Masson's Trichome Staining Research Articles

TAK-242 alleviates diabetic cardiomyopathy via inhibiting pyroptosis and TLR4/CaMKII/NLRP3 pathway.

Diabetic cardiomyopathy (DCM) is identified as a progressive disease that may lead to irreparable heart failure. Toll-like receptor (TLR) signaling is believed to be implicated in the pathogenesis of DCM. This study intended to explore the potential impact of Toll-like receptor 4 (TLR4) on DCM in vitro and in vivo. Streptozotocin and HG medium were utilized to induce diabetes in animal and cell models, respectively. Selective TLR4 inhibitor TAK-242 and calcium/calmodulin-dependent protein kinase-II (CaMKII) inhibitor KN-93 were employed to explore the involvement of TLR4/CaMKII in DCM. TLR4 expression was increased in DCM hearts, while inhibition of TLR4 activation by TAK-242 improved cardiac function, attenuated heart hypertrophy, and fibrosis, as well as reduced oxidative stress and proinflammatory cytokine levels in rats, which were confirmed by Doppler echocardiography, hematoxylin and eosin staining, and Masson Trichome staining and specific enzyme-linked immunosorbent assay kits. Besides, the expression of hypertrophy-related molecules and oxidative stress damage were also inhibited by TAK-242. Furthermore, TAK-242 treatment reduced CaMKII phosphorylation accompanied by decreased expression of NOD-like pyrin domain-containing protein 3, gasdermin D (GSDMD), The N-terminal domain of Gasdermin D (GSDMD-N), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and Caspase-1 both in vivo and in vitro. Similar positive impacts on HG-induced pyroptosis were also observed with KN-93 treatment, and this was achieved without affecting TLR4 expression. Collectively, our work suggested that TAK-242 demonstrated substantial benefits against DCM both in vivo and in vitro, potentially attributed to the suppression of the TLR4-mediated CaMKII/NLRP3 pathway activity.

Box A of HMGB1 Maintains the DNA Gap and Prevents DDR-induced Kidney Injury in D-galactose Induction Rats.

Disability and mortality rates for renal failure are still increasing. DNA damage and oxidative stress intoxication from body metabolism, high blood glucose, or the environment cause significant kidney damage. Recently, we reported that Box A of HMGB1 (Box A) acts as molecular scissors, producing DNA gaps that prevent DNA damage in kidney cell lines and ultimately reverse aging phenotypes in aging rat models. The present study aimed to demonstrate the potency of Box A in preventing D-galactose (D-gal)-induced kidney injury. A Box A expression plasmid was constructed and administered to a rat model. D-gal was injected subcutaneously for eight weeks. Serum was collected to study renal function, and white blood cells were collected for DNA gap measurement. Kidney tissue was also collected for γ-H2AX and NF-κB immunostaining; Senescence-associated (SA)-beta-gal staining; and analysis of the mRNA expression of p16INK4A, TNF-α, and IL-6. Moreover, histopathology analysis was performed using hematoxylin & eosin and Masson trichome staining. Pretreatment with Box A administration prevented the reduction of DNA gaps and the consequences of the DNA damage response, which include elevated serum creatinine; high serum BUN; an increased positive SA-beta-gal staining area; overexpression of p16INK4A, NF-κB and senescence-associated secretory phenotype molecules, including IL-6, TNF-α; and histological alterations, including tubular dilation and collagen accumulation. Box A effectively prevents DNA gap reduction and all D-gal-induced kidney pathological changes at the molecular, histological, and physiological levels. Therefore, Box A administration is a promising novel therapeutic strategy to prevent DNA-damaging agent-induced kidney failure.

Resveratrol treatment ameliorates hepatic damage via the TGF-β/SMAD signaling pathway in a phenobarbital/CCl4-induced hepatic fibrosis model.

Liver fibrosis is a wound healing response characterized by excessive accumulation of extracellular matrix proteins. This study aimed to investigate the effects of resveratrol treatment on the TGF-β/SMAD signaling pathway and related biochemical parameters, apoptosis, and liver regeneration phenobarbital-CCl4 induced hepatic fibrosis rat model. This model was created through phenobarbital and CCl4 (0.2-0.35 ml/kg). Resveratrol (1 mg/kg/day) was administered to the fibrosis and control groups. Immunohistochemical staining was performed to evaluate αSMA, TGF-β1, and PCNA in liver tissue. The TUNEL method and Masson's Trichome staining were used to determine apoptosis and collagen accumulation. AST, ALP, ALT, total protein, and total bilirubin levels were measured to determine biochemical status. SMAD2, SMAD3, SMAD4, and SMAD7 expression levels were measured to determine TGF-β1 related hepatic fibrosis. The SMAD2, SMAD3, and SMAD4 mRNA expression levels were increased and the SMAD7 mRNA expression level was decreased in the fibrosis control group. The SMAD7 mRNA expression level was higher in the phenobarbital-CCl4 induced resveratrol treated group. Increased biochemical parameters indicating hepatic damage, increased number of apoptotic cells, and collagen accumulation surrounding the central vein were observed in the fibrosis group compared with the other groups. It was concluded that administration of resveratrol ameliorates the adverse effects of hepatic fibrosis by regulating biochemical parameters, controlling TGF-β1/SMAD signaling, enhancing tissue regeneration, and reducing apoptosis in liver cells. Resveratrol can be a beneficial option for the prevention of liver damage in a phenobarbital-CCl4 induced hepatic fibrosis.

Carbon quantum dot-nanocomposite hydrogel as Denovo Nexus in rapid chondrogenesis

The incapability of cartilage to naturally regenerate and repair chronic muscular injuries urges the development of competent bionic rostrums. There is a need to explore faster strategies for chondrogenic engineering using mesenchymal stem cells (MSCs). Along these lines, rapid chondrocyte differentiation would benefit the transplantation demand affecting osteoarthritis (OA) and rheumatoid arthritis (RA) patients. In this report, a de novo nanocomposite was constructed by integrating biogenic carbon quantum dot (CQD) filler into synthetic hydrogel prepared from dimethylaminoethyl methacrylate (DMAEMA) and acrylic acid (AAc). The dominant structural integrity of synthetic hydrogel along with the chondrogenic differentiation potential of garlic peel derived CQDs led to faster chondrogenesis within 14 days. By means of extensive chemical and morphological characterization techniques, we illustrate that the hydrogel nanocomposite possesses lucrative features to influence rapid chondrogenesis. These results were further corroborated by bright field imaging, Alcian blue staining and Masson trichome staining. Thus, this stratagem of chondrogenic engineering conceptualizes to be a paragon in clinical wound care for the rapid manufacturing of chondrocytes.

Macrophage KLF15 prevents foam cell formation and atherosclerosis via transcriptional suppression of OLR-1

BackgroundMacrophage-derived foam cells are a hallmark of atherosclerosis. Scavenger receptors, including lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR-1), are the principal receptors responsible for the uptake and modification of LDL, facilitating macrophage lipid load and the uptake of oxidized LDL by arterial wall cells. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates the expression of genes by binding to the promoter during transcription. Therefore, this study aimed to investigate the precise role of macrophage KLF15 in atherogenesis. MethodsWe used two murine models of atherosclerosis: mice injected with an adeno-associated virus (AAV) encoding the Asp374-to-Tyr mutant version of human PCSK9, followed by 12 weeks on a high-fat diet (HFD), and ApoE−/−‐ mice on a HFD. We subsequently injected mice with AAV-KLF15 and AAV-LacZ to assess the role of KLF15 in the development of atherosclerosis in vivo. Oil Red O, H&E, and Masson's trichome staining were used to evaluate atherosclerotic lesions. Western blots and RT-qPCR were used to assess protein and mRNA levels, respectively. ResultsWe determined that KLF15 expression was downregulated during atherosclerosis formation, and KLF15 overexpression prevented atherosclerosis progression. KLF15 expression levels did not affect body weight or serum lipid levels in mice. However, KLF15 overexpression in macrophages prevented foam cell formation by reducing OLR-1-meditated lipid uptake. KLF15 directly targeted and transcriptionally downregulated OLR-1 levels. Restoration of OLR-1 reversed the beneficial effects of KLF15 in atherosclerosis. ConclusionMacrophage KLF15 transcriptionally downregulated OLR-1 expression to reduce lipid uptake, thereby preventing foam cell formation and atherosclerosis. Thus, our results suggest that KLF15 is a potential therapeutic target for atherosclerosis.

Breast Milk Mesenchymal Stem Cells and/or Derived Exosomes Mitigated Adenine-Induced Nephropathy via Modulating Renal Autophagy and Fibrotic Signaling Pathways and Their Epigenetic Regulations.

Chronic kidney disease (CKD), a global health concern, is highly prevalent among adults. Presently, there are limited therapeutic options to restore kidney function. This study aimed to investigate the therapeutic potential of breast milk mesenchymal stem cells (Br-MSCs) and their derived exosomes in CKD. Eighty adult male Sprague Dawley rats were randomly assigned to one of six groups, including control, nephropathy, nephropathy + conditioned media (CM), nephropathy + Br-MSCs, nephropathy + Br-MSCs derived exosomes (Br-MSCs-EXOs), and nephropathy + Br-MSCs + Br-MSCs-EXOs. Before administration, Br-MSCs and Br-MSCs-EXOs were isolated, identified, and labeled with PKH-26. SOX2, Nanog, and OCT3/4 expression levels in Br-MSCs and miR-29b, miR-181, and Let-7b in both Br-MSCs and Br-MSCs-EXOs were assayed. Twelve weeks after transplantation, renal function tests, oxidative stress, expression of the long non-coding RNA SNHG-7, autophagy, fibrosis, and expression of profibrotic miR-34a and antifibrotic miR-29b, miR-181, and Let-7b were measured in renal tissues. Immunohistochemical analysis for renal Beclin-1, LC3-II, and P62, Masson trichome staining, and histopathological examination of kidney tissues were also performed. The results showed that Br-MSCs expressed SOX2, Nanog, and OCT3/4, while both Br-MSCs and Br-MSCs-EXOs expressed antifibrotic miR-181, miR-29b, and Let-7b, with higher expression levels in exosomes than in Br-MSCs. Interestingly, the administration of Br-MSCs + EXOs, EXOs, and Br-MSCs improved renal function tests, reduced renal oxidative stress, upregulated the renal expression of SNHG-7, AMPK, ULK-1, Beclin-1, LC3, miR-29b, miR-181, Let-7b, and Smad-7, downregulated the renal expression of miR-34a, AKT, mTOR, P62, TGF-β, Smad-3, and Coli-1, and ameliorated renal pathology. Thus, Br-MSCs and/or their derived exosomes appear to reduce adenine-induced renal damage by secreting antifibrotic microRNAs and potentiate renal autophagy by modulating SNHG-7 expression.

#6227 SPONTANEOUS REMISSION OF MEMBRANOUS VARIANT OF PGNMID WITH MONOCLONAL IGG2κ

Abstract Background and Aims The clinical significance of the morphological patterns of glomerular injury and of each IgG subclass in proliferative glomerulonephritis (GN) with monoclonal immunoglobulin deposits (PGNMID) is not well understood. Literature suggests that PGNMID with certain histological features such as membranous or mesangio-proliferative features or non-IgG3 subclass staining may be associated with a more favourable renal prognosis. We present a patient with membranous variant of PGNMID with monoclonal IgG2κ who had spontaneous remission without recurrence over a 2-year follow-up. Method Not applicable Results A 62-year-old Malay gentleman presented with new onset nephrotic range proteinuria, on a background of type 2 diabetes mellitus with diabetic duration of 5 years, hypertension and hyperlipidemia. He was mildly hypertensive (blood pressure 148/87) and non-edematous at presentation. Investigations revealed preserved kidney function [serum creatinine (sCr) 66µmol/L], bland urinalysis, and 24-hour urinary total protein (UTP) of 3.74g/day. Serum albumin (sAlb) was 35g/L. Serum complements were not low. Autoimmune markers inclusive of anti-double stranded DNA antibodies, anti-neutrophil cytoplasmic antibodies, and anti-phospholipase A2 receptor (PLA2R) antibodies were negative. Viral serologies were also unremarkable. No monoclonal band was detected on serum electrophoresis, while serum immunofixation was not performed. Kidneys were normal sized. Renal biopsy performed demonstrated 28 glomeruli, of which only 1 was globally sclerosed. Glomeruli capillary walls were diffusely thickened by vacuolations. Capillary loops were mostly single contoured although occasional focal double contouring was seen. Masson trichome stain revealed fuchsinophilic subepithelial immune deposits while periodic acid methenamine silver stain showed argyrophilic basement membrane spikes. Variable mesangial expansion and hypercellularity was observed, as well as mesangiolysis. Focal leukocyte margination was present, but significant endocapillary proliferation was not seen. There were no Kimmelstiel-Wilson lesions. Tubular atrophy with interstitial fibrosis was overall mild (10% of parenchymal area) and there were no tubulointerstitial infiltrates. Immunofluoresence showed granular staining of IgG (1-2+), C3 (2-3+) along glomerular capillary walls, as well as presence of kappa light chain restriction (κ 1-2+; λ negative in glomeruli). Immunostaining for PLA2R was negative. IgG subclass analysis performed in view of kappa restriction revealed isolated deposition of IgG2. Electron microscopy was not available. Overall findings were most consistent with membranous variant of PGNMID with monoclonal IgG2κ. Age-appropriate and symptom-directed malignancy screening did not reveal significant abnormalities. Given that the patient was minimally symptomatic, a trial of conservative therapy was initiated with optimized non-immunosuppressive anti-proteinuric therapy using renin-angiotensin-aldosterone and sodium-glucose co-transporter-2 inhibitors. Gradual spontaneous remission was observed without recurrence over 2 years of follow-up (sCr 77µmol/L; sAlb 40g/L; 24-hour UTP 0.22g/day). Conclusion Our case adds to the literature that a membranous variant of PGNMID with monoclonal IgG2κ may be associated with better renal outcomes. Despite the lack of proliferative changes, it is possible that membranous variants may share similar underlying disease mechanisms as the other proliferative variants. Large international registries to allow the correlation of morphological features and IgG subclasses with clinical outcomes are required to confirm our observation.

Effect of vinpocetine alone and in combination with enalapril in experimental model of diabetic cardiomyopathy in rats: possible involvement of PDE-1/TGF-β/ Smad 2/3 signalling pathways.

Diabetic cardiomyopathy (DC) is one of the severe secondary complications of diabetes mellitus in humans. Vinpocetine is an alkaloid having pleiotropic pharmacological effects. The present study is designed to investigate the effect of vinpocetine in DC in rats. Rats were fed a high-fat diet for nine weeks along with single dose of streptozotocin after the second week to induce DC. The haemodynamic evaluation was performed to assess the functional status of rats using the Biopac system. Cardiac echocardiography, biochemical, oxidative stress parameters and inflammatory cytokine level were analysed in addition to haematoxylin-eosin and Masson's trichome staining to study histological changes, cardiomyocyte diameter and fibrosis, respectively. Phosphodiesterase-1 (PDE-1), transforming growth factor-β (TGF-β) and p-Smad 2/3 expression in cardiac tissues were quantified using western blot/RT-PCR. Vinpocetine treatment and its combination with enalapril decreased the glucose levels compared to diabetic rats. Vinpocetine improved the echocardiographic parameters and cardiac functional status of rats. Vinpocetine decreased the cardiac biochemical parameters, oxidative stress, inflammatory cytokine levels, cardiomyocyte diameter and fibrosis in rats. Interestingly, expressions of PDE-1, TGF-β and p-Smad 2/3 were ameliorated by vinpocetine alone and in combination with enalapril. Vinpocetine is a well-known inhibitor of PDE-1 and the protective effect of vinpocetine in DC is exerted by inhibition of PDE-1 and subsequent inhibition of the expression of TGF-β/Smad 2/3.

Establishing a Xenograft Model with CD-1 Nude Mice to Study Human Skin Wound Repair.

A significant gap exists in the translatability of small-animal models to human subjects. One important factor is poor laboratory models involving human tissue. Thus, the authors have created a viable postnatal human skin xenograft model using athymic mice. Discarded human foreskins were collected following circumcision. All subcutaneous tissue was removed from these samples sterilely. Host CD-1 nude mice were then anesthetized, and dorsal skin was sterilized. A 1.2-cm-diameter, full-thickness section of dorsal skin was excised. The foreskin sample was then placed into the full-thickness defect in the host mice and sutured into place. Xenografts underwent dermal wounding using a 4-mm punch biopsy after engraftment. Xenografts were monitored for 14 days after wounding and then harvested. At 14 days postoperatively, all mice survived the procedure. Grossly, the xenograft wounds showed formation of a human scar at postoperative day 14. Hematoxylin and eosin and Masson trichome staining confirmed scar formation in the wounded human skin. Using a novel artificial intelligence algorithm using picrosirius red staining, scar formation was confirmed in human wounded skin compared with the unwounded skin. Histologically, CD31 + immunostaining confirmed vascularization of the xenograft. The xenograft exclusively showed human collagen type I, CD26 + , and human nuclear antigen in the human scar without any staining of these human markers in the murine skin. The proposed model demonstrates wound healing to be a local response from tissue resident human fibroblasts and allows for reproducible evaluation of human skin wound repair in a preclinical model. Radiation-induced fibrosis is a widely prevalent clinical phenomenon without a well-defined treatment at this time. This study will help establish a small-animal model to better understand and develop novel therapeutics to treat irradiated human skin.

Protein Hydrolysate of Green Peas Bromelain Attenuates Kidney Fibrosis in Cisplatin-Induced Nephrotoxicity Rats: Emphasis on Anti-Inflammatory Activities

Massive tubule fibrosis is a histopathology hallmark of chronic renal failure (CRF). The previous study of protein hydrolysate of green pea (Pisum sativum) bromelain (PHGPB) showed promising results as an antifibrosis in glucose-induced renal mesangial culture cells, by decreasing their TGF-β levels. In this experiment the anti-inflammatory, anti-fibrotic effect of PHGPB in Cisplatin-induced chronic renal failure (CRF) rats was measured. The purpose of this study is to evaluate the anti-inflammatory and antifibrosis effect of PHGPB in Cisplatin-induced chronic renal failure rats. Five groups consisted of five rats: negative control, Cisplatin control, and three groups of Cisplatin+PHGPB (dose of 100, 200, and 400 mg/kg BW/day) treated for 56 days. The examined parameters are Beta-2-microglobulin (β2MG), hs-CRP, histopathological observations of Masson's trichrome stain, and IHC. The level of β2MG and hs-CRP in the Cisplatin+PHGPB group lowered and was highly different from Cisplatin control (p<0.05) depending on doses. In histopathology, the Cisplatin+PHGPB 400 mg/kg BW group showed less fibrosis and no significant difference with the negative control in Masson trichome staining. While in IHC histopathology, PHGPB treatment slightly ameliorated TGF-β expression and intensity of TGF-β values convincingly. In conclusion, PHGPB can relieve kidney fibrosis in cisplatin-induced nephrotoxicity rats: Emphasis on anti-inflammatory activities.

Pithecellobium dulce inhibits pulmonary metastasis induced by B16F10 melanoma cells in C57BL/6 via regulating EGFR/STAT/ NFκB /AKT signaling axis.

In this present study we analyzed anti-metastatic efficacy of fruit extract of Pithecellobium dulce (FPD) against B16F10 induced pulmonary metastatic model. FPD administration significantly (p < .01) reduced lung fibrosis, as evidenced by histochemical collagen analysis by Masson's trichome staining, total collagen, hexosamine, and uronic acid. Results showed that FPD treatment significantly attenuated the expression of EGFR mediated P38 and STAT1/3 expression, thus reduced NFκB mediated signaling cascade. Further, the expression of PIP3CA mediated activation of the AKT survival signaling pathway has been analyzed. Interestingly, in FPD treated group, the expression of AKT pathway has also downregulated. Further, we analyzed the downstream regulators of NFκB and AKT signaling pathways, which include, inflammatory genes (iNOS, COX2, NFκB, TGFβ1, IL5, IL1β, IFNγ, IL6, IL10, MCP1, GMCSF), anti-apoptotic genes (BCL2 and BCLXL), cell cycle regulators (cyclin D1 and Ki67), fibrosis activator (CT1α1), angiogenesis promoter (VEGF), metastatic promoter (MMP2/9, N CADH), mucin (MUC5AC), pro-apoptotic genes (Bax, CAS3 and CAS9) and metastasis inhibitors (TIMP1/2, E CADH, p53, PTEN). The expression of inflammatory, anti-apoptotic, cell cycle regulators, fibrosis activator, angiogenesis and metastasis promoter, and mucins were markedly reduced by FPD administration. Interestingly, the level of expression of anti-metastatic genes were markedly elevated in FPD administrated group. Lung histopathology further confirmed the anti-metastatic efficacy of FPD. PRACTICAL APPLICATIONS: Different parts of P. dulce has long been used as a folklore medicine against different diseases. This study demonstrated that bioactive constituents present in the fruit extract of P. dulce (FPD) significantly attenuated proliferation via regulating EGFR/STAT/NFκB/AKT signaling axis.

The modulatory effect of sodium molybdate against cisplatin-induced CKD: Role of TGF-β/Smad signaling pathway

AimsChronic kidney disease (CKD) is a global non-communicable health problem. Fibrosis is considered the base and the fate of CKD; thus, the goal of this study was to evaluate the effects of sodium molybdate on cisplatin-induced CKD model and demonstrate the possible involved mechanisms. Materials and methodsIn cisplatin model, Wistar rats were challenged with cisplatin (1 mg/kg, i.p.) twice weekly for ten weeks. Sodium molybdate (100 and 200 mg/kg, orally) was given one-week prior cisplatin and was continued daily for the next ten weeks. Key findingsAdministration of sodium molybdate amended kidney function and significantly reduced the pathological changes in renal histopathological sections. Moreover, it modulates oxidative stress parameters by increasing the levels of SOD and GSH and decreasing the level of MDA. Likewise, in renal homogenate, sodium molybdate attenuated inflammation that was revealed by NF-κB and TNF-α levels significant reduction. Additionally, it ameliorated fibrosis that was indicated by decreased Masson's trichome staining in cortex and medulla. Immunohistochemical staining of renal sections against TGF-β1 showed reduction of positive glomerular and tubular protein expression. Additionally, it decreased profibrotic Smad3 level and increased antifibrotic Smad7 level. SignificanceAdministration of sodium molybdate demonstrated a hopeful suppressor effect on cisplatin-induced CKD/fibrosis in rat model. This effect may be through the inhibition pof TGF-β/Smad signaling pathway and up-regulation of Smad7 expression leading to decreased extracellular collagen accumulation. Moreover, they could ameliorate the inflammation through NF-κB and TNF-α levels reduction, decrease in ROS, and retrieval of antioxidant defenses.

Progressive ascending telangiectasias

Progressive ascending telangiectasias

Effect of crude Ganoderma applanatum polysaccharides as a renoprotective agent against carbon tetrachloride-induced early kidney fibrosis in mice.

Background and Aim:Interstitial fibrosis is the final stage of chronic kidney injury, which begins with an inflammatory process. Crude Ganoderma applanatum polysaccharides are known to have anti-inflammatory properties. The potential role of crude G. applanatum polysaccharides in renal fibrosis through pro-inflammatory cytokines needs further investigation. This study aimed to determine the renoprotective effect of crude G. applanatum polysaccharide extract in mice with carbon tetrachloride (CCL4)-induced early kidney fibrosis.Materials and Methods:This study was conducted for 4 weeks using 24 male BALB/c mice selected for their metabolic stability. The mice were randomly divided into six groups, including control (CG), model (MG), silymarin group and crude G. applanatum polysaccharide extract groups comprising doses of 25, 50, and 100 mg/kg body weight. After sacrificing the mice, whole blood was analyzed for urea and creatine levels, and kidney tissue was prepared to assess tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), hyaluronic acid (HA), and laminin levels, both using enzyme-linked immunosorbent assay. Kidney histology was determined using hematoxylin and eosin staining, while the extracellular matrix (ECM) components were stained using Masson’s trichome staining. The α-smooth muscle actin (α-SMA) concentration was determined using immunohistochemistry. These parameters were measured to determine the effectiveness of the crude G. applanatum polysaccharide extract in preventing interstitial fibrosis.Results:Administration of crude G. applanatum polysaccharides effectively prevented increases in kidney weight and physiological enzymes, pro-inflammatory cytokines, and ECM production compared with those in the MG, as evidenced by the low levels of urea, creatinine, TNF-α, IL-6, HA, and laminin. Histopathological results also showed that crude G. applanatum polysaccharides prevented the occurrence of inflammatory infiltration, desquamated nuclei, cytoplasm debris, rupture at the brush border, dilatation of the glomeruli space and lumen of the proximal tubule, and necrotic cells compared with the MG. Masson’s trichrome staining revealed lower collagen levels in the interstitial tubules of kidney tissue than those in the MG. Immunohistochemical analysis revealed low α-SMA expression in the crude G. applanatum polysaccharides treatment groups than that in the MG.Conclusion:The crude polysaccharide extract of G. applanatum has a protective effect that prevents the progression of kidney fibrosis in mice.

Ochratoxin A treated rat derived urinary exosomes enhanced cell growth and extracellular matrix production in normal kidney cells through modulation of TGF-β1/smad2/3 signaling pathway

AimsKidney is the main target organ for ochratoxin A (OTA) toxicity; however, the mechanism(s) involved in OTA-induced nephrotoxicity is not fully understood. Recently, exosomes, nano-sized vesicles have been found to play an important role in promotion and progression of disease as well as environmental toxicant-induced patho-physiology of toxicity. Hence, we aimed to investigate the role of exosomes in OTA-mediated nephrotoxicity. Main methodsMale Wistar rats were divided in to two groups. Rats of one group were treated with OTA (210 μg/kg b. wt) and another with vehicle control through oral gavage (5 days/week) for 270 days. At the end of experiment, exosomes concentrations from rat's urine were measured. To examine the OTA-induced nephrotoxicity, histopathology was performed using H & E, Masson's trichome and PAS staining. For mechanistic study, normal rat kidney (NRK52E) cells were exposed with either vehicles treated rat's urinary exosomes (NEx) or OTA treated rat's urinary exosomes (OEx) and effects on cell proliferation, cell growth, extracellular matrix production and TGF-β1/smad2/3 pathway was analyzed. Key findingsOTA treatment to Wistar rats caused histopathological changes such as tubular degeneration, glomeruli shrinkage and hypercellularity in kidney tissue. Interestingly, OTA treated rat's urine has more exosomes secretion. Moreover, treatment of NRK52E cells with OEx caused increased cell proliferation, cell growth, enhanced the expression of extracellular matrix proteins and activation of TGF-β1/smad2/3 pathway. SignificanceOur investigations exogenous exposure of OTA derived urinary exosomes caused TGF-β1/smad2/3 pathway-mediated activation of pro-fibrotic changes in kidney will helpful for deeper understanding the OTA-induced nephrotoxicity.

Treg/Th17 Ratio Regulation May Play an Important Role in Epigallocatechin-3-Gallate-Mediated Attenuation of Increased Afterload-Induced Cardiac Hypertrophy.

:The aim of this study was to investigate whether Treg/Th17 ratio regulation plays an important role in epigallocatechin-3-gallate (EGCG) in attenuating increased afterload-induced cardiac hypertrophy. Three-month-old male C57BL/6 mice were divided into sham + vehicle, abdominal aortic constriction (AAC) + vehicle, and AAC + EGCG groups. Intraperitoneal EGCG (50 mg/kg/d) administration was conducted. Cardiac structure and function were examined by ultrasonography. Pathology was examined by hematoxylin and eosin staining, wheat germ agglutinin staining, and Masson's trichome staining. T-lymphocyte subtypes were analyzed using immunofluorescence and flow cytometry assays. Ultrasonography showed that the ventricular wall in the AAC + vehicle group was thicker than that in the sham + vehicle group (P < 0.05). Hematoxylin and eosin staining revealed cardiomyocyte hypertrophy accompanied by a small amount of inflammatory cell infiltration in the AAC + vehicle group. The results of wheat germ agglutinin staining demonstrated the presence of hypertrophic cardiomyocytes in the AAC + vehicle group (P < 0.01). Masson's trichome staining showed cardiac fibrosis in the AAC + vehicle group, and the immunofluorescence assay revealed infiltration of CD4+ cells in both AAC + vehicle and AAC + EGCG groups. Splenic flow cytometry showed a significant increase in the proportion of Treg cells in the AAC + EGCG group (P < 0.05). The proportion of Th17 cells in the AAC + vehicle group was significantly higher than that in the sham + vehicle group (P < 0.05). In conclusion, changes in the Treg/Th17 ratio are associated with the occurrence of myocardial hypertrophy caused by increased afterload. Moreover, regulation of the Treg/Th17 ratio by EGCG may play an important role in the attenuation of myocardial hypertrophy.

Combined effect of hydrogen sulfide and mesenchymal stem cells on mitigating liver fibrosis induced by bile duct ligation: Role of anti-inflammatory, anti-oxidant, anti-apoptotic, and anti-fibrotic biomarkers.

Objective(s):Liver fibrosis eventually develops into cirrhosis and hepatic failure, which can only be treated with liver transplantation. We aimed to assess the potential role of hydrogen sulfide (H2S) alone and combined with bone marrow-derived mesenchymal stem cells (BM-MSCs) on hepatic fibrosis induced by bile-duct ligation (BDL) and to compare their effects to silymarin.Materials and Methods:Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and alkaline phosphatase (ALP) were investigated in serum. Gene expression levels of CBS (cystathionine β-synthase), CSE (cystathionine γ-lyase), and alpha-smooth muscle actin (α- SMA) were measured in liver tissues using RT-PCR. Hepatic protein kinase (Akt) was assessed by Western blot assay. Liver oxidative stress markers, malondialdehyde (MDA), and reduced glutathione (GSH) were analyzed by the colorimetric method. Lipocalin-2 (LCN2) and transforming growth factor-β (TGF-β) were measured using ELIZA. Liver tissues were examined by H&E and Masson trichome staining for detection of liver necrosis or fibrosis. Caspase 3 expression was evaluated by immunohistochemistry. Results:H2S and BM-MSCs ameliorated liver function and inhibited inflammation and oxidative stress detected by significantly decreased serum ALT, AST, ALP, TB, and hepatic MDA, Akt, TGF-β, LCN2, and α-SMA expression and significantly increased CBS and CSE gene expression levels. They attenuated hepatic apoptosis evidenced by decreased hepatic caspase expression.Conclusion:Combined treatment with H2S and BM-MSCs could attenuate liver fibrosis induced by BDL through mechanisms such as anti-inflammation, anti-oxidation, anti-apoptosis, anti-fibrosis, and regenerative properties indicating that using H2S and MSCs may represent a promising approach for management of cholestatic liver fibrosis.

Pathological investigations and correlation research of microfibrillar-associated protein 4 and tropoelastin in oral submucous fibrosis

BackgroundOral submucous fibrosis (OSF), distinguished by abnormal collagen deposition, is a potentially malignant disorder with 4.2% (95% CI 2.7–5.6%) of malignant transformation and rising global prevalence. However, the precise pathogenesis and effective treatment remain elusive and controversial despite the abundance of literature on this topic. Therefore, it is crucial to explore the clinicopathological characteristics and potential markers for the diagnosis and prognosis of OSF. The objective of this study was to evaluate the influence and correlation of Microfibrillar-associated protein 4 (MFAP4) and tropoelastin (TE) in the development of OSF patients.Material and methodsClinicopathological factors, hematoxylin–eosin (HE) and Masson trichome staining, immunohistochemical characteristics and the correlation between MFAP4 and TE were recorded and compared among different stages of OSF progression among cases (n = 60) and controls (n = 10). Student's t test, ANOVA analysis, and the chi-square test were performed to compare the categorical variables for clinicopathological characteristics and the expression level of MFAP4 and TE between the fibrotic and normal tissues. Correlation analysis of MFAP4 and TE was performed using Pearson's correlation test and linear regression.ResultsMFAP4 and TE proteins are upregulated and increased gradually in patients with varying stages of OSF, relative to the control group. Furthermore, statistical analyses revealed that the expression level of MFAP4 was positively associated with TE, with a Pearson correlation coefficient of 0.3781 (p = 0.0048). Clinically, we found that OSF affected more males than females, with a ratio of 29:1. The age range was 16–60 years, and the mean age was 36.25 ± 10.25 years. In patients younger than 40 years, the positive expression rate of MFAP4 and TE was higher than in those over 40 years. All OSF cases had chewed areca nut, with 51.67% smoking tobacco.ConclusionsOur study elucidates that the accumulation of MFAP4 and TE proteins may play a vital role in the occurrence and development of OSF and may be promising candidate moleculars for prevention, diagnosis, and treatment strategies for OSF in the future.

Marantodes pumilum (blume) Kuntze (Kacip Fatimah) leaves aqueous extract prevents downregulation of Wnt/β-catenin pathway and upregulation of apoptosis in osteoblasts of estrogen-deficient, diabetes-induced rats

Ethnopharmacological relevanceMarantodes pumilum (Blume) Kuntze has been claimed to be beneficial in protecting the bone against loss in post-menopausal women. In view of increased incidence of diabetes mellitus (DM) in post-menopausal period, M. pumilum ability to overcome the detrimental effect of estrogen-deficiency and DM on the bones were identified. Aim of the studyTo identify the mechanisms underlying protective effect of MPLA on the bone in estrogen-deficient, diabetic condition. MethodsAdult female, estrogen-deficient, diabetic rats (225 ± 10 g) were divided into untreated group and treated with M. pumilum leaf aqueous extract (MPLA) (50 mg/kg/day and 100 mg/kg/day) and estrogen for 28 days (n = 6 per group). Fasting blood glucose (FBG) levels were weekly monitored and at the end of treatment, rats were sacrificed and femur bones were harvested. Bone collagen distribution was observed by Masson's trichome staining. Levels of bone osteoblastogenesis, apoptosis and proliferative markers were evaluated by Realtime PCR, Western blotting, immunofluorescence and immunohistochemistry. ResultsMPLA treatment was able to ameliorate the increased in FBG levels in estrogen deficient, diabetic rats. In these rats, decreased bone collagen content, expression level of osteoblastogenesis markers (Wnt3a, β-catenin, Frizzled, Dvl and LRP-5) and proliferative markers (PCNA and c-Myc) and increased expression of anti-osteoblastogenesis marker (Gsk-3β) and apoptosis markers (Caspase-3, Caspase-9 and Bax) but not Bcl-2 were ameliorated. Effects of 100 mg/kg/day MPLA were greater than estrogen. ConclusionMPLA was able to protect against bone loss, thus making it a promising agent for the treatment of osteoporosis in women with estrogen deficient, diabetic condition.

Protective effects of paclitaxel on thioacetamide-induced liver fibrosis in a rat model.

Liver fibrosis is a public health burden that is highly associated with morbidity and mortality. Therefore, this study aims to explore the anti-fibrotic effects of low dose of paclitaxel (PTX) against thioacetamide (TAA)-induced liver fibrosis in rats and the possible mechanisms involved. TAA was administered at a dose of 200 mg/kg twice weekly for 6 weeks in rats to induce liver fibrosis similar to that in humans. Liver dysfunction was shown by increased alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transferase, along with histopathological changes. Liver fibrosis was confirmed by Masson's Trichome staining, increased collagen content, and elevated α-smooth muscle actin (α-SMA) protein expression. In addition, TAA induced liver apoptosis as indicated by the increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in liver tissues. This study demonstrated that the administration of PTX (0.3 mg/kg/i.p.) three times a week for 6 weeks significantly alleviated functional and biochemical changes induced by TAA in addition to improving the liver architecture. PTX attenuated liver fibrosis as reflected by the decreased collagen content and α-SMA protein expression. Additionally, PTX attenuated liver apoptosis as indicated by the decreased TUNEL-positive cells. Moreover, PTX prevented TAA-induced elevation of transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels in liver tissues. These findings suggest that the low dose of PTX prevented TAA-induced liver fibrosis in rats, possibly byinhibiting the expression of TGF-β1 and PDGF-BB and subsequently suppressing the apoptosis and the expression of TIMP-1.