Probenecid induces the recovery of renal ischemia/reperfusion injury via the blockade of Pannexin 1/P2X7 receptor axis.

Renal ischemia/reperfusion injury (RI/RI) is one of the main driving causes of acute kidney injury. However, effective treatment to limit injury and promote recovery and/or survival is still unavailable. Probenecid (PBN), a drug indicated for refractory gout, exhibits protective activities against several preclinical diseases including cerebral and myocardial I/RI via Pannexin 1 (Panx1) and P2X7 receptors' (P2X7R) inhibition. However, its protective role against RI/RI has not been previously addressed. Accordingly, we subjected rats to bilateral RI/RI with/or without PBN treatment. Twenty-four hours post-reperfusion, PBN showed mild tubular injury and reduced serum nephrotoxicity indices, gene and protein expression levels of Panx 1 and P2X7R, and ATP and pro-inflammatory cytokines' levels. The nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome signaling was also downregulated, as demonstrated by reduced gene and protein expression of NLRP3 and caspase-1, along with suppressed IL-1ß maturation. Furthermore, PBN enhanced Tregs activity as indicated by elevated FoxP3 gene expression, IL-10, and TGF-ß renal levels. On day 5 post-reperfusion, PBN noticeably enhanced renal recovery, as demonstrated by intact tubular epithelium and restored nephrotoxicity indices, Panx 1 and P2X7R gene and protein expression levels, ATP and pro-inflammatory cytokine levels, and NLRP3 inflammasome signaling. Besides, renal Tregs activity was also significantly increased. Our study elaborates for the first time the effectiveness of PBN in recovering post-ischemic renal injury through synergistic inhibition in Panx1/P2X7R axis leading to inactivation of NLRP3 inflammasome signaling and activation of Tregs in ischemic renal tissues. Therefore, PBN can be considered a promising drug for RI/RI treatment.

Co-treatment with Esculin and erythropoietin protects against renal ischemia-reperfusion injury via P2X7 receptor inhibition and PI3K/Akt activation.

Renal ischemia/reperfusion (RI/R) is a critical clinical outcome with slightly reported improvement in mortality and morbidity. Effective therapies are still crucially required. Accordingly, the therapeutic effects of esculin (ESC, LCESI-MS/MS-isolated compound from Vachellia farnesiana flowers extract, with reported P2X7 receptor inhibitor activity) alone and in combination with erythropoietin (EPO) were investigated against RI/R injury and the possible underlying mechanisms were delineated. ESC and EPO were administered for 7 days and 30 min prior to RI, respectively. Twenty-four hour following reperfusion, blood and kidney samples were collected. Results revealed that pretreatment with either ESC or EPO reduced serum nephrotoxicity indices, renal oxidative stress, inflammatory, and apoptosis markers. They also ameliorated the renal histopathological injury on both endothelial and tubular epithelial levels. Notably, ESC markedly inhibited P2X7 receptors and NLRP3 inflammasome signaling (downregulated NLRP3 and Caspase-1 gene expressions), whereas EPO significantly upregulated PI3K and Akt gene expressions, also p-PI3K and p-Akt levels in renal tissues. ESC, for the first time, demonstrated effective protection against RI/R-injury and its combination with EPO exerted maximal renoprotection when compared to each monotherapy, thereby representing an effective therapeutic approach via inhibiting oxidative stress, inflammation, renal tubular and endothelial injury, apoptosis, and P2X7 receptors expression, and activating PI3K/Akt pathway.

6-Paradol alleviates Diclofenac-induced acute kidney injury via autophagy enhancement-mediated by AMPK/AKT/mTOR and NLRP3 inflammasome pathways.

Diclofenac (DIC)-induced acute kidney injury (AKI) causes high morbidity and mortality. With the absence of satisfactory treatment, we investigated the protective effects of 6-Paradol (PDL) against DIC-induced AKI, with focus on renal autophagy and NLRP3 inflammasome pathways . PDL has anti-inflammatory, antioxidant and AMPK-activation properties. PDL was administered to DIC-challenged rats. Nephrotoxicity, oxidative stress, inflammatory, and autophagy markers and histopathological examinations were evaluated. Compared to DIC, PDL restored serum nephrotoxicity, renal oxidative stress and pro-inflammatory markers. PDL almost restored renal architecture, upregulated renal Nrf2 pathway via enhancing Nrf2 mRNA expression and HO-1 levels. PDL suppressed renal NF-κB mRNA expression, and NLRP3 inflammasome pathway expression. Moreover, PDL enhanced renal autophagy through upregulating LC3B, AMPK and SIRT-1, and suppressed mTOR, p-AKT mRNA expressions and phosphorylated-p62 levels. Our study confirmed that autophagy suppression mediates DIC-induced AKI via AMPK/mTOR/AKT and NLRP3-inflammasome pathways. Also, PDL's nephroprotective effects could provide a promising therapeutic approach against DIC-induced AKI.

Zein/Phospholipid Composite Nanoparticles for Successful Delivery of Gallic Acid into aHSCs: Influence of Size, Surface Charge, and Vitamin A Coupling.

PURPOSE: Zein/phospholipid composite nanoparticles (CNPs) were developed as a delivery platform for gallic acid (GA), a polyphenolic compound with reported preclinical antifibrotic activities. However, the therapeutic applicability of GA is hampered owing to its low bioavailability and rapid clearance. Accordingly, we developed GA-loaded CNPs. The effect of their size, surface charge and targeting strategies was investigated and optimized, with the aim of enhancing their ability to deliver GA to the activated hepatic stellate cells (aHSCs) in order to suppress hepatic fibrosis progression. METHODS: Different CNP systems were prepared and characterized with regard to their particle size, zeta potential, and GA entrapment efficiency (EE%). Also, they were statistically optimized via response surface methodology. The optimized systems were investigated with regard to their in vitro GA release, in vitro efficacy on aHSCs, and in vivo biodistribution in healthy rats. RESULTS: The GA-loaded cationic CNPs coupled with vitamin A (GA-CACNP/VA; 192 nm) showed high GA EE% (60% w/w), highest cellular internalization via active targeting, and more selective hepatic distribution, relative to free GA solution, GA-loaded anionic, and GA-loaded cationic systems. Furthermore, GA-CACNP/VA markedly triggered the apoptosis of aHSCs, repressed collagen deposition, and inhibited HSCs' activation to a lesser extent. CONCLUSION: The GA-CACNP/VA was shown to be a promising candidate for specific and controlled delivery of GA to aHSCs, which may provide an effective antifibrotic therapeutic approach.

Impact of Reverse Micelle Loaded Lipid Nanocapsules on the Delivery of Gallic Acid into Activated Hepatic Stellate Cells: A Promising Therapeutic Approach for Hepatic Fibrosis.

PURPOSE: Gallic acid (GA) is a polyphenolic compound with proven efficacy against hepatic fibrosis in experimental animals. However, it suffers from poor bioavailability and rapid clearance that hinders its clinical investigation. Accordingly, we designed and optimized reverse micelle-loaded lipid nanocapsules (RMLNC) using Box-Behnken design that can deliver GA directly into activated-hepatic stellate cells (aHSCs) aiming to suppress hepatic fibrosis progression. METHODS: GA-RMLNC was prepared using soft energy, solvent free phase inversion temperature method. Effects of formulation variables on particle size, zeta potential, entrapment efficiency (EE%) and GA release were studied. In-vivo biodistribution of GA-RMLNC in rats and in-vitro activities on aHSCs were also explored. RESULTS: Nano-sized GA-RMLNCs (30.35 ± 2.34 nm) were formulated with high GA-EE% (63.95 ± 2.98% w/w) and physical stability (9 months). The formulated system showed burst GA release in the first 2 h followed by sustained release profile. In-vivo biodistribution imaging revealed that RMLNC-loaded with rhodamine-B accumulated mainly in rats' livers. Relative to GA; GA-RMLNC displayed higher anti-proliferative activities, effective internalization into aHSCs, marked down-regulation in pro-fibrogenic biomarkers' expressions and elevated HSCs' apoptosis. CONCLUSIONS: These findings emphasize the promising application of RMLNC as a delivery system in hepatic fibrosis treatment, where successful delivery of GA into aHSCs was ensured via increased cellular uptake and antifibrotic activities.

Chemical Profiling of Polyphenolics in Eucalyptus globulus and Evaluation of Its Hepato-Renal Protective Potential Against Cyclophosphamide Induced Toxicity in Mice.

Cyclophosphamide (CP) is a potent anti-neoplastic and immunosuppressive agent; however, it causes multi-organ toxicity. We elucidated the protective activities of Eucalyptus globulus (EG) leaf extract against CP-induced hepato-renal toxicity. Mice were treated with EG for 15 days plus CP on day 12 and 13 of the experiment. Using HPLC-DAD-ESI-MS/MS, 26 secondary metabolites were identified in EG leaf extract. Out of them, 4 polyphenolic compounds were isolated: (1) 4-(O-ß-d-xylopyranosyloxy)-3,5-di-hydroxy-benzoic acid, (2) 4-(O-α-l-rhamnopyranosyloxy)-3,5-di-hydroxy-benzoic acid, (3) gallic acid, and (4) methyl gallate. Effects of EG extract on biochemical parameters, gene expression, and immune-histopathological changes were assessed in comparison to mesna positive control. Results showed that EG improved CP-increased serum ALT, AST, creatinine, and blood urea nitrogen levels. The hepatic and renal tissue levels of MDA, nitric oxide, protein carbonyl, TNF-α, IL-6, and immunohistochemical expression of nuclear factor kappa-B (NF-kB) and caspase-3 were reduced. Also, hepatic and renal GSH contents, and nuclear factor E2-related factor 2 (NRf2)/ hemoxygenase-1 (HO-1) signaling levels were increased. Histopathological findings supported our findings where hepatic and renal architecture were almost restored. Results revealed the protective effects of EG against CP-induced hepato-renal toxicity. These effects may be related to EG antioxidant, anti-inflammatory, and anti-apoptotic properties coupled with activation of Nrf2/HO-1 signaling.

Rosmarinic acid attenuates hepatic fibrogenesis via suppression of hepatic stellate cell activation/proliferation and induction of apoptosis.

OBJECTIVE: To investigate the antifibrotic role of rosmarinic acid (RA), a natural polyphenolic compound, on HSCs activation/proliferation and apoptosis in vitro and in vivo. METHODS: The impact of RA on stellate cell line (HSC-T6) proliferation, activation and apoptosis was assessed along with its safety on primary hepatocytes. In vivo, rats were divided into: (i) normal; (ii) thioacetamide (TAA)-intoxicated rats for 12 weeks; (iii) TAA + silymarin or (iv) TAA + RA. At the end of experiment, liver functions, oxidative stress, inflammatory and profibrogenic markers, tissue inhibitor metalloproteinases type-1 (TIMP-1) and hydroxyproline (HP) levels were evaluated. Additionally, liver histopathology and immunohistochemical examinations of alpha-smooth muscle actin (α-SMA), caspase-3 and proliferation cellular nuclear antigen (PCNA) were determined. RESULTS: RA exhibited anti-proliferative effects on cultured HSCs in a time and concentration dependent manner showing an IC50 of 276 µg/mL and 171 µg/mL for 24 h and 48 h, respectively, with morphological reversion of activated stellate cell morphology to quiescent form. It significantly improved ALT, AST, oxidative stress markers and reduced TIMP-1, HP levels, inflammatory markers and fibrosis score (S1 vs S4). Furthermore, reduction in α-SMA plus elevation in caspase-3 expressions of HSCs in vitro and in vivo associated with an inhibition in proliferation of damaged hepatocytes were recorded. CONCLUSIONS: RA impeded the progression of liver fibrosis through inhibition of HSCs activation/proliferation and induction of apoptosis with preservation of hepatic architecture.

Anti-inflammatory/anti-fibrotic effects of the hepatoprotective silymarin and the schistosomicide praziquantel against Schistosoma mansoni-induced liver fibrosis.

BACKGROUND: Praziquantel (PZQ) is an isoquinoline derivative (2-cyclohexylcarbonyl-1, 2, 3, 6, 7, 11b-hexahydro-4H-pyrazino{2,1-a}-isoquinoline-4-one), and is currently the drug of choice for all forms of schistosomiasis. Silymarin, a standardized milk thistle extract, of which silibinin is the main component, is known for its hepatoprotective, anti-inflammatory, antioxidant activities, and hepatocyte regeneration. This study investigates the anti-inflammatory/anti-fibrotic effects of silymarin and/or PZQ on schistosomal hepatic fibrosis. METHODS: Schistosoma mansoni-infected mice were divided into two large groups (I & II), each with four subgroups and were run in parallel. (i) Infected untreated; (ii) treated with silymarin, starting from the 4th (3 weeks before PZQ therapy) or 12th (5 weeks after PZQ therapy) weeks post infection (PI); (iii) treated with PZQ in the 7th week PI; and (iv) treated with silymarin, as group (ii) plus PZQ as group (iii). Comparable groups of uninfected mice run in parallel with the infected groups. Mice of groups I and II were killed 10 and 18 weeks PI, respectively. Hepatic content of hydroxyproline (HYP), serum levels and tissue expression of matrix metalloproteinase-2 (MMP-2), transforming growth factor-ß1 (TGF-ß1) and number of mast cells were determined. In addition, parasitological, biochemical and histological parameters that reflect disease severity and morbidity were examined. RESULTS: Silymarin caused a partial decrease in worm burden; hepatic tissue egg load, with an increase in percentage of dead eggs; modulation of granuloma size, with significant reduction of hepatic HYP content; tissue expression of MMP-2, TGF-ß1; number of mast cells, with conservation of hepatic reduced glutathione (GSH). PZQ produced complete eradication of worms, eggs and alleviated liver inflammation and fibrosis. The best results were obtained, in most parameters studied, in groups of mice treated with silymarin in addition to PZQ. CONCLUSIONS: Our results point to silymarin as a promising anti-inflammatory and anti-fibrotic agent; it could be introduced as a therapeutic tool with PZQ in the treatment of schistosomal liver fibrosis, but further studies on mechanisms of silymarin and PZQ in chronic liver diseases may shed light on developing therapeutic methods in clinical practice.