Chlorogenic acid may improve memory function and decrease inflamation of frontal lobe in diabetic rat.

INTRODUCTION: Diabetes Mellitus (DM) is a chronic disease with many complications, one of which is diabetic encephalopathy which is characterised by memory dysfunction. Hyperglycaemia that occurs in DM will activate inflammatory pathways in neurons, including NF-κB pathway. Activation of this pathway produce proinflammatory agents such as MCP-1 and IL-6, which activate glial cells. Activation of glial cells is characterised by Glial Fibrillary Acid Protein (GFAP). Chlorogenic acid (CGA) has been reported to have anti-inflammatory effects and can improve memory function. This research aimed to determine the effect of CGA as anti-inflammation, its effect on memory function, mRNA expression of NF-κB, MCP-1, IL- 6, and GFAP of frontal lobe. MATERIALS AND METHODS: A total of 24 male rats were randomly divided into six groups: control, DM 1.5 month (DM1.5), DM 2 months (DM2) and the group with three different doses of CGA 12.5 (CGA1), 25 (CGA2), and 50 (CGA3) mg/KgBW. Frontal lobe tissue is taken for analysis of mRNA expression for NF-κB, MCP-1, IL-6, and GFAP using Reverse Transcriptase PCR (RT-PCR). Samples were also taken for histopathology preparation and stained by immunohistochemistry method using anti-GFAP antibodies to observe glial cell activation in frontal lobe tissue. RESULTS: The group that was given CGA at all doses have statistically significant better memory function, i.e. DM2 versus CGA1 (p = 0.036), CGA2 (p = 0.040), and CGA3 (p = 0.021). The result of mRNA expression in NF-κB was lower in the group given CGA, i.e. DM2 compared to CGA2 (p = 0.007). mRNA expression of MCP-1 was significantly lower in all CGA treatment groups compared to the non-CGA group (p = 0.000). IL-6 mRNA expression was lower than the group not given CGA, DM compared to CGA2 (p = 0.028). GFAP mRNA expression was lower than the group given CGA in DM, DM2 group compared to CGA1 (p = 0.04) and CGA3 (p = 0.004). CONCLUSION: Administration of CGA can improve memory function at all doses given, and can reduce brain inflammatory activity, especially in the CGA2 group.

Vascular remodeling and association with inflammation in the heart of obesity model.

INTRODUCTION: Obesity alters several metabolic activities, subsequently leading to the development of cardiovascular diseases. The insulin resistance-induced obesity stimulates vasodilatation and vasoconstriction imbalance, which ends up in cardiac vascular remodeling. Therefore, we aimed to investigate the effect of obesity in cardiac diseases with a focus on inflammatory mediators associated with endothelial dysfunction. MATERIALS AND METHODS: Rats (3 months old, weighing 200 g) were divided into control (n=6) and the obese groups, which included rats fed on a high-fat diet (HFD, n=6 in each subgroup) for 1 month (OB1), 2 months (OB2), and 4 months (OB4). Then, the rats were sacrificed, and their hearts were harvested for histological quantification as well as the quantification of the mRNA expression of inflammatory mediators, eNOS, and ppET-1 by reverse transcriptasepolymerase chain reaction (RT-PCR). Sirius Red staining was performed to assess vascular remodeling, while immunohistochemistry of CD68 was performed to assess the localization of macrophage. RESULTS: HFD-induced obesity was significantly manifested in the obese groups relative to that in the control group. It was followed by an increase in the mRNA expression of inflammatory mediators in the obese groups when compared to that in the control group. Long-term obesity promoted vascular remodeling, which was noted in the OB4 group, along with downregulation of the eNOS mRNA expression and the upregulation of the ppET-1 mRNA expression. CONCLUSION: Obesity associated with inflammation and vascular remodeling in the heart.

Liver fibrosis associated with adipose tissue and liver inflammation in an obesity model.

INTRODUCTION: Obesity, the main risk factor for type 2 diabetes mellitus (T2DM), affects the secretion of various hormones that lead to change in metabolism. Visceral adipose tissue accumulation may contribute to Non-alcoholic Fatty Liver Disease (NAFLD) and induce liver injury. This study was aimed to investigate the association between adipose tissue inflammation and liver fibrosis. MATERIALS AND METHODS: Wistar male rats (3 months old, 160- 230 grams) were divided into 4 groups that consisted of six rats in each group. The obesity model was induced through the administration of high-fat diet for a month (OB1), two months (OB2), and four months (OB4). Standard chow was provided for the control group for four months. After the specified date the rats were euthanized and the liver and retroperitoneal white adipose tissue (RWAT) were harvested. We performed RT-PCR to assess the mRNA expressions involved in proinflammatory mediators, fibrosis and antifibrosis signaling. Sirius red staining was performed to assess liver fibrosis. Data were analyzed with SPSS 23 for Windows with significance set as p<0.05. RESULTS: Obesity-induced high-fat diet stimulated an increase of body mass index (BMI) in the OB groups (p<0.05) compared to the control group. Increased BMI was followed by upregulation of proinflammatory mediators (MCP-1, CD68, TLR4, and NFκB) of the RWAT and liver in the obese groups (p<0.05), which promoted hepatic fibrosis in triad portal areas and upregulation of TGFß (p<0.05) mRNA expression as well as downregulation of HGF and c-Met (p<0.05). In addition, hepatic ppET1 and EDNRB mRNA level expressions (p<0.05) were obviously upregulated in the obese groups followed by downregulation of eNOS (p<0.05) mRNA expressions. CONCLUSION: Obesity enhanced inflammation in RWAT and was associated with inflammation and fibrosis of liver.

Upregulation of Megalin, Cubilin, NGAL mRNA expression in kidney may represent tubular injury and apoptosis in chronic condition of rat diabetic model.

INTRODUCTION: Diabetes mellitus (DM) leads to microvascular injury development and produces diabetes nephropathy (DN) with proteinuria, tubular injury, apoptosis and autophagy with upregulation of Bax, BASP and mTORC-1. Megalin, Cubilin and Neutrophil Gelatinase Associated Lipocalin (NGAL) play role in acute pathological condition of kidney injury, however its expression in chronic and slowly progressive kidney injury such as DN has not been elucidated yet. This study focuses upregulation of Megalin, Cubilin and NGAL in association with tubular injury and apoptosis in DN condition. MATERIALS AND METHODS: Diabetic condition was performed with intraperitoneal injection of Streptozotocin 60 mg/kg body weight (BW) in Sprague Dawley rats (2 months old, n=24), and were kept for 1, 2, and 4 months (DM1, DM2, and DM4, respectively). Control group was injected with NaCl 0.9%. Serum glucose level and proteinuria score were assessed, furthermore tubular injury score was quantified based on Periodic-Acid Schiff (PAS) staining. Reverse Transcriptase-PCR (RT-PCR) was carried out for NGAL, Megalin, Cubilin, m-TOR, Bax, and BASP-1 mRNA expression. Data were analyzed using SPSS 22 software. RESULTS: DM led to kidney injury in this model with significant higher glucose level, proteinuria and tubular injury, especially in DM4 group which represented chronic phase of DN and CKD. These findings associated with upregulation of Megalin,Cubilin and NGAL mRNA expression in DM groups, especially in DM4 group. DM4 group also revealed higher expression of Bax, BASP and mTOR mRNA expression which demonstrated apoptosis. CONCLUSION: Megalin, Cubilin and NGAL upregulation may represent tubular injury and apoptosis as progression of DN.

Chlorogenic acid attenuates kidney fibrosis via antifibrotic action of BMP-7 and HGF.

BACKGROUND: Kidney fibrosis, characterised by tubulointerstitial fibrosis, is a histological landmark of chronic kidney disease. The body attempts to compensate for progressive detrimental process of kidney fibrosis by producing antifibrotic substances, such as bone morphogenetic protein-7 (BMP-7) and hepatocyte growth factor (HGF). Chlorogenic acid is known to have renoprotective and antifibrotic properties. This study aims to evaluate the effect of chlorogenic acid on unilateral ureteral obstruction (UUO)-induced kidney fibrosis mice model. METHODS: This study was a quasi-experimental with posttestonly control group design. Twenty-five adult male Swiss Webster mice were randomly divided into five groups: shamoperated group (SO), UUO-control day-7 (U7), UUO-control day-14 (U14), UUO-chlorogenic acid day-7 (UC7), and UUOchlorogenic acid day 14 (UC14). Myofibroblasts were identified by immunohistochemical staining of alphasmooth muscle actin (α-SMA) while collagen fibers were identified by Sirius Red staining. Both data were presented as area fraction. BMP-7 and HGF mRNA expressions were assessed by reverse transcription PCR (RT-PCR). Data were quantified using ImageJ software. RESULTS: UUO-control groups (U7 and U14) showed higher α- SMA-immunopositive (6.52±1.33, 18.24±1.39 vs. 0.22±0.01; p<0.05) and Sirius Red-positive area fractions (6.61±0.8, 12.98±2.31 vs. 0.62±0.10; p<0.05), lower BMP-7 (1.02±0.47, 1.18±0.65 vs. 2.09±0.87; p<0.05) and HGF mRNA expressions (1.06±0.31, 0.89±0.14 vs. 1.88±0.81; p<0.05) compared to SO group. UUO-chlorogenic acid groups (UC7 and UC14) showed lower α-SMA-immunopositive (1.24±0.37, 4.58±0.61; p<0.05) and Sirius Red-positive area fractions (4.76±1.03, 3.72±0.54; p<0.05), higher BMP-7 (1.84±0.49, 2.19±0.43; p<0.05) and HGF (1.58±0.38; p>0.05, 1.84±0.42; p<0.05) mRNA expressions compared to UUO-control groups. UUOchlorogenic acid groups showed BMP-7 and HGF mRNA expressions that were not significantly different from the SO group. CONCLUSION: Chlorogenic acid administration prevents kidney fibrosis in UUO mice model through modulating antifibrotic pathway.

Uric acid induces liver fibrosis through activation of inflammatory mediators and proliferating hepatic stellate cell in mice.

INTRODUCTION: Uric acid is associated with cardiometabolic risk factor and severity of liver damage. The mechanism of uric acid inducing liver damage is still elusive. This study elucidates the development of liver fibrosis under hyperuricemia. METHODS AND MATERIALS: Hyperuricemia model was performed in male Swiss Webster mice. Intraperitoneally injection of uric acid (125mg/kg body weight) was done for 7 and 14 days (UA7 and UA14 groups). Meanwhile, the UAL groups were injected with uric acid and followed by the administration of allopurinol (UAL7 and UAL14 groups). On the due date, mice were sacrificed, and liver was harvested. Uric acid, SGOT, SGPT, and albumin level were measured from the serum. The mRNA expression of TLR4, MCP1, CD68, and collagen1 were assessed through RT-PCR. Liver fibrosis was quantified through Sirius red staining, while the number of hepatic stellates cells (HSCs) and TLR4 were assessed through IHC staining. RESULTS: Uric acid induction for 7 and 14 days stimulated an increase of both SGOT and SGPT serum levels. Followed by enhanced inflammatory mediators: Toll-like receptor-4 (TLR- 4), Monocyte Chemoattractant Protein-1 (MCP-1) and Cluster of Differentiation 68 (CD68) mRNA expression in the liver (p<0.05). The histological findings showed that the UA7 and UA14 groups had higher liver fibrosis scores (p<0.05), collagen I mRNA expression (p<0.05), and the number of HSCs (p<0.05) compared to Control group. Administration of allopurinol showed amelioration of uric acid and liver enzymes levels which followed by inflammatory mediators, liver fibrosis and collagen1, and hepatic stellate cells significantly. CONCLUSION: Therefore, uric acid augmented the liver fibrosis by increasing the number of hepatic stellate cells.

Effect of kidney ischemia/reperfusion injury on proliferation, apoptosis, and cellular senescence in acute kidney injury in mice.

INTRODUCTION: Kidney ischemia/reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI). Kidney IRI demonstrated apoptosis of epithelial cells in acute phase followed by proliferation of interstitial cells in chronic episode, and cellular senescence may contribute to development of AKI, however, its occurrence within acute or chronic episodes is still not completely understood. METHODS: Kidney IRI was performed with bilateral pediculus clamping in Swiss Background mice (3 months, 30-40g). Mice were euthanised on day one (I/R1, n=6), day eight (I/R8, n=6), and day twelve (I/R12, n=6) to exam acute and chronic episodes. Sham operation procedure was performed in the control. Tubular injury was assessed based on periodic acid- Schift (PAS) staining. Reverse transcriptase PCR (RT-PCR) was done to quantify mRNA expression of Bax, Bcl-2, and p16. Immunohistostaining (IHC) was performed to examine localisation of apoptosis (p53) and proliferation (Bcl-2). RESULTS: RT-PCR analysis showed upregulation of mRNA expression of Bcl-2, Bax, and p16 (p<0.05). The data showed that ischemia/reperfusion induces upregulation of Bax (p=0.20), Bcl-2 (p=0.45), p16 (p=0.18). Apoptosis and proliferation occurred in the epithelial cells in acute episodes, but occurred in interstitial areas in chronic episodes. CONCLUSIONS: Ischemia/reperfusion injury induces upregulation proliferation, apoptosis, and cellular senescence in acute kidney injury. Apoptosis reached its peak on day 1, proliferation on day 8, and cellular senescence on day 12.

Vitamin D upregulates endothelin-1, ETBR, eNOS mRNA expression and attenuates vascular remodelling and ischemia in kidney fibrosis model in mice.

We examined the upregulation of ET-1/ETBR/eNOS signaling in renoprotective effect of vitamin D in kidney fibrosis model in mice using unilateral ureteral obstruction (UUO). One group was treated with intraperitoneal injection of 0.125 mg/kg of Calcitriol (UUO+VD). Vascular remodeling was quantified based on lumen area and lumen/wall area ratio (LWAR) of intrarenal arteries using Sirius Red staining. ET-1, ETBR, eNOS, CD31 and VEGF mRNA expressions were quantified using qRT-PCR. Focusing on endothelin-1 (ET-1) signaling in endothelial cells (EC), siRNA of ET-1 was performed in human umbilical vein endothelial cells (HUVEC) for reducing ET-1 expression. Then HUVECs were treated with and without 100 nM Calcitriol treatment in hypoxic and normoxic conditions to elucidate ET-1/eNOS signaling. Our in vivo study revealed vascular remodeling and renal ischemia attenuation after Calcitriol treatment. Vascular remodeling was attenuated in the UUO+VD group as shown by increasing lumen areas and LWAR in intrarenal arteries. These findings were associated with significant higher CD31 and VEGF mRNA expression compared to the UUO group. Vitamin D treatment also increased ET-1, ETBR and eNOS mRNA expressions. Our in vitro study demonstrated Calcitriol induced ET-1 and eNOS mRNA expressions upregulation in HUVEC under normoxic and hypoxic condition. Meanwhile, siRNA for ET-1 inhibited the upregulation of eNOS mRNA expression after Calcitriol treatment. Vitamin D ameliorates kidney fibrosis through attenuating vascular remodeling and ischemia with upregulating ET-1/ETBR and eNOS expression.

Endothelial-derived endothelin-1 promotes pulmonary vascular remodeling in bleomycin-induced pulmonary fibrosis.

Endothelin-1 (ET-1) induces pulmonary vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. Given that endothelial cells are the main source of ET-1 and ET-1 from other cells may encounter difficulty penetrating vascular compartments, we hypothesize that endothelial-derived ET-1 promotes vascular remodeling secondary to pulmonary fibrosis. We used vascular endothelial ET-1 knock-out (VEETKO) and Wild type mice for this research. They were given intratracheal bleomycin and euthanized at day 28. We quantified pulmonary fibrosis, measured lung ET-1 and its receptors' expression, and assessed pulmonary vascular remodeling by calculating medial wall index, muscularization index, adventitial collagen and adventitial fibroblast and macrophage accumulation. Right ventricle remodeling was also assessed. Both VEETKO and Wild type mice developed comparable pulmonary fibrosis and similar fibrosis-related gene expression. Compared to Wild type mice, bleomycin-induced VEETKO mice had lower ET-1 peptide levels (15.4 pg/mg vs. 31.2 pg/mg, p<0.01). Expression of both ET-1 receptors mRNAs were increased in fibrosis models. Bleomycin-induced fibrosis VEETKO mice had significantly less muscularized arterioles, lower muscularization index and attenuated adventitial collagen, fibroblast and macrophage accumulation as compared to that of Wild type mice. Right ventricular pressure, hypertrophy and fibrosis did not increase both in VEETKO and Wild type mice despite the more enhanced vascular remodeling in Wild type. In conclusion, endothelial-derived endothelin-1 promotes pulmonary vascular remodeling secondary to bleomycin-induced pulmonary fibrosis.