TGF-β1 Gene Expression Research Articles

P2X4 receptors mediate induction of antioxidants, fibrogenic cytokines and ECM transcripts; in presence of replicating HCV in in vitro setting: An insight into role of P2X4 in fibrosis.

Major HCV infections lead to chronic hepatitis, which results in progressive liver disease including fibrosis, cirrhosis and eventually hepatocellular carcinoma (HCC). P2X4 and P2X7 are most widely distributed receptors on hepatocytes. Full length P2X4 (1.7kb) (Rattus norvegicus) was sub cloned in mammalian expression vector pcDNA3.1+. Two stable cell lines 293T/P2X4 (experimental) and 293T/ NV or null vector (control) were established. Both cell lines were inoculated with high viral titers human HCV sera and control human sera. Successfully infected cells harvested on day 5 and day 9 of post infection were used for further studies. The results revealed a significant increase in gene expression of P2X4 on day 5 and day 9 Post -infection in cells infected with HCV sera compared with cells inoculated with control sera. Quantitative real time PCR analysis revealed that HO-1 was significantly upregulated in presence of P2X4 in HCV infected cells (P2X4/HCV) when compared with control NV/HCV cells. A significant decrease was observed in expression of Cu/ZnSOD in presence of P2X4 in HCV infected cells compared to control NV/HCV cells. However, expression of both antioxidants was observed unaltered in cells harvested on day 9 post infection. Gene expression of angiotensin II significantly increased in HCV infected cells in presence of P2X4 on day 5 and day 9 of post infection when compared with control NV/HCV cells. A significant increase in gene expression of TNF-α and TGF-β was observed in HCV infected cells in presence of P2X4 on day 9 post infection in comparison with control (NV/HCV cells). However, gene expression of adipokine leptin was not affected in both experimental (P2X4/HCV) and control (NV/HCV) groups on day 5 and day 9 of post infection. Extracellular matrix proteins, laminin and elastin genes expression also significantly increased in presence of P2X4 (HCV/P2X4) on day 9 of post-infection compared to control group NV/HCV cells. In conclusion, these findings constitute the evidence that P2X4 receptors in the presence of HCV play a significant role in the regulation of key antioxidant enzymes (HO-1, Cu/ZnSOD), in the induction of proinflammatory. cytokine (TNF-α), profibrotic cytokine (TGF-β) vasoactive cytokine (angiotensin II). P2X4 also increases the expression of extracellular matrix proteins (laminin and elastin) in the presence of HCV.

Evaluation of IGF-1, TNF-α, and TGF-β Gene Expression after Oral Vitamin D Supplementation in School-Aged Children with Chronic Bronchial Asthma

BACKGROUND: Airway remodeling in children with bronchial asthma is due to the effect of inflammatory mediators and growth factors on the bronchial epithelium. Vitamin D (VitD) has immunomodulatory effect in many inflammatory diseases as bronchial asthma. The ant-inflammatory and anti-fibrotic role of VitD could prevent or improve air way remodeling in asthmatic patients. AIM: The study investigated the effect of VitD supplementation on the expression of transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and insulin growth factor 1(IGF-1) and to correlate them with asthma severity and level of control. METHODS: The serum level of VitD and the mRNA expression of IGF-1, TGF-β, and TNF-α were estimated in 50 patients and 20 healthy controls control subjects using quantitative PCR in real-time. Asthmatic patients with VitD deficiency received VitD supplementation for 2 months followed by remeasurement of serum VitD and the genes expression TGF-β, TNF-α, and IGF-1. RESULT: Pre-intake of VitD and serum level of VitD were lower in all patients than control subjects (p = 0.005). VitD level was directly correlated with IGF-1 mRNA expression, which was indirectly correlated with TGF-β, r = 0.5 and −0.57; p = 0.0001 and 0.002, respectively. After VitD supplementation, the expression of the TGF-β mRNA gene was the only gene that decreased significantly (p = 0.04) together with improved asthma control and spirometric parameters. CONCLUSIONS: VitD supplementation down regulated the gene expression of TGF-β and improved asthma control level, but it did not significantly affect the gene expression of TNF-α and IGF-1.

The Effect of Aerobic Exercise Training and Eugenol Supplementation on the Wnt, TGF-β, and Beta-Catenin gene Expression in Myocardial Tissue of Poisoned Rats Induced by Chlorpyrifos

: Chlorpyrifos is an organophosphate toxin used to control agricultural pests that cause oxidative stress, apoptosis, and cell destruction in various body tissues, including the heart. The expression of Wnt, TGFβ, and beta-catenin plays a crucial role in cardiac fibrosis development. No study has investigated changes in the function of these genes following physical activity and eugenol supplementation in chlorpyrifos intoxication. The present study investigated the independent and interactive effect of aerobic exercise and eugenol supplementation on the Wnt, TGFβ, and beta-catenin gene expression in the heart tissue of rats poisoned with chlorpyrifos. Forty-two male rats were divided into seven groups: healthy control, healthy receiving DMSO as chlorpyrifos toxin solvent, healthy receiving corn oil as eugenol solvent, poisoned control, aerobic exercise poisoning, eugenol poisoning, aerobic poisoning, and eugenol poisoning. The expression of the desired genes in the hearts of rats was measured after providing the intervention. Data were analyzed using the one-way analysis of variance and Tukey's post hoc tests. Statistical significance was considered when P ≤ 0.05. Poisoning significantly increased the Wnt, TGFβ, and beta-catenin gene expression in the heart tissue. The use of exercise and supplementation reduced the Wnt, TGFβ, and beta-catenin gene expression, each independently, in the hearts of poisoned rats. Exercise-eugenol interaction did not significantly reduce the gene expression than exercise or supplementation alone. Overall, the present study showed the positive role of exercise and eugenol in reducing chronic cardiac toxicity on Wnt, TGFβ, and beta-catenin gene expression.

Use of paclitaxel carried in solid lipid nanoparticles to prevent peritoneal fibrosis in rats.

Progressive fibrous thickening of peritoneal membrane (PM) is a major complication of long-term peritoneal dialysis. TGF-β/SMAD pathway activation, inflammation and neoangiogenesis have an important role in PM changes induced by peritoneal dialysis. Here, we investigated the effects of paclitaxel (PTX) carried in lipid core nanoparticles (LDE) on the development of peritoneal fibrosis (PF) in rats. To induce PF, 21 male Wistar rats (300-350g) were injected with chlorhexidine gluconate for 15 consecutive days and randomly assigned to three groups: 1)PF, n = 5: no treatment; 2)LDE, n = 8: treated with LDE only, 3/3 days during 15 days; 3)LDE-PTX, n = 8: treated with PTX (4mg/kg) associated with LDE, 3/3 days during 15 days. A Control group without PF induction (n = 5) was designed, received saline solution, 3/3 days. Peritoneum function tests were performed, and anterior abdominal wall samples of the PM were collected for analyses of peritoneal thickness, immunohistochemitry, and gene expression. LDE-PTX treatment preserved the membrane function, maintaining the ultrafiltration rate and mass transfer of glucose at normal levels. LDE-PTX also prevented PM thickening induced by chlorhexidine gluconate injections. LDE-PTX treatment reduced the number of myofibroblasts infiltrating PM and inhibited the cell proliferation. Gene expression of fibronectin, FSP-1, VEGF, TGF-β, and SMAD3 were reduced by LDE-PTX. LDE-PTX was effective to prevent development of PF and preserve the PM filtration capacity in this rat model, with clear-cut actions on pro-fibrotic mechanisms. Thus, LDE-PTX can be candidate for future clinical trials as adjuvant to peritoneal dialysis to prevent PF development, since this preparation is devoid of toxicity as shown previously.

Exosomes Derived from BM-MSCs Mitigate the Development of Chronic Kidney Damage Post-Menopause via Interfering with Fibrosis and Apoptosis.

The rate of chronic kidney disease (CKD) is increasing globally, and it is caused by continuous damage to kidney tissue. With time the renal damage becomes irreversible, leading to CKD development. In females, post-menopause lack of estrogen supply has been described as a risk factor for CKD development, and studies targeting post-menopause CKD are scarce. In the present study, we used exosomes isolated from bone marrow mesenchymal stem/stromal cells (BM-MSCs) to test their therapeutic potential against the development of CKD. At first, the menopause model was achieved by surgical bilateral ovariectomy in female albino rats. After that, 100 µg of exosomes was given to ovariectomized rats, and the study continued for 2 months. Changes in urine volume, urine protein content, kidney function biochemical parameters (creatinine and BUN), kidney antioxidant parameters (SOD, GPx and CAT), histological changes, immunohistochemical levels of caspase 3, and the gene expression of NGAL (related to kidney damage), TGFβ1 and αSMA (related to fibrosis and EMT), and caspase 3 (related to apoptosis) were studied. After the ovariectomy, the occurrence of CKD was confirmed in the rats by the drastic reduction of serum estrogen and progesterone levels, reduced urine output, increased urinary protein excretion, elevated serum creatinine and BUN, reduced GPx SOD, and CAT in kidney tissue, degenerative and fibrotic lesions in the histopathological examination, higher immunohistochemical expression of caspase 3 and increased expression of all studied genes. After exosomes administration, the entire chronic inflammatory picture in the kidney was corrected, and a near-normal kidney structure and function were attained. This study shows for the first time that BM-MSCs exosomes are potent for reducing apoptosis and fibrosis levels and, thus, can reduce the chronic damage of the kidneys in females that are in their menopause period. Therefore, MSCs-derived exosomes should be considered a valuable therapy for preserving postmenopausal kidney structure and function and, subsequently, could improve the quality of females’ life during menopause.

Pancreatic cancer cell-derived exosomes induce epithelial-mesenchymal transition in human pancreatic cancer cells themselves partially via transforming growth factor β1.

Distant metastasis is a dismal prognostic factor of pancreatic cancer. Metastasis is established in several steps, but the mechanism underlying the very early stages remains unclear. Epithelial-mesenchymal transition (EMT) is involved in these stages. Although signaling molecules have been reported to induce EMT, the mechanism underlying their origin is unclear. In this study, we hypothesized that pancreatic cancer cell-derived exosomes induce EMT in cancer cells themselves, a notion we entertained because we found EMT in in vitro three-dimensional colonies of cancer cells, with vimentin-positive cells observed in some of the budding pancreatic cancer cells and in single cells outside the colony as well. First, we clarified that pancreatic cancer cell-derived exosomes induce EMT in cancer cells themselves. Next, we examined the involvement of transforming growth factor-β1 (TGF-β1), and TGF-β1 knock-down in pancreatic cancer cells with TGF-β1 siRNA significantly suppressed TGF-β1 gene expression in cancer cells, and exosomal TGF-β1 was significantly reduced in the secretory exosomes. Exosomes from TGF-β1 knock-down cells suppressed EMT induction in cancer cells themselves and TGF-β1 protein expression in target cells. Taken together, these findings suggest that TGF-β1 is involved in EMT induction via exosomes, results that may support the production of effective metastasis inhibitors.

Hepatoprotective activity of Picrorhiza kurroa and Berberis lycium is mediated by inhibition of COX-2 and TGF-β expression in lantadenes-induced sub-chronic toxicity in guinea pigs

BackgroundThe toxicity of Lantana camara weed is common in grazing livestock throughout the world. Specific treatment for the toxicity is lacking. However, herbal plants could be investigated for their effectiveness in inhibiting hepatic damage caused by lantadenes of L. camara. Therefore, the extracts Berberis lycium and Picrorhiza kurroa, which are known to exhibit multiple useful effects were assessed for their hepatoprotective action in sub-chronic lantadene toxicity. PurposeThe focus of the study was to investigate the molecular pathogenesis of sub-chronic lantadene toxicity and, the mechanism of hepatoprotection by freeze-dried methanolic extracts of the Berberis lycium root bark and Picrorhiza kurroa rhizome in lantadenes-induced sub-chronic hepatopathy in guinea pig laboratory animal model. MethodsIsolation of lantadenes from L. camara leaves, followed by its characterization and quantification by UPLC-MS method was done. The methanolic extracts of ameliorating plant parts (root bark of B. lycium, rhizome of P. kurroa) were prepared and quantification of berberine and picroside was carried out. The in vivo sub-chronic toxicity and amelioration experiment in guinea pigs was conducted for 90 days by distributing them into 7 groups with 6 animals in each group. At the end of the experiment, serum biochemical analysis, oxidation stress levels in the liver and kidneys were determined. Gross pathology and microscopic observations in different organs, Masson’s trichome staining for fibrous collagenous tissue deposition assessment, and immunohistochemical expression of the TGF-β antigen in the liver of animals were done. The effect of lantadenes on pro-inflammatory cytokines and the level of α-smooth muscle actin in the liver was estimated by real-time RT-PCR and ELISA, respectively. ResultsSub-chronic lantadene intoxication increased serum ALT, AST, ALP, bilirubin, creatinine, total proteins and lipid peroxidation in liver tissue; decreased catalase, superoxide dismutase and reduced glutathione activity in liver tissue; caused hepatic necrosis with bile duct proliferation and TGF-β antigen expression in the periportal regions; upregulated the IL-1β, IL-6, TGF-β, and COX-2 pro-inflammatory cytokine gene expression and increased the titre of α-SMA in the liver. The P. kurroa extract at 200 mg/kg bw and B. lycium extract at 200 mg/kg bw produced favourable effects against lantadenes-induced hepatic toxicity and significantly reversed these changes to near normal. ConclusionsP. kurroa and B. lycium extracts at 200 mg/kg bw can be used to ameliorate the hepatotoxicity produced by sub-chronic exposure to lantadenes. The findings of the molecular pathogenesis of sub-chronic lantadene toxicity and its amelioration in guinea pig laboratory animal model are novel. Further investigations are needed to study the in-depth mechanism of hepatoprotection of P. kurroa and B. lycium in lantana intoxicated grazing (host) animals.

Moringa oleifera Leaf Extract Promotes Healing of Infected Wounds in Diabetic Rats: Evidence of Antimicrobial, Antioxidant and Proliferative Properties.

Moringa oleifera is known to possess wound healing activity. The present study evaluated the healing properties of methanolic extract of M. oleifera leaves in excision wounds infected with methicillin-resistant Staphylococcus aureus (MRSA) or P. aeruginosa in diabetic rats. An in vitro study was also carried out to determine the gene expression of VEGF and TGF-β1. Preliminary phytochemical and GC-MS analyses were carried out to determine different chemical constituents present in the extract. M. oleifera was applied locally as an ointment at two different concentrations. Wound contraction, period of epithelization, antioxidant enzyme activities and histological changes were determined. For the gene expression study, HaCaT cell lines were used. The formulation of M. oleifera extract improved wound contraction and decreased the period of epithelization, which was associated with an increase in antioxidant enzyme activities, epithelization, capillary density and collagen formation in MRSA-infected diabetic rats. However, this effect was reduced in diabetic animals infected with P. aeruginosa. An increase in the expression of VEGF and TGF-β1 was observed in HaCaT cell lines. M. oleifera extract promotes the healing of infected wounds in MRSA-infected diabetic rats but is less effective in the healing of wounds infected with P. aeruginosa in diabetic rats.

Pathological and immunological evaluation of different regimens of praziquantel treatment in a mouse model of Schistosoma mansoni infection.

One of the considerable challenges of schistosomiasis chemotherapy is the inefficacy of praziquantel (PZQ) at the initial phase of the infection. Immature schistosomes are not susceptible to PZQ at the curative dose. Here, we investigated the efficacy of different PZQ regimens administered during the initial stage of Schistosoma mansoni infection in mice. Two months-old mice were individually infected with 80 S. mansoni cercariae and divided into one infected-untreated control group (IC) and four PZQ-treated groups: PZQ at 100 mg/kg/day for five consecutive days (group PZQ1), PZQ at 100 mg/kg/day for 28 days (group PZQ2), PZQ at 18 mg/kg/day for 28 days (group PZQ3) and a single dose of PZQ at 500 mg/kg (group PZQ4). The treatment started on day one post-infection (p.i), and each group of mice was divided into two subgroups euthanized on day 36 or 56 p.i, respectively. We determined the mortality rate, the parasitological burden, the hepatic and intestinal granulomas, the serum levels of Th-1, Th-2, and Th-17 cytokines, and gene expression. The treatment led to a significant (p < 0.001) reduction of worm burden and egg counts in the intestine and liver in groups PZQ2 and PZQ3. On 56th day p.i, there was a significant reduction (p < 0.001) of the number and volume of the hepatic granulomas in groups PZQ2 and PZQ3 compared to group PZQ1 or PZQ4. Moreover, in group PZQ3, the serum levels of IFN-γ, TNF-α, IL-13, and IL-17 and their liver mRNA expressions were significantly reduced while IL-10 and TGF-β gene expression significantly increased. The highest mortality rate (81.25%) was recorded in group PZQ2. This study revealed that the administration of PZQ at 18 mg/kg/day for 28 consecutive days was the optimal effective posology for treating S. mansoni infection at the initial stage in a murine model.

SMAD4 TGF-β-independent function preconditions naive CD8+ T cells to prevent severe chronic intestinal inflammation.

SMAD4, a mediator of TGF-β signaling, plays an important role in T cells to prevent inflammatory bowel disease (IBD). However, the precise mechanisms underlying this control remain elusive. Using both genetic and epigenetic approaches, we revealed an unexpected mechanism by which SMAD4 prevents naive CD8+ T cells from becoming pathogenic for the gut. Prior to the engagement of the TGF-β receptor, SMAD4 restrains the epigenetic, transcriptional, and functional landscape of the TGF-β signature in naive CD8+ T cells. Mechanistically, prior to TGF-β signaling, SMAD4 binds to promoters and enhancers of several TGF-β target genes, and by regulating histone deacetylation, suppresses their expression. Consequently, regardless of a TGF-β signal, SMAD4 limits the expression of TGF-β negative feedback loop genes, such as Smad7 and Ski, and likely conditions CD8+ T cells for the immunoregulatory effects of TGF-β. In addition, SMAD4 ablation conferred naive CD8+ T cells with both a superior survival capacity, by enhancing their response to IL-7, as well as an enhanced capacity to be retained within the intestinal epithelium, by promoting the expression of Itgae, which encodes the integrin CD103. Accumulation, epithelial retention, and escape from TGF-β control elicited chronic microbiota-driven CD8+ T cell activation in the gut. Hence, in a TGF-β–independent manner, SMAD4 imprints a program that preconditions naive CD8+ T cell fate, preventing IBD.

Hepatic Epigenetic Reprogramming After Liver Resection in Offspring Alleviates the Effects of Maternal Obesity.

Obesity has become a public health problem in recent decades, and during pregnancy, it can lead to an increased risk of gestational complications and permanent changes in the offspring resulting from a process known as metabolic programming. The offspring of obese dams are at increased risk of developing non-alcoholic fatty liver disease (NAFLD), even in the absence of high-fat diet consumption. NAFLD is a chronic fatty liver disease that can progress to extremely severe conditions that require surgical intervention with the removal of the injured tissue. Liver regeneration is necessary to preserve organ function. A range of pathways is activated in the liver regeneration process, including the Hippo, TGFβ, and AMPK signaling pathways that are under epigenetic control. We investigated whether microRNA modulation in the liver of the offspring of obese dams would impact gene expression of Hippo, TGFβ, and AMPK pathways and tissue regeneration after partial hepatectomy (PHx). Female Swiss mice fed a standard chow or a high-fat diet (HFD) before and during pregnancy and lactation were mated with male control mice. The offspring from control (CT-O) and obese (HF-O) dams weaned to standard chow diet until day 56 were submitted to PHx surgery. Prior to the surgery, HF-O presented alterations in miR-122, miR-370, and Let-7a expression in the liver compared to CT-O, as previously shown, as well as in its target genes involved in liver regeneration. However, after the PHx (4 h or 48 h post-surgery), differences in gene expression between CT-O and HF-O were suppressed, as well as in microRNA expression in the liver. Furthermore, both CT-O and HF-O presented a similar regenerative capacity of the liver within 48 h after PHx. Our results suggest that survival and regenerative mechanisms induced by the partial hepatectomy may overcome the epigenetic changes in the liver of offspring programmed by maternal obesity.

MTA-Based Cements: Biocompatibility and Effects on the Gene Expression of Collagen Type 1 and TGF-β1.

Objective This study sought to evaluate the biocompatibility of Neomineral Trioxide Aggregate (Neo-MTA), MTA Repair High Plasticity (MTA-HP), and Mineral Trioxide Aggregate-Angelus white (MTA-Ang) in fibroblasts of human dental pulp. Materials and Methods Morphology was evaluated after 24 h of incubation. LIVE/DEAD assay and cell adhesion tests were performed at 24 h of treatment. Cell proliferation assays (MTSs) and Annexin V were performed at 48 h incubation with different treatments. The expression of Col-1 and TGF-β1 was tested by endpoint PCR at 5 days of treatment. Results Morphological changes were observed in all groups. Neo-MTA and MTA-Ang were associated with increased cell viability, and all materials induced apoptosis, with a higher percentage in the MTA-HP group than in the other groups. In the LIVE/DEAD assay, there was more damage to the cell membrane in the group of cells treated with MTA-HP than in the other groups. Conclusion Neo-MTA and MTA-Ang presented similar biocompatibility, and both showed greater biocompatibility than MTA-HP. MTA-HP and MTA-Ang increased Col-1A gene expression, and Neo-MTA and MTA-Ang increased TGF-β1 gene expression in a similar way.

Therapeutic Effects of Aloe vera (L.) Burm.f. and Vitis vinifera L. Combination Cream on Wound Healing in Second-Degree Burn Model in Rats: Quantification of Compounds and VEGF &amp; TGFβ Gene Expression

Finding more efficient agents with fewer side effects for the treatment of burns has been a concern for researchers. The current study aims to assess Aloe vera (L.) Burm.f. and Vitis vinifera L. extract combination (AVEC) effects on wound healing in rats compared with silver sulfadiazine (SSD). Identification of individual polyphenols of the plant extracts was performed by HPLC. The animals were randomly divided into twelve groups. Standard second-degree burn wounds were induced on the back. Groups were treated with Aloe vera (A.V) and Vitis vinifera (V.V) creams at concentrations of 0.5, 1, 1.5, and 2% with ED50 values of 1.5 ± 0.02 and 1.4 ± 0.08 %, respectively. The other group was treated with an AVEC cream (1.5%). The samples of burned skin tissue were collected from the rats for histopathological examination. To evaluate the expression of VEGF and TGFβ1, the real time-PCR method was used. Kaempferol was only detected in Aloe. This study revealed that AVEC cream exhibits significant wound healing activity. VEGF and TGFβ1 had a significant increase in the AVEC. Based on our findings, AVEC cream can be a therapy of choice for burn injuries.

Correlation between Transforming Growth Factor-β and Periapical Lesions in Patients with Chronic Apical Periodontitis.

Objective To examine the expression of transforming growth factor-β (TGF-β) in the periapical granulation tissue and serum of patients with chronic apical periodontitis and to conduct immunohistochemical analysis so as to explore the relationship between TGF-β and the degree of periapical lesions. Methods Periapical granulation tissues of 20 cases of chronic apical periodontitis were collected as the experimental group. Healthy gingival tissues without eruption of third molars of 5 cases were collected as the control group. Immunohistochemistry, enzyme-linked immunosorbent assay, and real-time PCR (RT-PCR) were utilized to determine the expression of TGF-β mRNA and protein, and the difference in the expression of TGF-β was compared between groups. In the experimental group, oral CBCT was taken to measure the periapical bone resorption area. Spearman's correlation method was applied to analyze the correlation between TGF-β protein and gene expression levels and periapical bone resorption area. Results Immunohistochemistry and enzyme-linked immunosorbent assay demonstrated that the expression of TGF-β protein in chronic apical periodontitis tissue and serum was higher than that in the controls (P < 0.05). RT-PCR revealed that the expression of TGF-β mRNA was higher in chronic apical periodontitis tissue than that of the controls (P < 0.05). Spearman's correlation analysis showed that in the experimental group, the mRNA expression of TGF-β was positively correlated with the periapical bone resorption area (P < 0.01), and the protein expression level was not correlated with the periapical bone resorption area (P < 0.05). Conclusion The increased expression of TGF-β in the periapical granulation tissue and serum of patients with chronic apical periodontitis has a certain correlation with the progression of periapical periodontitis. The correlation between TGF-β at the mRNA level and the degree of early stage disease as well as the high expression of TGF-β in inflammatory cells in immunohistochemistry have confirmed that TGF-β promotes bone resorption in early periapical periodontitis, and its mechanism of action deserves further investigation.

Influence of exposure to microbial ligands, immunosuppressive drugs and chronic kidney disease on endogenous immunomodulatory gene expression in feline adipose-derived mesenchymal stem cells.

Feline autologous mesenchymal stem cells (MSCs) show promise for immunomodulatory activity, but the functional impact of chronic kidney disease (CKD), concurrent immunosuppressive drug administration or infection is unknown. The study objectives compare endogenous cytokine gene expression (interleukin [IL]-6, IL-10, IL-12p40, IL-18 and transforming growth factor beta [TGF-β]) in adipose-derived MSCs (aMSCs) from cats with and without CKD, following in vitro exposure to microbial ligands and treatment with common immunosuppressive drugs. Previously obtained aMSCs, phenotype CD44+, CD90+, CD105+ and MHCII-, from cats with (n = 6) and without (n = 6) CKD were compared via real-time PCR (RT-PCR) for immunomodulatory gene expression. aMSCs were exposed in vitro to lipopolysaccharide (LPS), peptidoglycan or polyinosinic:polycytidylic acid (Poly I:C), simulating bacterial or viral exposure, respectively. aMSCs were also exposed to ciclosporin, dexamethasone or methotrexate. Gene expression was measured using RT-PCR, and Cq was utilized after each run to calculate the delta cycle threshold. aMSCs isolated from healthy and CKD cats showed no significant differences in gene expression in the five measured cytokines. No significant changes in measured gene expression after drug treatment or microbial ligand stimulation were observed between normal or CKD affected cats. Proinflammatory genes (IL-6, IL-12p40 and IL-18) showed altered expression in aMSCs from both groups when compared with the same cells in standard culture after exposure to methotrexate. Poly I:C altered IL-6 and TGF-β gene expression in aMSCs from both healthy and CKD cats when compared with the same cells in standard culture. The five genes tested showed no statistical differences between aMSCs from healthy or CKD cats. There was altered cytokine gene expression between the control and treatment groups of both healthy and CKD cats suggesting feline aMSCs have altered function with immunosuppressive treatment or microbial ligand exposure. Although the current clinical relevance of this pilot study comparing brief exposure to select agents in vitro in aMSCs from a small number of cats is unknown, the study highlights a need for continued investigation into the effects of disease and concurrent therapies on use of cell-based therapies in feline patients.

Effects of High Starch and Supplementation of an Olive Extract on the Growth Performance, Hepatic Antioxidant Capacity and Lipid Metabolism of Largemouth Bass (Micropterus salmoides).

An 8-week feeding trial was conducted to investigate the effects of high-starch diets and the supplementation of an olive extract (OE) on the growth performance, liver health and lipid metabolism of largemouth bass (Micropterus salmoides). Four isonitrogenous and isolipidic diets were prepared: two basal diets containing low (9.0%) and high (14.4%) levels of starch (named as LS and HS), and 0.125% OE was supplemented to each basal diet (named LSOE and HSOE). The results show that high-starch diets had significant negative effects on growth performance, with lower FR, SGR and higher FCR, whereas OE significantly lowered FCR, determined by two-way ANOVA analysis. High-starch diets induced oxidative stress, inflammatory response and liver function injury, with significant increases in the content of plasmatic AKP, AST, ALT, hepatic SOD and MDA, and up-regulation of hepatic TNFα, IL1β, and TGFβ1 gene expression. In addition, a high-starch diet decreased the phosphorylation of AMPK and upregulated the expression of SREBP, together with higher hepatic liver lipid and HSI. The oxidative stress and lipid metabolism disorders indicate metabolic liver disease (MLD) of largemouth bass fed high-starch diets. Feeding on OE-supplemented diets increased the hepatic antioxidant capacity by decreasing the content of MDA and SOD. Fish fed the HSOE diet had an activated phosphorylation of JNK and decreased expression of pro-inflammatory IL1β compared with those fed the HS diet, which strongly indicated that the degree of inflammatory responses was reduced after OE supplementation. Interestingly, this study demonstrated that OE regulates hepatic lipid metabolism in fish by inhibiting the expression of hepatic lipogenesis genes (ACC1 and FASN) and promoting lipolysis (ATGL) and β-oxidation (CPT1α) to prevent TG accumulation. In conclusion, high-starch feed induced oxidative stress and lipid metabolic disorder of largemouth bass, while supplementation with OE improved its antioxidant capacity, anti-inflammatory responses and lipid metabolism. However, hepatic histopathological results suggested that OE supplementation could not completely repair the MLD caused by the high level of starch in largemouth bass.

Hsa-miR-142-3p reduces collagen I in human scleral fibroblasts by targeting TGF-β1 in high myopia

High myopia has been continually increasing globally until now and often results in visual impairment. Scleral extracellular matrix (ECM) remodeling is considered a common factor contributing to progression of myopia. However, the role of microRNAs (miRNAs) in regulating scleral ECM organization is not well understood. We aimed to explore the effect and regulatory mechanism of hsa-miR-142-3p on collagen I in human scleral fibroblasts in high myopia. First, next-generation sequencing was conducted to identify 37 miRNAs differentially expressed in the aqueous humor of high myopia samples and control samples. Furthermore, hsa-miR-142-3p in the aqueous humor was found to positively relate to the ocular axial length. Besides, the results of immunofluorescence and Western blot assay indicated that hsa-miR-142-3p overexpression decreased collagen I expression in the human fetal scleral fibroblasts (HFSFs); while hsa-miR-142-3p downregulation increased collagen I. Moreover, hsa-miR-142-3p targets TGFβ-1 gene expression. Quantitative polymerase chain reaction (qPCR) and Western blot analysis showed that miRNA 142-3p reduced TGFβ-1 expression while an inhibitor had an opposite effect. Therefore, there is an inverse relationship between changes in miR-142-3p expression levels and those of collagen1a1 in human scleral fibroblasts. Such a dependence suggests that miR-142-3p may be a target to improve therapeutic management of this condition.

Protective and therapeutic role of mango pulp and eprosartan drug and their anti-synergistic effects against thioacetamide-induced hepatotoxicity in male rats

The present study was done to evaluate the protective and therapeutic role of mango pulp (M), eprosartan drug (E), and their co-administration (EM) against hepatotoxicity induced by thioacetamide (T). Seven groups of rats were prepared as follows: the control (C) group (normal rats), T group (the rats were injected with T), T-M group (the rats were injected with T, and then treated with M), T-E group (the rats were injected with T, and then treated with E), T-EM group (the rats were injected with T, and then treated with E and M), M-TM-M group (the rats were administered with M before, during, and after T injection), and M group (the healthy rats were administered with M only). Firstly, the characterizations of M were determined. Also, the markers of hepatic oxidative stress [malondialdehyde (MDA) and glutathione (GSH) levels and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GSR)], inflammation and fibrosis [(tumor necrosis factor-α (TNF-α) and platelet-derived growth factor-BB (PDGF-BB) levels and gene expression of transforming growth factor-beta1(TGF-β1)], and liver functions and microscopic examination were evaluated. The present results revealed that M contains 419 ± 1.04 μg total phenolics as gallic acid equivalent and 6.8 ± 0.05 μg total flavonoids as quercetin equivalent. The analysis of phenolics and flavonoids showed the presence of chlorogenic, caffeic, 2,5-dihydroxy benzoic, 3,5-dicaffeoylquinic, 4,5-dicaffeoylquinic, tannic, cinnamic acidS, and catechin, phloridzin, and quercetin with different concentrations. Also, M contains various minerals with different concentrations involving potassium, calcium, magnesium, sodium, iron, copper, zinc, and manganese. The current results showed that the total antioxidant capacity of 1 g of M was 117.2 ± 1.16 as μg ascorbic acid equivalent. Our biochemical studies showed that all treatments significantly reduced T-induced hepatotoxicity and liver injuries, as the oxidative stress and inflammatory and fibrotic markers were diminished where MDA level and the activities of GST, GSSG, and GR were decreased when compared with T group. In contrast, GSH level and the activities of SOD and GPx and GSH/GSSG ratio were increased. In addition, TNF-α and PDGF-BB levels were reduced, and the gene expression of TGF-β1 was down-regulated. Consequently, the liver functions were significantly improved. In conclusion, each E, M, and EM has a therapeutic effect against T-induced hepatotoxicity via the reduction of the OS, inflammation, and fibrosis. Unfortunately, treatment with M and E simultaneously revealed the less effectiveness than the treatment with M or E demonstrates the presence of anti-synergistic effect between them. Additionally, M-TM-M treatment showed a better effect than T-M treatment against T-induced hepatotoxicity revealing the prophylactic role of M. The administration of healthy rats with M for 12 weeks has no side effect.Graphical abstract

Topical Mentha piperita Effects on Cutaneous Wound Healing: A Study on TGF-β Expression and Clinical Outcomes.

BACKGROUNDWound healing is a critical clinical concept. We aimed to evaluate the effects of topical Mentha piperita essence on cutaneous wound healing. METHODSThis randomized controlled trial was conducted in Tehran University of Medical Sciences, Tehran, Iran in 2019. Square-shaped 1.5×1.5 cm wounds were made on the neck of 60 male Wistar rats in a sterile condition. Samples were randomly divided into a control group and three experimental groups. Group A treated with M. piperita essence and Vaseline. The second group received the M. piperita essence, and the third group received Vaseline. Histological specimens were obtained in 4th, 7th, and 14th days and were explored for fibroblasts, epithelial cells, inflammatory cells, and vessels. RT-PCR was performed for molecular and gene expression evaluation of TGF-β. RESULTSThe M. piperita essence increases TGF-β gene expression as an important factor in wound healing. After 14 d, group A, who received M. piperita and Vaseline, had 99.73% of wound healing with the mean wound size of 0.006 cm2 while wound healing in the control group was only 52%. Samples treated with M. piperita have 74.58% wound healing following by group treated with Vaseline, which was 67.02% (P<0.05), respectively.CONCLUSIONThe application of the M. piperita essence for wound healing accelerates the process and improves outcomes.

Evaluating the effect of methotrexate on the rate of renal fibrosis by elastography and fibrosis-related gene expression.

Methotrexate is mainly used to treat diseases such as rheumatoid arthritis (RA), but its potential for nephrotoxicity has always been a significant concern on the use of this medication. This study aimed to determine the rate of renal fibrosis using transient elastography and its relationship with cumulative dose and duration of drug use in patients with rheumatoid arthritis treated with methotrexate. TGFβ gene expression was also assessed for further evaluation. Patients with rheumatoid arthritis who received methotrexate for more than six months were included. Renal fibrosis was determined by measuring the stiffness of the kidney by elastography (FiberScan Device). RA patients were divided into two groups based on kidney stiffness measurement with and without renal fibrosis, and demographic, clinical, and biochemical parameters were compared to investigate the relationship between cumulative dose and duration of methotrexate treatment and renal fibrosis. Also, in this study, 50 controls (healthy people) and 50 cases (RA patients) were used to evaluate the expression of the TGFβ gene by real-time PCR method. The existence of kidney fibrosis was observed in 10 patients. There was no significant relationship between renal fibrosis and the cumulative dose (P = 0.21) and duration of methotrexate (P = 0.30). Multivariate regression analysis showed that the chances of developing renal fibrosis in patients increase with increasing serum ALT levels (P = 0.01). The results of the TGFβ gene expression showed that the expression of this gene in the group of RA patients with fibrosis was higher than the control group (healthy people) and the group of RA patients without fibrosis (P <0.01). These results showed that evaluation of renal fibrosis by elastography method is recommended for scanning RA patients while they are being treated with methotrexate, which is also confirmed by the results of the fibrosis-related-gene expression.