Monocyte Chemoattractant Protein-1 mRNA Expression Research Articles

Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1β, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1β in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1β, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1β in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.

Effect of Flavonoids on MCP-1 Expression in Human Coronary Artery Endothelial Cells and Impact on MCP-1-Dependent Migration of Human Monocytes.

The monocyte chemoattractant protein-1 (MCP-1), also known as chemokine (CC motif) ligand 2 (CCL2), is involved in the formation, progression, and destabilization of atheromatous plaques. Flavonoids, found in fruits and vegetables, have been associated with various health-promoting properties, including antioxidant, anti-inflammatory, and cardioprotective effects. In the present study, the flavonoids quercetin, kaempferol, and luteolin, but not cannflavin A, were shown to substantially inhibit interleukin (IL)-1β-induced MCP-1 mRNA and protein expression in human coronary artery endothelial cells (HCAEC). At the functional level, conditioned medium (CM) from IL-1β-stimulated HCAEC caused an increase in the migration of THP-1 monocytes compared with CM from unstimulated HCAEC. However, this induction was suppressed when IL-1β-treated HCAEC were coincubated with quercetin, kaempferol, or luteolin. The functional importance of MCP-1 in IL-1β-induced monocyte migration was supported by experiments showing that neutralization of MCP-1 in the CM of IL-1β-treated HCAEC led to a significant inhibition of migration. In addition, a concentration-dependent induction of monocyte migration in the presence of recombinant MCP-1 was demonstrated. Collectively, the flavonoids quercetin, kaempferol, and luteolin were found to exert potential antiatherogenic effects in HCAEC, challenging further studies with these compounds.

Tetramethylpyrazine inhibits keratitis and neovascularization induced by corneal alkali burn by suppressing the TLR4/NF-κB pathway activation and NLRP1/NLRP3 inflammasomes in rats

The role and related mechanisms of tetramethylpyrazine (TMP) in corneal alkali burn in rats were expected to be explored in this article. After construction of corneal alkali burn rat models, TMP eye drops were given four times daily for consecutive 7 days. H&E staining was utilized for observing the histopathological changes of corneas on the 3rd and 7th days of treatment; immunohistochemistry for detecting the Nestin protein expression changes; qRT-PCR for determining the expression changes of genes correlated with neovascularization [C-X-C Motif Chemokine Ligand 1 (CXCL-1), vascular endothelial growth factor A (VEGFA) and CD31] and inflammation-related factors [monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), tumour necrosis factor α (TNF-α), and IL-6]; Western blot for testing NLR Family Pyrin Domain Containing 1 (NLRP1)/NLRP3 inflammasomes and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway-related protein expression changes. All above trials were completed based on rat corneal tissue. TMP ameliorated the pathological damage in alkali-burned rat corneal tissue. Specifically, TMP treatment decreased Nestin-positive cell expression and the CXCL-1, VEGFA and CD31 mRNA expression in alkali-burned rat corneal tissue dose-dependently. TMP also down-regulated the IL-1β, TNF-α, MCP-1 and IL-6 mRNA expression and inhibited the NLRP1, caspase-1, NLRP3, pro-IL-1β and mature IL-1β protein expression in the alkali-burned rat corneal tissue. In addition, TMP treatment down-regulated the myeloid differentiation factor 88 (MyD88) and TLR4 protein expression and decreased the p–NF–κB p65/NF-κB p65 ratio in the alkali-burned rat corneal tissue. The mechanism of TMP relieving the inflammatory response and inhibiting neovascularization caused by corneal alkali burn in rats might have a correlation with the suppression of acitivation of TLR4/NF-κB pathway and NLRP1/NLRP3 inflammasomes in rats.

Oral contraceptive pill (OCP) treatment alters the gene expression of intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) in polycystic ovary syndrome (PCOS) women compared to drug-naive PCOS women

BackgroundPolycystic ovary syndrome (PCOS) presents clinical symptoms of menstrual abnormalities, excessive hair growth (hirsutism), scalp hair loss, acne and infertility. Metabolic abnormalities such as obesity, insulin resistance, glucose intolerance and cardiovascular problems constitute an essential part of PCOS, all of which can have significant long-term health consequences. Low-grade chronic inflammation demonstrated by persistent moderately elevated serum levels of inflammatory and coagulatory markers plays a critical role in the pathogenesis of PCOS. Oral contraceptive pills (OCPs) constitute the mainstay of pharmacologic therapy for women with PCOS to regularize cyclicity and ameliorate androgen excess. On the other hand, OCP use is associated with various venous thromboembolic and proinflammatory events in the general population. PCOS women always carriers the increased lifetime risk of these events. The studies on the effect of OCPs on inflammatory, coagulation and metabolic parameters in PCOS are less robust. Therefore in this study, we investigated and compared the messenger RNA (mRNA) expression profiles of genes implicated in inflammatory and coagulation pathways between drug-naive and OCP-treated PCOS women. The selected genes include intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1). Furthermore, the correlation between the selected markers and various metabolic indices in the OCP group has also been explored.MethodThe relative amounts of ICAM-1, TNF-α, MCP-1 and PAI-1 mRNA in peripheral blood mononuclear cells from 25 drug-naive PCOS subjects (controls) and 25 PCOS subjects who received OCPs containing 0.03 mg-ethinyl-estradiol and 0.15 mg-levonorgestrel for at least six months (cases) were estimated using real-time qPCR. The statistical interpretation was conducted using SPSS version 20.0 (SPSS, Inc, Chicago, IL), Epi Info version 2002 (Disease Control and Prevention Centres, Atlanta, GA) and GraphPad Prism 5 (GraphPad Software, La Jolla, CA) software.ResultSix months of OCP therapy enhanced the expression of inflammatory genes viz ICAM-1, TNF-α and MCP-1 mRNA in PCOS women by 2.54, 2.05 and 1.74 folds, respectively, in this study. However, PAI-1 mRNA in the OCP group showed no significant increase. Furthermore, in cases, ICAM-1 mRNA expression positively correlated with body mass index (BMI) (p = 0.01), fasting insulin (p = 0.01), insulin 2 h p = 0.02), glucose 2 h (p = 0.01) and triglycerides (p = 0.01). TNF-α mRNA expression positively correlated with fasting insulin (p = 0.0007). MCP-1 mRNA expression positively correlated with (BMI) (p = 0.002).ConclusionOCPs helped reduce clinical hyperandrogenism and regularise menstrual cycles in women with PCOS. However, OCP use was associated with increased fold expression of inflammatory markers which positively correlated with metabolic abnormalities.

Inhibition of LPS-Induced Microglial Activation by the Ethyl Acetate Extract of Pueraria mirifica.

Microglial activation has been found to play a crucial role in various neurological disorders. Proinflammatory substances overproduced by activated microglia, such as cytokines, chemokines, reactive oxygen species, and nitric oxide (NO), can result in neuroinflammation that further exacerbates the course of the diseases. This study aimed to explore the anti-inflammatory effect of the ethyl acetate extract of Pueraria mirifica on microglial activation. Lipopolysaccharide (LPS)-induced inflammation was used as a model to investigate the effects of P. mirifica on HAPI (highly aggressive proliferating immortalized), a rat microglial cell line. Administration of ethyl acetate extract from the tuberous roots of P. mirifica to HAPI cells dose-dependently reduced NO production and iNOS expression induced by LPS. Attenuation of IRF-1 (interferon regulatory factor-1) induction, one of the transcription factors governing iNOS expression, suggested that the inhibitory effect on NO production by the plant extract was at least partially mediated through this transcription factor. In addition, LPS-stimulated mRNA expression of MCP-1 (monocyte chemoattractant protein-1), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α) was also suppressed with P. mirifica extract pretreatment. This study indicates that the ethyl acetate extract of P. mirifica could potentially serve as an anti-inflammatory mediator and may be useful in relieving the severity of neurological diseases where microglia play a role.

Effects of 3'-isovaleryl-4'-senecioylkhellactone from Peucedanum japonicum Thunberg on PMA-Stimulated Inflammatory Response in A549 Human Lung Epithelial Cells.

Peucedanum japonicum Thunberg (PJT) has been used in traditional medicine to treat colds, coughs, fevers, and other inflammatory diseases. The goal of this study was to investigate whether 3'-isovaleryl-4'-senecioylkhellactone (IVSK) from PJT has anti-inflammatory effects on lung epithelial cells. The anti-inflammatory effects of IVSK were evaluated using phorbol 12-myristate 13-acetate (PMA)-stimulated A549 cells and regular human lung epithelial cells as a reference. IVSK reduced the secretion of the inflammatory mediators interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1), and the mRNA expression of IL-6, IL-8, MCP-1, and IL-1β. Additionally, it inhibited the phosphorylation of IκB kinase (IKK), p65, Iκ-Bα, and mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK in A549 cells stimulated with PMA. Moreover, the binding affinity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) was significantly reduced in the luciferase assay, while nuclear translocation was markedly inhibited by IVSK in the immunocytochemistry. These findings indicate that IVSK can protect against inflammation through the AP-1 and NF-κB pathway and could possibly be used as a lead compound for the treatment of inflammatory lung diseases.

Antiatherosclerotic effects of corilagin via suppression of the LOX-1/MyD88/NF-κB signaling pathway in vivo and in vitro.

Corilagin, a natural polyphenol compound isolated from Phyllanthus urinaria L., exerts various pharmacological effects, such as antihyperglycemic, antitumor, and antioxidative stress properties, but the mechanisms underlying the antiatherosclerotic effects of corilagin have not been entirely elucidated. In the present study, we investigated the antiatherosclerotic effects of corilagin using a high-fat diet (HFD)-induced atherosclerotic rabbit model and ox-LDL-induced vascular smooth muscle cells (VSMCs) and explored the underlying molecular mechanisms. The serum lipid levels were measured through an enzymatic colorimetric assay. A histological analysis of rabbit aortas was performed after hematoxylin-eosin and oil red O staining. The proliferation of ox-LDL-induced VSMCs was detected using MTT assays, and the migration of cells was determined by wound scratch assays. In addition, the mRNA and protein expression levels of lectin-like ox-LDL receptor-1 (LOX-1), myeloid differentiation factor 88 (MyD88), nuclear factor-kappa B (NF-κB), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor α (TNF-α) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assays. Our results indicate that corilagin significantly reduced the serum levels of TC, TG and LDL-C, increased the HDL-C levels, decreased the intimal thickening in the thoracic aorta, and reduced the formation of foam cells in an HFD-induced rabbit atherosclerosis model. Moreover, corilagin suppressed the proliferation and migration of ox-LDL-induced VSMCs and reduced LOX-1, MyD88, NF-κB, MCP-1, and TNF-α mRNA and protein expression in vivo and in vitro. These data demonstrate that corilagin exerts antiatherosclerotic effects in vivo and in vitro and that the mechanisms may be closely associated with downregulation of the LOX-1/MyD88/NF-κB pathway.

Microcystin-LR incorporated into colonic cells through probenecid-sensitive transporters leads to upregulated MCP-1 expression induced by JNK activation

Harmful algae that inhabit eutrophic lakes produce cyanotoxic microcystins. Therefore, the relationship between chronic exposure to microcystins via drinking water and organ disorders has been investigated. The present study aimed to determine whether representative microcystin-LR is involved in increased monocyte chemoattractant protein-1 (MCP-1) expression in rat colonic mucosa and enterocyte-like differentiated Caco-2 cells. The mRNA expression of MCP-1 was increased in the colons of rats administered with microcystin-LR, compared with controls. Furthermore, mRNA levels of MCP-1 expression significantly and positively correlated with those of Adhesion G Protein-Coupled Receptor E1 (ADGRE1; EMR1; F4/80), an indicator of macrophage infiltration, suggesting that increased MCP-1 expression induced by microcystin-LR promotes macrophage infiltration into the colon. Microcystin-LR increased MCP-1 expression in enterocyte-like differentiated Caco-2 cells, by activating c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) or p38. The findings of transporter inhibitors indicated that microcystin-LR is incorporated into cells via ATP Binding Cassette (ABC) or solute carrier (SLC) transporters other than the organic anion transporting polypeptides (OATPs)1B1, 1B3, 2B1, and 1A2, which this leads to increased MCP-1 expression in the colon through activating JNK. Thus, increased MCP-1 expression induced by microcystin-LR might be a trigger for initiating tumorigenesis with inflammation in the colon because increased MCP-1 expression induces inflammation associated with macrophage infiltration into the colon, and chronic inflammation is associated with the initiation of tumorigenesis.

Total flavonoids from Lagerstroemia speciosa (L.) Pers inhibits TNF-α-induced insulin resistance and inflammatory response in 3T3-L1 adipocytes via MAPK and NF-кB signaling pathways

Adipocyte tissue inflammation orchestrates the occurrence and development of diseases induced by obesity. Here, the effects of Lagerstroemia speciosa (L.) Pers total flavonoids (LTF) on TNF-α-induced insulin resistance (IR) and inflammatory response in 3T3-L1 adipocytes and underlying mechanisms were studied. Cell glucose uptake test confirmed that TNF-α could induce IR in 3T3-L1 adipocytes. The sensitivity of 3T3-L1 adipocytes to insulin was increased by LTF treatment. The phosphorylation of IRS1 and Akt in TNF-α-treated 3T3-L1 adipocytes was markedly restored by LTF treatment. The results revealed that interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) mRNA expression was elevated in TNF-α-administered 3T3-L1 adipocytes, whereas the mRNA expression of adiponectin (ADPN) was considerably decreased. When treated with 50 μg/mL LTF, the IL-6 and MCP-1 mRNA expression levels were drastically reduced, and the ADPN mRNA expression level was significantly raised. Moreover, the concentration of IL-6 in the supernatant of the medium was decreased after LTF treatment compared with the model group. Western blotting results showed that the phosphorylation of p38, JNK, and ERK of mitogen-activated protein kinase (MAPK) signaling pathways and IкB of NF-кB signaling pathway were inhibited. It suggested that LTF improves TNF-α-induced IR and inflammatory response in 3T3-L1 adipocytes via inhibiting MAPK and NF-кB signaling pathways.

Potentilla discolor ameliorates LPS-induced inflammatory responses through suppressing NF-κB and AP-1 pathways

Potentilla discolor Bunge (PD) is a traditional Chinese medicine which has been widely used for the treatment of various inflammatory diseases (e.g., diarrhea, fever and furuncle). However, few studies focused on its effect on classical inflammation. This study aimed to investigate the anti-inflammatory effect and potential mechanism of the ethanol extract of the whole herbs of PD (EPD) in lipopolysaccharide (LPS)-induced inflammatory models. The obtained results showed that EPD decreased supernatant NO, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in LPS-activated RAW264.7 cells and mouse peritoneal macrophages. Moreover, its effect on NO was attributed to the suppression of iNOS expression rather than its activity. At the transcriptional level, EPD suppressed iNOS, TNF-α and MCP-1 mRNA expressions in LPS-stimulated RAW264.7 cells. Further study showed that EPD didn’t affect the phosphorylation and degradation of IκBα, but yet impeded the nuclear translocation of p65 to inhibit NF-κB activation. Meanwhile, it also prevented JNK, ERK1/2 and p38 phosphorylation to dampen the activation of AP-1. In endotoxemia mouse model, EPD not only decreased interleukin-6, TNF-α and MCP-1 levels in serum, but also potently ameliorated diarrhea. These findings provide the theoretical basis for PD to treat inflammatory diseases, especially intestinal inflammation.

Monocyte chemotactic protein-1 concentrations and expression of women with endometriosis undergoing IVF cycles

To research the local action of endometriosis on follicular inflammatory microenvironment of patients undergoing in vitro fertilization (IVF) cycles. A prospective case control study was organized at Kamal AL-Samarai IVF center (Baghdad/Iraq), January 2021 - May 2021. The study included (80) infertile women undergoing IVF cycles were split to two groups: Patients with endometriosis group (n = 40) and Patients without endometriosis group (n = 40). Follicular fluid (FF) and blood samples were gathered at the period of oocyte pick-up. Levels of MCP-1 mRNA were investigated with the reverse transcription polymerase chain reaction (RT-PCR) technique. Concentrations of cytokines (monocyte chemo attractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10)) were investigated in FF by sandwich enzyme-linked immunosorbent assay (ELISA). The mRNA expression of MCP-1 was significantly (P = 0.00) higher in patients with endometriosis group (1.95 ± 0.69) compared with patients without endometriosis group (0.40 ± 0.15). The studied cytokines concentrations in FF of women with endometriosis group were significantly (P = 0.00) higher than those without endometriosis group: MCP-1 (548.07 ± 199.43 pg/ml vs. 45.98 ± 18.72 pg/ml), TNF-α (912.15 ± 316.12 pg/ml vs. 261.89 ± 90.69 pg/ml), IL-10 (258.94 ± 68.82 pg/ml vs. 52.12 ± 14.46 pg/ml). The immune milieu of FF of patients with endometriosis is modified, which could explain the effect of endometriosis on infertility. Furthermore, elevated MCP-1 expression in endometriosis patients suggesting systemic inflammatory changes.

Impairment of Whole Kidney Blood Flow Autoregulation in the Transition from Acute to Chronic Kidney Disease in Ischemia‐Reperfusion Rats

Efficient autoregulation of renal blood flow (RBF) is an essential mechanism of renal microcirculation to protect against glomerular injury. Patients surviving an episode of acute kidney injury (AKI) develops chronic kidney disease (CKD). Renal ischemia-reperfusion (IR) is a major cause of AKI and triggers inflammation and reactive oxygen species (ROS) accumulation. We hypothesized that IR‑induced inflammation and ROS accumulation leads to a persistent reduction of RBF and impairment of RBF autoregulation contributing to CKD progression. Whole kidney RBF autoregulation was assessed in vivo in Sprague-Dawley male IR induced by bilateral arterial ischemia for 60 minutes followed by 24 hours, 1-week and 4‑weeks of reperfusion. Plasma creatinine increased in IR at 24 hours (3.9±0.2 vs. 1.0±0.1 mg/dl in sham, P<0.05, n=6-7/group) and week 1 (1.4±0.1 vs. 1.1±0.0 mg/dl in sham, P<0.05, n=7-8/group). Systolic blood pressure was similar between sham and IR over the 4 weeks post-IR (113-135 mmHg). IR reduced baseline RBF (5.8±0.5 vs. 9.1±0.5 ml/min.gkw in sham, P<0.05, n=6/group) at 24 hours and further decreased RBF at week 1 (3.7±0.7 vs. 9.1±1.1 ml/min.gkw in sham, n=5-6/group). At week 4, RBF was still significantly low in IR (6.9±0.4 vs. 10.4±0.3 ml/min.gkw in sham, P<0.05, n=6/group). RBF remained stable during step reduction of renal perfusion pressure (RPP) from 120 to 100 mmHg in all shams with autoregulatory index (AI) less than 0.2, indicating intact autoregulation. RBF autoregulation, however, was completely impaired in IR at 24 hours, week 1 and week 4 as RBF passively decreased during reduction of RPP, yielding AI of 0.8±0.2, 1.1±0.2 and 0.9±0.3, respectively. Preglomerular microvessels (PGMV) were collected for mRNA measurement (n=6-8/group). IR elevated mRNA expression of p67phox 7‑fold at 24 hours and 2‑fold at week 1 (P<0.05 vs. respective shams). IR also elevated gp91phox mRNA expression 5-fold at 24 hours and 2-fold at week 1 (P<0.05 vs. shams). IR tended to elevate p47phox mRNA expression at 24 hours and reached a significance at week 1 (2-fold, P<0.05 vs. sham). Inflammatory mediators, monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1), were also assessed in PGMV. MCP-1 mRNA expression was increased in IR with a peak of 10-fold at 24 hours and then decreased at week 1 but still elevated compared to shams (2-fold, P<0.05). VCAM-1 mRNA expression remained unchanged at 24 hours but increased 2-fold at week 1 (P<0.05). We also assessed profibrotic markers (TGF-β, collagen IV and fibronectin). TGF-β mRNA expression started to increase in IR at 24 hours (2-fold) and reached a significance at week 1 (3-fold, P<0.05 vs. sham) and further increased at week 4 (4-fold). IR also increased mRNA expression of collagen IV 5-fold at week 1 and 4-fold at week 4 (P<0.05). Fibronectin mRNA expression was also increased in IR 4-fold at week 1 and remained at the same level at week 4 (P<0.05). In conclusion, these results demonstrate that IR leads to a persistent reduction of RBF and impairment of RBF autoregulation in male rats and suggest that renal hemodynamic dysfunction might be associated with activation of NADPH oxidase, MCP-1 and TGF-β in PGMV of IR.

Centella asiatica Extract Attenuates Kidney Fibrosis Through Reducing Mesenchymal Transition and Inflammation in Ureteral Ligation Model in Mice

Background: Kidney fibrosis is the common final pathway of chronic kidney disease (CKD), and is characterized by inflammation, mesenchymal transition with myofibroblast formation and epithelial to mesenchymal transition (EMT). Centella asiatia (CeA) is an herb that has a reno-protective effect. However, its mechanism of action in kidney fibrosis has not been elucidated. Aim: To elucidate the effect of CeA in amelioration of kidney fibrosis in a unilateral ureteral obstruction (UUO) model and focus on mesenchymal transition and inflammation. Methods: Unilateral ureteral obstruction was performed in male Swiss-background mice (age: 2–3 months, weight: 30–40 g, UUO group n = 6) to induce kidney fibrosis. Two doses of CeA extract with oral administration, 210 and 840 mg/kg body weight were added in UUO (U+C210 and U+C840 groups, each n = 6). The sham operation procedure was performed for the control group (SO, n = 6). The mice were euthanized at day-14 after operation. Tubular injury and interstitial fibrosis area fractions in kidney tissues of the mice were quantified based on periodic acid-Schiff (PAS) and Sirius Red (SR) staining. Immunostaining was performed for examination of fibroblast (PDGFR-β), myofibroblast (α-SMA), Monocyte Chemoattractant Protein-1 (MCP-1) and macrophage (CD68), meanwhile double immunofluorescence was performed with PDGFR-β and α-SMA. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to examine mRNA expression of TGF-β, Collagen-1, Snail, E-cadherin, vimentin, fibroblast-specific protein 1 (FSP-1), CD68, toll-like receptor 4 (TLR4), and MCP-1. Results: We observed a significantly higher interstitial fibrosis area fraction and tubular injury (p < 0.001) with fibroblast expansion and myofibroblast formation in the UUO group than in the SO group. These findings were associated with higher mRNA expression of TGF-β, Collagen-1, Snail, vimentin, FSP-1, CD68, TLR4, and MCP-1 and lower mRNA expression of E-cadherin. The U+C840 group had a significantly lower tubular injury score and interstitial fibrosis area fraction, which associated with downregulation of mRNA expression of TGF-β, Collagen-1, Snail, vimentin, FSP-1, CD68, TLR4, and MCP-1, with upregulation of mRNA expression of E-cadherin. Immunostaining observation revealed the U+C840 group demonstrated reduction of macrophage infiltration and myofibroblast expansion. Conclusion: CeA treatment with dose-dependently ameliorates mesenchymal transition and inflammation in kidney fibrosis in mice.

Cardio-protective and Anti-atherosclerosis Effect of Crocetin on Vitamin D3 and HFD-induced Atherosclerosis in Rats.

Cardiovascular disease (CVD) is a chronic disease and causes the highest rate of death globally. CVD-related deaths account for 80% of all deaths in low and middle-income countries, such as China. Crocetin (CT), a carotenoid phytoconstituent already confirm their anti-inflammatory and antioxidant effects in various diseases animal models. In the study, we make effort to access the cardio-protective effect of Crocetin against vitamin D3 and high fat induced atherosclerosis in rats and scrutinize the underlying mechanism. Sprague Dawley (SD) rats were used in this study and rats were divided into different groups and high fat diet and vitamin D was used for induction the atherosclerosis. The rats were received oral administration of crocetin (5, 10 and 15 mg/kg) and simvastatin (0.5 mg/kg) until 30 days. At the end of the experimental period, lipid, cardiac markers, anti-inflammatory, antioxidant, pro-inflammatory cytokines and atherogenic index were estimated. The mRNA expression of Intercellular adhesion molecule-1 (ICAM-1), Monocyte Chemoattractant Protein-1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) in aortic tissue of the atherosclerotic rats. Crocetin significantly reduced the aortic membrane thickness and platelet aggregation rates. Crocetin also dose-dependently reduced total cholesterol (TC), very low-density lipoprotein (VLDL), triacylglycerol (TG), low-density lipoprotein (LDL) and augmented the level of high-density lipoprotein (HDL) level. Additionally, Crocetin significantly (p < 0.001) abridged the level of malonaldehyde (MDA) and augmented the level of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and glutathione peroxidase (GPx). Furthermore, Crocetin significantly (p < 0.001) dose-dependently reduced the levels of pro-inflammatory cytokines and inflammatory mediators. Crocetin attenuated mRNA expression of VCAM-1, ICAM-1 and MCP-1. Crocetin had anti-atherosclerosis and cardio-protective effects on vitamin D3 and high fat induced atherosclerosis in rats through anti-inflammatory and antioxidant mechanisms.

Interleukin-6 via Toll-Like Receptor 3 Signaling Attenuates the Expression of Proinflammatory Chemokines in Human Podocytes

Background: Although toll-like receptor 3 (TLR3) signaling is involved in the development of certain chronic kidney diseases, the specific molecular mechanisms underlying inflammatory reactions via activation of TLR3 signaling in human podocytes remain unclear. Interleukin (IL)-6 is a pleiotropic cytokine associated with innate and adaptive immune responses; however, little is known about the implication of IL-6 via the activation of regional TLR3 signaling in the inflammatory reactions in human podocytes. Methods: We treated immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), an authentic viral double-stranded RNA, and assessed the expression of IL-6, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against IFN-β and IL-6. Results: We found that the activation of TLR3 induced expression of IL-6, MCP-1, CCL5, and IFN-β in human podocytes. RNA interference experiments revealed that IFN-β was involved in the poly IC-induced expression of IL-6, MCP-1, and CCL5. Interestingly, IL-6 knockdown markedly increased the poly IC-induced expression of MCP-1 and CCL5. Further, treatment of cells with IL-6 attenuated the expression of CCL5 and MCP-1 mRNA and proteins. Conclusion: IL-6 induced by TLR3 signaling negatively regulates the expression of representative TLR3 signaling-dependent proinflammatory chemokines in human podocytes.

Enhancement of Porphyromonas gingivalis-Lipopolysaccharide Induced MCP-1 Expression by High Glucose in Human Endothelial Cells

To investigate the synergistic effects of periodontal disease and diabetes mellitus on atherosclerosis, we evaluated the monocyte chemoattractant protein-1 (MCP-1) expression of endothelial cells induced by Porphyromonas gingivalis-lipopolysaccharide (LPS) and high glucose. We also tested whether antioxidants could inhibit the MCP-1 expression induced by the simultaneous stimulation of LPS and high glucose. Human umbilical vein endothelial cells (HUVECs) were stimulated with various concentrations of P. gingivalis-LPS (0.1, 1.0, and 10 µg/mL) in normal glucose (5.5 mM), with high glucose (10 mM and 20 mM), and with 0.1 µg/mL P. gingivalis-LPS in high glucose. MCP-1 expressions were measured by realtime RT-PCR and ELISA. Vitamin C (100 µM) and vitamin E (50 µM) were administered before simultaneous stimulation with 0.1 µg/mL P. gingivalis-LPS and 20 mM glucose. LPS ≥ 1.0 μg/mL increased the expression of MCP-1 mRNA and protein compared to unstimulated HUVECs. High glucose in the culture medium increased the MCP-1 mRNA expression slightly but significantly at 2 hr of incubation, but the MCP-1 protein level was not increased. Simultaneous stimulation with 0.1 μg/mL LPS and high glucose increased the expression of MCP-1 mRNA and protein compared to unstimulated HUVECs. By contrast, pre-incubation of vitamin C or E inhibited the increase of MCP-1 mRNA expression induced by 0.1 μg/mL LPS and 20 mM glucose. Our finding that high glucose enhanced the MCP-1 expression with even a low level of LPS suggests that caution is advisable regarding the atherogenicity of diabetic patients with periodontal disease.

Endothelin converting enzyme-1 (ECE-1) deletion in association with Endothelin-1 downregulation ameliorates kidney fibrosis in mice

Kidney fibrosis is a common final pathway of chronic kidney diseases, which are characterized by renal architecture damage, inflammation, fibroblast expansion and myofibroblast formation. Endothelin converting enzyme-1 (ECE-1) contributes to activation of Endothelin-1 (ET-1), a potent vasoconstrictor and pro-fibrotic substance. This study elucidated the effect of ECE-1 knockout in kidney fibrosis model in mice in association of ET-1 downregulation. Kidney fibrosis was performed in ECE-1 knockout (ECE-1 KO) and vascular endothelial derived ET-1 KO (VEETKO) mice (2 months, 20–30 g, n = 30) and their wild type (WT) littermates using unilateral ureteral obstruction (UUO) procedure. Mice were euthanized on day-7 and day-14 after UUO. Histopathological analysis was conducted for fibrosis and tubular injury. Immunostainings were done to quantify macrophages (F4/80), fibroblasts (FSP-1) and myofibroblasts (α-SMA). Monocyte Chemoattractant Protein-1 (MCP-1), ECE-1 and preproET-1 (ppET-1) mRNA expression were quantified with qRT-PCR, while Transforming Growth Factor-β1 (TGF-β1) and α-SMA protein level were quantified with Western blot. ECE-1 KO mice demonstrated reduction of ECE-1 and ppET-1 mRNA expression, attenuation of kidney fibrosis, tubular injury, MCP-1 mRNA expression and macrophage number compared to WT. Double immunostaining revealed fibroblast to myofibroblast formation after UUO, while ECE-1 KO mice had significantly lower fibroblast number and myofibroblast formation compared to WT, which were associated with significantly lower TGF-β1 and α-SMA protein levels in day-14 of UUO. VEETKO mice also demonstrated attenuation of ET-1 protein level, fibrosis and myofibroblast formation. In conclusion, ECE-1 knockout and ET-1 downregulation attenuated kidney fibrosis.

Vitamin D receptor and monocyte chemoattractant protein-1 expressions in peripheral blood mononuclear cells in patients with systemic lupus erythemaotsus

To detect the expressions of vitamin D receptor (VDR) in peripheral blood monocytes (PBMCs) and its association with monocyte chemoattractant protein-1 (MCP-1) in patients with systemic lupus erythemaotsus (SLE). We examined the expressions of VDR and MCP-1 mRNAs in the PBMCs in 60 SLE patients and 28 healthy individuals using real- time quantitative PCR. We also detected the expression of VDR protein in the PBMCs using Western blotting and peripheral blood MCP-1 level using ELISA for these participants. The correlation of VDR and MCP-1 expressions with the disease activity index of SLE (SLEDAI) of the patients were analyzed. The expressions of VDR mRNA and protein in the PBMCs were significantly lower in patients with SLE than in the healthy individuals (P < 0.01), and that in the patients with active disease was lower than in remission (P < 0.05). The MCP-1 mRNA expression in the PBMCs and its serum levels were significantly increased in SLE patients as compared with the healthy individuals (P < 0.01), and the increase was significantly more obvious in the patients with active disease than in those in remission (P < 0.01). Pearson correlation analysis showed that VDR mRNA in the PBMCs was negatively correlated with SLEDAI (r=-0.417, P=0.001); negative correlations were also found between VDR mRNA and MCP-1 mRNA(r=-0.554, P=0.000) and between VDR protein expression and serum MCP-1 level (r=-0.400, P=0.028). The down-regulation of VDR expression in the PBMC is negatively correlated with the disease activity of SLE. VDR may play an important role in the pathogenesis of SLE by affecting the expression of MCP-1.

Expression of genes involved in the NF-κB-dependent pathway of the fibrosis in the mare endometrium

Equine endometrosis is a multifactorial chronic degenerative condition, considered to be one of a major causes of equine infertility. The formation of periglandular fibrosis seems to be linked to chronic inflammation of the mare endometrium in a paracrine way and in a response to numerous forms of inflammatory stimuli elicit the net deposition of extracellular matrix (ECM) around the endometrial glands and stroma. We hypothesized some of these stimuli, such as monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and hyaluronan synthases (HASs), may share the nuclear factor-κB (NF-κB) dependent activation pathway. This study aimed to determine whether mRNA expression of MCP-1, IL-6, HASs, and proteins of canonical (RelA/NK-κβ1) and noncanonical (NK-κβ2) signaling pathways for NF-kB would change in subsequent categories of endometrosis during the estrous cycle. The expression of selected genes was established in mare endometrium (n = 80; Kenney and Doig categories I, IIA, IIB, III), obtained in the follicular phase (FLP) and mid-luteal phase (MLP). The high expression of RelA mRNA was observed in III, whereas of NK-κβ1 and NK-κβ2 also in IIA, and IIA and IIB, respectively. The expression of MCP-1 mRNA occurred constantly, regardless of the category, whereas IL-6 mRNA was low in IIA, IIB, and III. The expression of HAS 1 was high in IIA and HAS 3 in IIA, IIB, and III. All those changes were observed in FLP, but not MLP. Our results suggest that NF-κB may be involved in progression of the chronic degenerative condition of the mare endometrium, on both canonical and noncanonical pathways. The most important changes in target genes expression were observed only in FLP, which may suggest the hormone-dependent activation of the NF-κB-dependent fibrosis pathway.

Effect of Ursolic Acid on Insulin Resistance and Hyperinsulinemia in Rats with Diet-Induced Obesity: Role of Adipokines Expression

Excess of visceral adipose tissue (VAT) characteristic of obesity leads to a proinflammatory state disrupting the insulin signaling pathway, triggering insulin resistance (IR) and inflammation, the main processes contributing to obesity comorbidities. Ursolic acid (UA), a pentacyclic triterpenoid occurring in a variety of plant foods, exhibits anti-inflammatory properties. The aim of this study was to evaluate UA effects on IR, hyperinsulinemia, and inflammation in experimental diet-induced obesity. Forty male Wistar rats were randomly assigned to eight groups (n = 5). One group was used for time 0. Three groups were labeled as OBE (control): receiving high-fat diet (HFD; fat content 45.24% of energy) during 3, 6, or 9 weeks; three groups UA-PREV: exposed to simultaneous HFD and UA during 3, 6, or 9 weeks to evaluate UA preventive effects; one group UA-REV: receiving HFD for 6 weeks, followed by simultaneous HFD and UA for three additional weeks to analyze UA reversal effects. Measurements were performed after 3, 6, or 9 weeks of treatment. Adiposity was calculated by weighing VAT after sacrifice. Serum markers were quantified through colorimetric and enzyme-linked immunosorbent assay methods. VAT adipokines RNAm expression was evaluated by quantitative reverse transcriptase-polymerase chain reaction. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests. UA significantly decreased adiposity, IR, hyperinsulinemia, triacylglycerides, and cholesterol levels, and also VAT mRNA expression of MCP-1 (monocyte chemoattractant protein-1), IL (interleukin)-1β and IL-6, concomitantly increasing adiponectin levels. UA metabolic effects demonstrated in this study support its potential therapeutic utility to improve IR, hyperinsulinemia, and inflammation observed in obesity and diabetes.