioxidant impact of Bacterial Protease (BP) produced by a new mutant strain of bacteria (Bacillus cereus S6-3/UM90) and 4,4′-(2,5-dimethoxy-1,4-phenylene) bis (1-(3-ethoxy phenyl)-1H-1,2,3-triazole) (DPET) in the treatment of early stage of liver fibrosis induced by thioacetamide (TAA). Sixty male rats were divided into six groups, ten rats each as follows: (1) Control group, (2) BP group, (3) TAA group, (4) TAA-Silymarin (S) group, (5) TAA-BP group, and (6) TAA-DPET group. Liver fibrosis significantly elevated liver function ALT, AST, and ALP, as well as anti-inflammatory interleukin 6 (IL-6) and VEGF. The oxidative stress parameters (MDA, SOD, and NO) were significantly increased with a marked reduction in GSH. Expression of MAPK and MCP-1 was unregulated in the TAA group, with downregulation of Nrf2 was observed. TAA caused histopathological alterations associated with hepatic vacuolation and fibrosis, increasing collagen fibers and high immuno-expression of VEGF. On the other hand, treatment with BP successfully improved the severe effects of TAA on the liver and restored histological architecture. Our study concluded the protective potentials of BP for attenuating liver fibrosis and could be used as adjuvant therapy for treating hepatic fibrosis.

A potent and selective CXCR2 antagonist improves neuroimmune dysregulation through the inhibition of NF-κB and notch inflammatory signaling in the BTBR mouse model of autism.

Autism comprises a broad range of neurodevelopmental disorders characterized by social communication deficits and repetitive and stereotyped behaviors. Chemokine receptor CXCR2 is expressed on neurons and is upregulated in neurological disorders. BTBR T+ Itpr3tf/J (BTBR) mice, a model for autism that shows the core features of ASD. Here, we studied the anti-inflammatory effect of a potent and selective CXCR2 antagonist SB332235 in the BTBR mice. The CXCR2 antagonist represents a promising therapeutic agent for several neuroinflammatory disorders. In this study, we investigated the effects of SB332235 administration on NF-κB-, Notch-1-, Notch-3-, GM-CSF-, MCP-1-, IL-6-, and IL-2- and TGF-β1-expressing CD40+ cells in BTBR and C57BL/6 (C57) mice in the spleen cells by flow cytometry. We further assessed the effect of SB332235 treatment on NF-κB, Notch-1, GM-CSF, MCP-1, IL-6, and IL-2 mRNA expression levels in the brain tissue by RT-PCR. We also explored the effect of SB332235 administration on NF-κB, GM-CSF, IL-6, and TGF-β1 protein expression levels in the brain tissue by western blotting. The SB332235-treated BTBR mice significantly decreases in CD40+NF-κB+, CD40+Notch-1+, CD40+Notch-3+, CD40+GM-CSF+, CD40+MCP-1+, CD40+IL-6+, and CD40+IL-2+, and increases in CD40+TGF-β1+ in the spleen cells. Our results further demonstrated that BTBR mice treated with SB332235 effectively decreased NF-κB, Notch-1, GM-CSF, MCP-1, IL-6, and IL-2, increasing TGF-β1 mRNA and protein expression levels in the brain tissue. In conclusion, these results indicate that SB332235 elicits an anti-inflammatory response by downregulating the inflammatory mediators and NF-κB/Notch inflammatory signaling in BTBR mice. This could represent a promising novel therapeutic target for autism treatment.

Oscillating Glucose Induces the Increase in Inflammatory Stress through Ninjurin-1 Up-Regulation and Stimulation of Transport Proteins in Human Endothelial Cells

Clinical data implicate fluctuations of high levels of plasma glucose in cardiovascular diseases. Endothelial cells (EC) are the first cells of the vessel wall exposed to them. Our aim was to evaluate the effects of oscillating glucose (OG) on EC function and to decipher new molecular mechanisms involved. Cultured human ECs (EA.hy926 line and primary cells) were exposed to OG (5/25 mM alternatively at 3 h), constant HG (25 mM) or physiological concentration (5 mM, NG) for 72 h. Markers of inflammation (Ninj-1, MCP-1, RAGE, TNFR1, NF-kB, and p38 MAPK), oxidative stress (ROS, VPO1, and HO-1), and transendothelial transport proteins (SR-BI, caveolin-1, and VAMP-3) were assessed. Inhibitors of ROS (NAC), NF-kB (Bay 11-7085), and Ninj-1 silencing were used to identify the mechanisms of OG-induced EC dysfunction. The results revealed that OG determined an increased expression of Ninj-1, MCP-1, RAGE, TNFR1, SR-B1, and VAMP-3 andstimulated monocyte adhesion. All of these effects were induced bymechanisms involving ROS production or NF-kB activation. NINJ-1 silencing inhibited the upregulation of caveolin-1 and VAMP-3 induced by OG in EC. In conclusion, OG induces increased inflammatory stress, ROS production, and NF-kB activation and stimulates transendothelial transport. To this end, we propose a novel mechanism linking Ninj-1 up-regulation to increased expression of transendothelial transport proteins.

120-kDa FN-fragment Induces MCP-1 Production in Human Temporomandibular Joint Fibroblast-like Synoviocytes

Fibronectin is a component of the extracellular matrix in the synovium. Fibronectin fragments（FN-fragments）result from fibronectin breakdown by proteolytic enzymes released from an early inflammatory response. We examined the effect of a 120-kDa fibronectin fragment（120-kDa FN-fragment）in the inflammatory condition of temporomandibular joint（TMJ）synovitis. Fibroblast-like synoviocytes, which were isolated from the TMJ synovium using the outgrowth method, were treated with or without 120-kDa FN-fragment. The fibronectin fragment induced the expression of monocyte chemotactic protein（MCP）-1, -2, and -3 genes in fibroblast-like synoviocytes as seen with DNA microarray analysis and real-time PCR. Among 120-kDa FN-fragment response genes, MCP-1（also called CCL2）, which is the main MCP chemokine, was abundantly expressed. MCP-1 protein level was increased in the conditioned medium with fibroblast-like synoviocytes treated with 120-kDa FN-fragment using ELISA. Furthermore, experiments using inhibitors of signaling pathway revealed that 120-kDa FN-fragment up-regulated MCP-1 expression as a result of NF-κB activation. MCP-1 mainly induces monocyte/macrophage migration in inflammation caused by various diseases. These results suggest that production of the MCP chemokine by 120-kDa FN-fragment may be involved in promoting the inflammatory condition of TMJ.

Cardiomyocyte-specific deletion of TLR4 attenuates angiotensin II-induced hypertension and cardiac remodeling.

Toll-like receptor 4 (TLR4) is an integral factor in the initiation of the innate immune response and plays an important role in cardiovascular diseases such as hypertension and myocardial infarction. Previous studies from our lab demonstrated that central TLR4 blockade reduced cardiac TLR4 expression, attenuated hypertension, and improved cardiac function. However, the contribution of cardiac specific TLR4 to the development of hypertension and cardiac remodeling is unknown. Therefore, we hypothesized that cardiomyocyte specific knockdown of TLR4 would have beneficial effects on hypertension, cardiac hypertrophy, and remodeling. To test this hypothesis, cardiomyocyte-specific TLR4 knockdown (cTLR4KO) mice were generated by crossing floxed TLR4 mice with Myh6-Cre mice, and subjected to angiotensin II (Ang II, 1 µg/kg/min or vehicle for 14 days) hypertension model. Blood pressure measurements using radio telemetry revealed no differences in baseline mean arterial pressure between control littermates and cTLR4KO mice (103 ± 2 vs. 105 ± 3 mmHg, p > 0.05). Ang II-induced hypertension (132 ± 2 vs. 151 ± 3 mmHg, p < 0.01) was attenuated and cardiac hypertrophy (heart/body weight; 4.7 vs. 5.8 mg/g, p < 0.01) was prevented in cTLR4KO mice when compared with control mice. In addition, the level of myocardial fibrosis was significantly reduced, and the cardiac function was improved in cTLR4KO mice infused with Ang II. Furthermore, cardiac inflammation, as evidenced by elevated gene expression of TNF, IL-6, and MCP-1 in the left ventricle, was attenuated in cTLR4KO mice infused with Ang II. Together, this data revealed a protective role for cardiomyocyte-specific deletion of TLR4 against Ang II-induced hypertension and cardiac dysfunction through inhibition of proinflammatory cytokines.

Epidermal Growth Factor Receptor in Hepatic Endothelial Cells Suppresses MCP-1-Dependent Monocyte Recruitment in Diabetes.

Insulin resistance is a compromised response to insulin in target tissues such as liver. Emerging evidence shows that vascular endothelial cells (ECs) are critical in mediating glucose metabolism. However, how liver ECs can regulate inflammation in the setting of insulin resistance is still unknown. Using genome-wide transcriptome analysis of ECs isolated from diabetic mice, we found enrichment of the genes involved in epidermal growth factor receptor (Egfr) signaling. In line with this, hepatic sinusoidal ECs in diabetic mice had elevated levels of Egfr expression. Interestingly, we found an increased number of hepatic myeloid cells, especially macrophages, and systemic glucose intolerance in Cdh5Cre/+Egfrfl/fl mice lacking Egfr in ECs compared with littermate control mice with type II diabetes. Egfr deficiency upregulated the expression of MCP-1 in hepatic sinusoidal ECs. This resulted in augmented monocyte recruitment and macrophage differentiation in Cdh5Cre/+Egfrfl/fl mice compared with littermate control mice as determined by a mouse model of parabiosis. Finally, MCP-1 neutralization and hepatic macrophage depletion in Cdh5Cre/+Egfrfl/fl mice resulted in a reduced number of hepatic macrophages and ameliorated glucose intolerance compared with the control groups. Collectively, these results demonstrate a protective endothelial Egfr signaling in reducing monocyte-mediated hepatic inflammation and glucose intolerance in type II diabetic mice.

Potent anti-inflammatory responses: Role of hydrogen in IL-1α dominated early phase systemic inflammation.

Introduction: It has been proven that hydrogen has obvious anti-inflammatory effects in animal experiments and clinical practice. However, the early dynamic process of the inflammatory response caused by lipopolysaccharide (LPS) and the anti-inflammatory effect of hydrogen has not been definitively reported. Methods: Inflammation in male C57/BL6J mice or RAW264.7 cells was induced with LPS, for which hydrogen was immediately administered until samples were taken. Pathological changes in lung tissue were assessed using hematoxylin and eosin (HE) staining. Levels of inflammatory factors in serum were determined using liquid protein chip. The mRNA levels of chemotactic factors in lung tissues, leukocytes, and peritoneal macrophages were measured by qRT-PCR. The expression levels of IL-1α and HIF-1α were measured by immunocytochemistry. Results: Hydrogen alleviated LPS-induced inflammatory infiltration in the lung tissues of mice. Among the 23 inflammatory factors screened, LPS-induced upregulation of IL-1α etc. was significantly inhibited by hydrogen within 1 hour. The mRNA expression of MCP-1, MIP-1α, G-CSF, and RANTES was inhibited obviously by hydrogen at 0.5 and 1 h in mouse peritoneal macrophages. In addition, hydrogen significantly blocked LPS or H2O2-induced upregulation of HIF-1α, and IL-1α in 0.5 h in RAW264.7 cells. Discussion: The results suggested that hydrogen is potentially inhibitive against inflammation by inhibiting HIF-1α and IL-1α release at early occurrence. The target of the inhibitive LPS-induced-inflammatory action of hydrogen is chemokines in macrophages in the peritoneal cavity. This study provides direct experimental evidence for quickly controlling inflammation with the translational application of a hydrogen-assisted protocol.

The systemic deletion of interleukin-1α reduces myocardial inflammation and attenuates ventricular remodeling in murine myocardial infarction

Myocardial inflammation following myocardial infarction (MI) is crucial for proper myocardial healing, yet, dysregulated inflammation may promote adverse ventricular remodeling and heart failure. IL-1 signaling contributes to these processes, as shown by dampened inflammation by inhibition of IL-1β or the IL-1 receptor. In contrast, the potential role of IL-1α in these mechanisms has received much less attention. Previously described as a myocardial-derived alarmin, IL-1α may also act as a systemically released inflammatory cytokine. We therefore investigated the effect of IL-1α deficiency on post-MI inflammation and ventricular remodeling in a murine model of permanent coronary occlusion. In the first week post-MI, global IL-1α deficiency (IL-1α KO mice) led to decreased myocardial expression of IL-6, MCP-1, VCAM-1, hypertrophic and pro-fibrotic genes, and reduced infiltration with inflammatory monocytes. These early changes were associated with an attenuation of delayed left ventricle (LV) remodeling and systolic dysfunction after extensive MI. In contrast to systemic Il1a-KO, conditional cardiomyocyte deletion of Il1a (CmIl1a-KO) did not reduce delayed LV remodeling and systolic dysfunction. In conclusion, systemic Il1a-KO, but not Cml1a-KO, protects against adverse cardiac remodeling after MI due to permanent coronary occlusion. Hence, anti-IL-1α therapies could be useful to attenuate the detrimental consequences of post-MI myocardial inflammation.

Myeloid-specific fatty acid transport protein 4 deficiency induces a sex-dimorphic susceptibility for nonalcoholic steatohepatitis in mice fed a high-fat, high-cholesterol diet.

Newborns with FATP4 mutations exhibit ichthyosis prematurity syndrome (IPS), and adult patients show skin hyperkeratosis, allergies, and eosinophilia. We have previously shown that the polarization of macrophages is altered by FATP4 deficiency; however, the role of myeloid FATP4 in the pathogenesis of nonalcoholic steatohepatitis (NASH) is not known. We herein phenotyped myeloid-specific Fatp4-deficient (Fatp4M-/-) mice under chow and high-fat, high-cholesterol (HFHC) diet. Bone-marrow-derived macrophages (BMDMs) from Fatp4M-/- mice showed significant reduction in cellular sphingolipids in males and females, and additionally phospholipids in females. BMDMs and Kupffer cells from Fatp4M-/- mice exhibited increased LPS-dependent activation of proinflammatory cytokines and transcription factors PPARγ, CEBPα, and p-FoxO1. Correspondingly, these mutants under chow diet displayed thrombocytopenia, splenomegaly, and elevated liver enzymes. After HFHC feeding, Fatp4M-/- mice showed increased MCP-1 expression in livers and subcutaneous fat. Plasma MCP-1, IL4, and IL13 levels were elevated in male and female mutants, and female mutants additionally showed elevation of IL5 and IL6. After HFHC feeding, male mutants showed an increase in hepatic steatosis and inflammation, whereas female mutants showed a greater severity in hepatic fibrosis associated with immune cell infiltration. Thus, myeloid-FATP4 deficiency led to steatotic and inflammatory NASH in males and females, respectively. Our work offers some implications for patients with FATP4 mutations and also highlights considerations in the design of sex-targeted therapies for NASH treatment.NEW & NOTEWORTHY FATP4 deficiency in BMDMs and Kupffer cells led to increased proinflammatory response. Fatp4M-/- mice displayed thrombocytopenia, splenomegaly, and elevated liver enzymes. In response to HFHC feeding, male mutants were prone to hepatic steatosis, whereas female mutants showed exaggerated fibrosis. Our study provides insights into a sex-dimorphic susceptibility to NASH by myeloid-FATP4 deficiency.

Immunoregulatory effects of Tetrastigma hemsleyanum polysaccharide via TLR4-mediated NF-κB and MAPK signaling pathways in Raw264.7 macrophages

Polysaccharide of Tetrastigma hemsleyanum (THP) exert antioxidant, antibacterial, lipid-lowering, and anti-inflammatory properties, especially some evidences have highlighted the efficiency of it as an anti-tumor agent. However, as a biological macromolecule with bidirectional immune regulation, the immunological enhancement effects of THP on macrophages and its underlying mechanisms are still largely unknown. In the present study, THP was prepared and characterized, and then the effect of THP on Raw264.7 cell activation was investigated. Structural characteristics of THP showed that the average molecular weight was 370.26 kDa, and the main monosaccharide composition was galactose, glucuronic acid, mannose, and glucose at a ratio of 31.56: 25.15: 19.44: 12.60, with high viscosity causing by relative high uronic acid. For immunomodulatory activity investigation, THP promoted the production of NO, IL-6 and TNF-α, as well as the expression of IL-1β, MCP-1, iNOS and COX-2, which were almost completely inhibited by TLR4 antagonist. Further study showed that THP could activate NF-κB and MAPK signaling pathways, and thus enhanced the phagocytic activity of Raw264.7 macrophages. In conclusion, the present study provided evidences that THP could be served as a new immunomodulator in both functional foods and the pharmaceutical field.

Advanced Oxidation Protein Products Contribute to Chronic-Kidney-Disease-Induced Adipose Inflammation through Macrophage Activation.

Fat atrophy and adipose tissue inflammation can cause the pathogenesis of metabolic symptoms in chronic kidney disease (CKD). During CKD, the serum levels of advanced oxidation protein products (AOPPs) are elevated. However, the relationship between fat atrophy/adipose tissue inflammation and AOPPs has remained unknown. The purpose of this study was to investigate the involvement of AOPPs, which are known as uremic toxins, in adipose tissue inflammation and to establish the underlying molecular mechanism. In vitro studies involved co-culturing mouse-derived adipocytes (differentiated 3T3-L1) and macrophages (RAW264.7). In vivo studies were performed using adenine-induced CKD mice and AOPP-overloaded mice. Fat atrophy, macrophage infiltration and increased AOPP activity in adipose tissue were identified in adenine-induced CKD mice. AOPPs induced MCP-1 expression in differentiated 3T3-L1 adipocytes via ROS production. However, AOPP-induced ROS production was suppressed by the presence of NADPH oxidase inhibitors and the scavengers of mitochondria-derived ROS. A co-culturing system showed AOPPs induced macrophage migration to adipocytes. AOPPs also up-regulated TNF-α expression by polarizing macrophages to an M1-type polarity, and then induced macrophage-mediated adipose inflammation. In vitro data was supported by experiments using AOPP-overloaded mice. AOPPs contribute to macrophage-mediated adipose inflammation and constitute a potential new therapeutic target for adipose inflammation associated with CKD.

Luteolin Protects Against Obese Sarcopenia in Mice with High-Fat Diet-Induced Obesity by Ameliorating Inflammation and Protein Degradation in Muscles.

Although sarcopenia is mainly caused by aging, sarcopenia due to obesity has become an emerging issue given the increase in obesity among people of various ages. There are studies on obesity or sarcopenia, our understanding of obesity-mediated sarcopenia is insufficient. Luteolin (LU) has exhibited antiobesity effects, but no studies have investigated the LU effects on antisarcopenia. This study therefore investigated the effects of LU on obese sarcopenia in mice with high-fat diet (HFD)-induced obesity. To evaluate its inhibitory efficacy against obese sarcopenia, 5-week-old mice are fed an HFD supplemented with LU for 20weeks. LU exerts suppressive effects on obesity, inflammation, and protein degradation in the HFD-fed obese mice. It also inhibits lipid infiltration into the muscle and decreases p38 activity and the mRNA expression of inflammatory factors, including TNF-α, Tlr2, Tlr4, MCP1, and MMP2, in the muscle. The suppression of muscle inflammation by LU leads to the inhibition of myostatin, FoxO, atrogin, and MuRF expression. These effects of LU affect inhibition of protein degradation and improvement of muscle function. Here, it demonstrates that LU's antiobesity and antiinflammatory functionality affect inhibition of muscle protein degradation, and consequently, these interactions by LU exerts a protective effect against obese sarcopenia.

Exploration of the mechanism of NORAD activation of TGF-β1/Smad3 through miR-136-5p and promotion of tacrolimus-induced renal fibrosis

Background Tacrolimus is a potent immunosuppressant, but has various side effects, with nephrotoxicity being the most common. Renal fibrosis is an important process of tacrolimus nephrotoxicity. Therefore, it is important to identify the factors that contribute to renal fibrosis after tacrolimus nephrotoxicity, and control its development. Methods The present study aims to determine whether tacrolimus may speed up the course of renal fibrosis by upregulating noncoding RNA activated by DNA damage (NORAD) to compete with miR-136-5p, and activating the TGF-β1/Smad3 pathway. Furthermore, in vivo rat models and in vitro cell models were established. Then, the expression levels of NORAD and miR-136-5p were determined by RT-qPCR, while the expression of the TGF-β1/Smad3 pathway was determined by western blot and RT-qPCR. In order to investigate the interaction between NORAD and miR-136-5p, as well as miR-136-5p and SYK, two luciferase reporters were employed. The renal fibrosis of mice was observed using Masson and PAS staining. The expression of inflammatory factors IL-1, IL-6, MCP-1 and TNF-α was detected by ELISA. Results In the in vitro experiments, NORAD was upregulated, while miR-136-5p was downregulated after tacrolimus induction. The expression of the TGF-β1/Smad3 pathway correspondingly changed after the induction by tacrolimus. In the in vivo experiments, the expression of NORAD and miR-136-5p, and the trend for renal fibrosis were consistent with the results in the in vitro experiments. Furthermore, the inflammatory factors correspondingly changed with the severity of renal fibrosis. Moreover, the expression trend of the TGF-β1/Smad3 pathway in tacrolimus-induced rats was consistent with that in the in vitro experiments. Conclusion Through in vitro and in vivo experiments, the present study was able to successfully prove that tacrolimus upregulates NORAD to compete with miR-136-5p, resulting in a decrease in miR-136-5p expression, which in turn activates the TGF-β1/smad3 pathway, and finally induces the aggravation of renal fibrosis.

Shensong Yangxin Capsule Reduces the Susceptibility of Arrhythmia in db/db Mice via Inhibiting the Inflammatory Response Induced by Endothelium Dysfunction.

The aim of our study was to investigate the mechanism by which the Chinese compound Shensong Yangxin Capsule (SSYX) reduces susceptibility to arrhythmia in db/db mice. The db/db mice without drug treatment served as the model group. Other-treated db/db mice were administered SSYX for 8 weeks. Electrocardiogram (ECG), electrical mapping, pathological changes, immunofluorescence staining, real-time quantitative PCR, and Western blot analyses were then conducted. SSYX decreased arrhythmia susceptibility and shortened the abnormal ECG parameters of db/db mice. Meanwhile, SSYX restored irregular conduction direction and shortened the conduction time of the isolated heart. HE and Masson staining showed that SSYX alleviated inflammatory infiltration and collagen fiber deposition. Western blot showed that SSYX decreased the protein expression of ICAM-1, VCAM-1, and MCP-1 and increased the protein expression of occludin, ZO-1, eNOS, and Cx43. SSYX also increased the content of NO, decreased ET-1, TNF-α, IL-1β, IL-6, MCP-1, and CCR-2 mRNA expression, and increased Kv 4.2, Kv 4.3, Cav 1.2, and Nav 1.5 mRNA expression. Furthermore, SSYX decreased the fluorescence intensity of F4/80 and iNOS, increased the fluorescence intensity of CD31 and eNOS, and improved the Cx43 and α-actinin connection structure in cardiac tissues. The above therapeutic effects of SSYX were inhibited by L-NAME. SSYX reduced the susceptibility of db/db mice to arrhythmia by inhibiting the inflammatory response and macrophage polarization, and this effect of SSYX occurred through protection of endothelial cell function.

Bone Marrow Mesenchymal Stem Cells Combined with Bone Transport Improves Bone Defect in Rats

This study assessed the mechanism of BMSC combined with bone transport in improving bone defect. Fifty rats were divided into five sets randomly including NC set, DEX set, BMSC set and BT set. There were ten rats in each set. The BMSC was isolated using whole bone marrow adherent method and then cultivated. The general condition of rats in each set was observed and morphological parameter, pathological change in bone defect tissue was detected along with analysis of the expression of MCP-1, p53, TNF-αand STAT1 in bone tissue. The primary BMSC was cultivated for seven days and the fusiform BMSC was enlarged and the quantity of binucleate or multinucleate cells was increased after passage. The bone defect model was prepared successfully when the degree of fusion reached 100%. The mental condition was good. DEX set showed significantly reduced TBV and increased TRS compared with NC set. However, TBV was increased and TRS was reduced in BMSC set, BT set and BMSC+BT set significantly compared with DEX set. MCP-1 mRNA level in DEX set was lower and increased in the treatment group. In addition, p53, TNF-αand STAT1 was increased in DEX set but reduced in BMSC set, BT set and BMSC+BT set. In conclusion, MCP-1 in rats’ bone defect tissue is upregulated and the p53/TNF-α/STAT1 signal activity is restrained by BMSC combined with bone transport so as to treat the bone defect.

Endothelial cell CD36 mediates stroke-induced brain injury via BBB dysfunction and monocyte infiltration in normal and obese conditions

CD36 expressed in multiple cell types regulates inflammation, vascular function, and innate immunity. Specifically, CD36 in microvascular endothelial cells (ECs) signals to elicit inflammation and causes EC death. This study investigated roles for EC-CD36 on acute stroke pathology in normal and obese conditions. Obesity induced by a high-fat diet (HD) selectively increased CD36 expression in ECs, not in monocytes/macrophages, in the post-ischemic brain. Mice deficient CD36 in ECs (ECCD36−/−) showed reduced injury size and vascular permeability in normal conditions. While control mice fed a HD developed obesity and aggravated stroke injury, ECCD36−/− mice were resistant to develop an obesity phenotype. Subjecting ECCD36−/− mice to stroke resulted in reduced injury size and BBB disruption. Moreover, the mice had reduced MCP-1 and CCR2 gene expression, resulting in reduced monocyte trafficking with improved survival and acute motor function. Reduced MCP-1 and CCR2 expression was still evident in ECCD36−/− mice subjected to severe stroke, suggesting that monocyte trafficking is an infarct-independent metabolic effect associated with specific EC-CD36 deletion. Our findings demonstrate the importance of EC-CD36 in developing vascular comorbidities and suggest that targeting EC-CD36 is a potential preventative strategy to normalize vascular risk factors, leading to improved acute stroke outcomes.