Alantolactone Attenuates Renal Fibrosis via Inhibition of Transforming Growth Factor ß/Smad3 Signaling Pathway.

BACKGRUOUND: Renal fibrosis is characterized by the accumulation of extracellular matrix proteins and interstitial fibrosis. Alantolactone is known to exert anticancer, anti-inflammatory, antimicrobial and antifungal effects; however, its effects on renal fibrosis remains unknown. Here, we investigated whether alantolactone attenuates renal fibrosis in mice unilateral ureteral obstruction (UUO) and evaluated the effect of alantolactone on transforming growth factor (TGF) signaling pathway in renal cells. METHODS: To evaluate the therapeutic effect of alantolactone, cell counting kit-8 (CCK-8) assay, histological staining, Western blot analysis, and real-time quantitative polymerase chain reaction were performed in UUO kidneys in vivo and in TGF-ß-treated renal cells in vitro. RESULTS: Alantolactone (0.25 to 4 µM) did not affect the viability of renal cells. Mice orally administered 5 mg/kg of alantolactone daily for 15 days did not show mortality or liver toxicity. Alantolactone decreased UUO-induced blood urea nitrogen and serum creatinine levels. In addition, it significantly alleviated renal tubulointerstitial damage and fibrosis and decreased collagen type I, fibronectin, and α-smooth muscle actin (α-SMA) expression in UUO kidneys. In NRK-49F cells, alantolactone inhibited TGF-ßstimulated expression of fibronectin, collagen type I, plasminogen activator inhibitor-1 (PAI-1), and α-SMA. In HK-2 cells, alantolactone inhibited TGF-ß-stimulated expression of collagen type I and PAI-1. Alantolactone inhibited UUO-induced phosphorylation of Smad3 in UUO kidneys. In addition, it not only decreased TGF-ß secretion but also Smad3 phosphorylation and translocation to nucleus in both kidney cell lines. CONCLUSION: Alantolactone improves renal fibrosis by inhibiting the TGF-ß/Smad3 signaling pathway in obstructive nephropathy. Thus, alantolactone is a potential therapeutic agent for chronic kidney disease.

Gemigliptin exerts protective effects against doxorubicin-induced hepatotoxicity by inhibiting apoptosis via the regulation of fibroblast growth factor 21 expression.

AIM: Doxorubicin is a highly effective anticancer agent that causes hepatotoxicity and cardiotoxicity in patients. Fibroblast growth factor 21, a well-known regulator of glucose and lipid metabolism, exerts cardioprotective effects. Gemigliptin and dipeptidyl peptidase-4 inhibitors are widely used in the treatment of patients with type 2 diabetes. The protective effects of gemigliptin on hepatotoxicity via the increase in fibroblast growth factor 21 expression has not yet been elucidated. This study was designed to investigate the protective effects of gemigliptin against doxorubicin-induced hepatotoxicity via the upregulation of fibroblast growth factor 21 expression in the cultured murine hepatocyte cell line, AML12. METHODS: Murine hepatocyte AML12 cells were treated with doxorubicin, fibroblast growth factor 21 and gemigliptin in 0.5% fetal bovine serum medium for 24 h at the indicated doses. Cells were transfected with the fibroblast growth factor 21 small interfering RNA for 24 h, followed by protein isolation. RESULTS: Fibroblast growth factor 21 expression levels were increased during doxorubicin-induced hepatotoxicity in the murine hepatocyte AML12 cells. Fibroblast growth factor 21 treatment prevented doxorubicin-induced hepatotoxicity by attenuating apoptosis. Gemigliptin prevented doxorubicin-induced hepatotoxicity by upregulating fibroblast growth factor 21 expression. However, the protective effects of gemigliptin were blocked by fibroblast growth factor 21 inhibition in doxorubicin-treated AML12 cells. CONCLUSION: These results indicate that gemigliptin exhibits protective effects against doxorubicin-induced hepatotoxicity by upregulating the fibroblast growth factor 21 expression levels in the cultured murine hepatocyte AML12 cells.

Lin28a ameliorates glucotoxicity-induced ß-cell dysfunction and apoptosis.

An excessive and prolonged increase in glucose levels causes ß-cell dysregulation, which is accompanied by impaired insulin synthesis and secretion, a condition known as glucotoxicity. Although it is known that both Lin28a and Lin28b regulate glucose metabolism, other molecular mechanisms that may protect against glucotoxicity are poorly understood. We investigated whether Lin28a overexpression can improve glucotoxicityinduced ß-cell dysregulation in INS-1 and primary rat islet cells. INS-1, a rat insulinoma cell line was cultured and primary rat islet cells were isolated from SD-rats. To define the effect of Lin28a in chronic high glucose-induced ß-cell dysregulation, we performed several in vitro and ex-vivo experiments. Chronic exposure to high glucose led to a downregulation of Lin28a mRNA and protein expression, followed by a decrease in insulin mRNA expression and secretion in ß-cells. The mRNA and protein expression levels of PDX-1 and BETA2, were reduced; The levels of apoptotic factors, including c-caspase3 and the Bax/Bcl-2 ratio, were increased due to glucotoxicity. Adenovirusmediated Lin28a overexpression in ß-cells reversed the glucotoxicity- induced reduction of insulin secretion and insulin mRNA expression via regulation of ß-cell-enriched transcription factors such as PDX-1 and BETA2. Adenovirus-mediated overexpression of Lin28a downregulated the glucotoxicity-induced upregulation of c-caspase3 levels and the Bax/Bcl-2 ratio, while inhibition of endogenous Lin28a by small interfering RNA resulted in their up-regulation. Lin28a counteracted glucotoxicity-induced downregulation of p-Akt and p-mTOR. Our results suggest that Lin28a protects pancreatic ß-cells from glucotoxicity through inhibition of apoptotic factors via the PI3 kinase/Akt/mTOR pathway. [BMB Reports 2021; 54(4): 215-220].

Lin28a attenuates TGF-ß-induced renal fibrosis.

Lin28a has diverse functions including regulation of cancer, reprogramming and regeneration, but whether it promotes injury or is a protective reaction to renal injury is unknown. We studied how Lin28a acts in unilateral ureteral obstruction (UUO)-induced renal fibrosis following unilateral ureteral obstruction, in a mouse model. We further defined the role of Lin28a in transforming growth factor (TGF)-signaling pathways in renal fibrosis through in vitro study using human tubular epithelium-like HK-2 cells. In the mouse unilateral ureteral obstruction model, obstruction markedly decreased the expression of Lin28a, increased the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin. In TGF-ß-stimulated HK-2 cells, the expression of Lin28a was reduced and the expression of renal fibrotic markers such as type I collagen, α-SMA, vimentin and fibronectin was increased. Adenovirus-mediated overexpression of Lin28a inhibited the expression of TGF-ß-stimulated type I collagen, α-SMA, vimentin and fibronectin. Lin28a inhibited TGF-ß-stimulated SMAD3 activity, via inhibition of SMAD3 phosphorylation, but not the MAPK pathway ERK, JNK or p38. Lin28a attenuates renal fibrosis in obstructive nephropathy, making its mechanism a possible therapeutic target for chronic kidney disease. [BMB Reports 2020; 53(11): 594-599].

Arg-Gly-Asp-modified elastin-like polypeptide regulates cell proliferation and cell cycle proteins via the phosphorylation of Erk and Akt in pancreatic ß-cell.

OBJECTIVE: Enhancement of ß-cell proliferation plays an important role in maintaining ß-cell mass and function, and in improving pancreatic ß-cell survival before transplantation. Extracellular matrix (ECM) components increase the adhesion and proliferation of ß-cells, and the RGD-modified elastin-like polypeptide (RGD-ELP, REP) has been described as a bioactive matrix. In this study, we investigated whether REP could enhance ß-cell adhesion and proliferation and elucidated the signaling pathways involved. METHODS: We investigated the effect of REP on cell adhesion, proliferation and insulin secretion via assays using Rin-m and rat islets. Crystal violet, CCK-8, and BrdU assay, FACS, western blot, real time q-PCR analyses and insulin ELISA were examined. To explain the associated mechanisms, phosphorylation of Akt and extracellular signal-regulated kinase (Erk) were measured. RESULTS: REP more increased the adhesion, proliferation and survival of Rin-m cells compared to elastin-like poly peptide (ELP) without RGD-motif. The enhancement of ß-cell proliferation by REP was associated with increased cyclin D1, cyclin D2 and cdk6, and decreased p27 levels. When ß-cells were cultured on REP, Erk and the phosphatidylinositol 3-kinase (PI3-kinase) downstream effector, Akt was stimulated. Treatment with the Erk pathway inhibitor and PI3-kinase inhibitor decreased REP-induced ß-cell adhesion and proliferation, and regulated REP-induced cell cycle proteins. Additionally, REP increased the mRNA and protein levels of insulin and its transcription factor, PDX-1, and insulin secretion. CONCLUSIONS: Our results demonstrate that the up-regulation of the PI3K/Akt and Erk signaling pathways and the regulation of cell cycle proteins by REP could serve as effective strategies for improving pancreatic ß-cell adhesion and proliferation.

Enzymatic hydrolysis of perilla seed meal yields water-soluble dietary fiber as a potential functional carbohydrate source.

This study was conducted with an aim to produce a novel water-soluble fiber (WSF) by enzymatic hydrolysis of perilla seed meal (PSM), which could be used as a functional food material. The cellulose fraction (CF) and hemicellulose fraction (HF) derived from PSM were hydrolyzed using Celluclast 1.5 L and Viscozyme L, respectively. Although WSF produced from PSM had low acetylcholinesterase inhibitory activity, WSF exhibited excellent antioxidant, antidiabetic and anti-dementia activities, and effectively delayed the diffusion of glucose and bile acid from the dialysis membranes. In particular, WSF produced from CF showed a significantly higher bile acid retarding index than pectin, and WSF obtained from HF had low IC50 values for radical scavenging activity and reducing power. Thus, these results suggest that the WSF derived from PSM by enzymatic hydrolysis can be used as functional carbohydrate source such as additive and a dietary supplement in the food industry.

Effect of Rhus verniciflua Extract on IgE-Antigen-Mediated Allergic Reaction in Rat Basophilic Leukemic RBL-2H3 Mast Cells and Passive Cutaneous Anaphylaxis in Mice.

Rhus verniciflua is widely known for its antioxidant, antibacterial, anticancer, and antiaging efficacy and α-glucosidase inhibition. This study was designed whether Rhus verniciflua extracts inhibit the IgE-antigen-mediated allergic reaction in RBL-2H3 mast cells, and it further investigated the FcεRI- and arachidonate-signaling by which Rhus verniciflua extracts exert its antiallergic effects. IgE-antigen-sensitized RBL-2H3 mast cells were investigated for the cytotoxicity of Rhus verniciflua extracts and ß-hexosaminidase release, and inflammatory mediators (e.g., TNF-α, IL-4, IL-6, histamine, and PGD2) were then assessed. Additionally, we examined expressions of genes involved in arachidonate- and FcεRI-signaling pathway in RBL-2H3. Rhus verniciflua extracts inhibited ß-hexosaminidase release and production of the inflammatory mediators in RBL-2H3. Rhus verniciflua extracts reduced amounts of histamine and expressions of FcεRI signaling-related genes such as Lyn and Syk and phosphorylation of extracellular signal-regulated kinase in mast cells. Finally, in late allergic responses, Rhus verniciflua extracts reduced PGD2 release and COX-2 and cPLA2 phosphorylation expressions from IgE-antigen-mediated mast cells. Lastly, 250-500 mg/kg RVE significantly attenuated the Ag/IgE-induced passive cutaneous anaphylaxis (PCA) reaction in mice. These findings provide novel information on the molecular mechanisms underlying the antiallergy properties of Rhus verniciflua extracts in FcɛRI-mediated allergic reaction.