Protective effect of irbesartan against hepatic ischemia-reperfusion injury in rats: role of ERK, STAT3, and PPAR-γ inflammatory pathways in rats.

This study aimed to elucidate the possible hepatocellular protective role of irbesartan during hepatic ischemia-reperfusion injury (HIRI) and the probable underlying mechanisms. Wistar rats were allocated into four groups: sham; HIRI (control); irbesartan (50 mg/kg) + HIRI; irbesartan (100 mg/kg) + HIRI; irbesartan + GW9662 (1 mg/kg, i.p.) + HIRI. Rats pretreated orally with irbesartan or vehicle for 14 days underwent 45-min hepatic ischemia followed by 60-min reperfusion. Irbesartan preconditioning diminished alanine transaminase (ALT) and aspartate transaminase (AST) serum levels, and reduced extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3). Irbesartan decreased proapoptotic BAX (bcl-2-like protein 4), increased anti-apoptotic B-cell lymphoma 2 (BCL2) hepatic content, and thereby reduced BAX/BCL2 ratio. Moreover, irbesartan preconditioning reduced autophagy-related proteins Beclin1 and LC3 II, and elevated p62 (protein responsible for autophagosome degradation). It elevated proliferator-activated receptor γ (PPAR-γ), and reduced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) hepatic gene expression. Also, hepatic protein expressions of nuclear factor kappa-B p65 (NF-κB p65) and caspase-3 were lessoned by irbesartan pretreatment in HIRI rats. However, GW9662 abrogated irbesartan's effect on HIRI. The protective effect of irbesartan on HIRI may be mediated by alleviation of ERK, STAT3, and PPAR-γ inflammatory pathways, exerting anti-apoptotic and anti-autophagic effects in HIRI in rats.

Evaluation of the protective effect of losartan in acetaminophen-induced liver and kidney damage in mice.

Acetaminophen is widely used among humans as an antipyretic and analgesic. In this study, the protective effect of losartan in hepatotoxicity and nephrotoxicity induced by acetaminophen in mice was investigated owing to its anti-inflammatory and antioxidant effects. An injection of a single dose of 500 mg/kg (i.p.) acetaminophen was administered to induce hepatotoxicity and nephrotoxicity in Groups VI-X. Losartan at doses of 1 mg/kg (Group VII), 3 mg/kg (Group VIII), and 10 mg/kg (Groups III, V, IX, and X) was injected intraperitoneally twice, at 1 and 12 h after the acetaminophen injection. Additionally, a 4 mg/kg dose of GW9662 (peroxisome proliferator-activated receptor gamma (PPAR-γ) antagonist) was injected intraperitoneally 30 min before the losartan injections in Groups V and X. At the end of 24 h, the mice were euthanized, and blood, liver, and kidney tissue samples were collected. Levels of AST, ALT, creatinine, and oxidative stress markers including TBARS, SOD, CAT, GPx, TAS, TOS, GSH, and GSSG, along with pro-inflammatory cytokines IL-1ß, IL-6, IL-8, IL-10, IL-17, and TNF-α, were measured using ELISA kits. Additionally, a histological evaluation of the tissue samples was performed. Acetaminophen causes increases in the levels of AST, ALT, creatinine, TBARS, TOS, GSSG, IL-1ß, IL-6, IL-8, IL-10, IL-17, and TNF-α in serum, liver, and kidney tissue. Meanwhile, it led to a decrease in the levels of SOD, CAT, GPx, TAS, and GSH. Losartan injection reversed oxidative and inflammatory damage induced by acetaminophen. Histopathological changes in liver and kidney tissue were alleviated by losartan. The substance GW9662 increased the protective effect of losartan. In light of all the data obtained from our study, it can be said that losartan has a protective effect on liver and kidney damage induced by acetaminophen due to its antioxidant and anti-inflammatory effects. In terms of the study, losartan was found to be an alternative substance that could protect people from the harmful effects of acetaminophen.

Peroxisome proliferator activated receptor-gamma (PPAR-γ) ligand, pioglitazone, increases analgesic and anti-inflammatory effects of naproxen.

The aim of this study was the investigation of analgesic and anti-inflammatory activity of naproxen and pioglitazone following intra-plantar injection of carrageenan and assessment of the PPAR-γ receptor involvement in these effects. Rats were intra-plantarly injected with carrageenan (1%, 100 µl) to induce thermal hyperalgesia and paw inflammation. Different groups of rats were pre-treated intraperitoneally with naproxen (1 and 10 mg/kg) or pioglitazone (3 and 10 mg/kg) or GW9662 (a selective PPAR-γ antagonist, 100 µl/paw). The volume of the paw was evaluated using a plethysmometer, and the hot plate test was employed to assess the pain threshold in the animals. Finally, TNF-α, IL-1ß, IL-6, and myeloperoxidase (MPO) activity status were evaluated in the hind paw tissue. Naproxen and pioglitazone demonstrated analgesic and anti-inflammatory activity. Concurrent injection of an ineffective dose of naproxen (1 mg/kg) with an ineffective dose of pioglitazone (3 mg/kg) caused augmented analgesic and anti-inflammatory activity, significantly (p≤0.001 and p≤0.01, respectively). Additionally, intra-plantar injection of GW-9662 before naproxen or pioglitazone significantly suppressed their analgesic (p≤0.001) and anti-inflammatory activity (p≤0.01). Also, naproxen and pioglitazone (10 mg/kg) significantly (p≤0.001) reduced carrageenan-induced MPO activity and TNF-α, IL-6, and IL-1ß releasing. Furthermore, PPAR-γ blockade significantly prevented suppressive effects of naproxen and pioglitazone on the MPO activity and inflammatory cytokines. Pioglitazone significantly increased analgesic and anti-inflammatory effects of naproxen. This study proposes that concurrent treatment with naproxen and pioglitazone may be a substitute for overcome pain and inflammation clinically, in the future, particularly in patients with cardiovascular disorders and diabetes.

GW9662 ameliorates nonalcoholic steatohepatitis by inhibiting the PPARγ/CD36 pathway and altering the gut microbiota.

BACKGROUND & AIMS: Peroxisome proliferator-activated receptors (PPARs) are currently among the most focused-on therapeutic targets for non-alcoholic steatohepatitis (NASH), although no clinical transformation has been achieved to date. In this study, we aimed to evaluate the effects of GW9662 on choline-deficient, L-amino acid-defined high-fat diet (CDAA-HFD)-induced NASH mice and reveal the mechanism underlying this effect. METHODS: GW9662 (1 mg/kg) was administered in CDAA-HFD mouse model of NASH. The effect of GW9662 on hepatic lipid metabolism was investigated using liver RNA-seq and HepG2 cells induced by oleic acid and palmitic acid. In addition, 16S rRNA gene sequencing was performed to analyze the effects of GW9662 on the composition and function of the fecal microbiota. RESULTS: GW9662 improved the CDAA-HFD caused elevation in the levels of ALT, AST, hepatic free fatty acids and triglycerides. The liver pathological analysis indicated that GW9662 alleviated the hepatic steatosis and fibrosis. The NAFLD activity score and RNA-Seq revealed that GW9662 mainly regulated the fatty acids transport and lipid synthesis by inhibiting PPARγ, CD36, FABP1, FASN, and SCD1, and through the up-regulation of PPARα. Moreover, GW9662 reduced the epididymal fat weight. GW9662 reversed the gut microbiota disorder by increasing the abundance of the beneficial bacteria Dubosiella and Lactobacillus and decreasing the abundance of harmful bacteria Lachnospiraceae_NK4A136_group, Helicobacteraceae, Desulfovibriaceae, and Rickenaceae. CONCLUSIONS: GW9662 ameliorated lipid metabolism by inhibiting the PPARγ/CD36 pathway and altering the composition of the gut microbiota in NASH mice. Therefore, the PPARγ antagonist GW9662 deserves more attention as a potential therapeutic agent for NASH.

The anti-fibrotic efficacy of adelmidrol depends on hepatic PPARγ levels.

Adelmidrol, an anti-inflammatory small-molecule compound, can treat inflammatory diseases like arthritis and colitis in a PPARγ-dependent manner. Effective anti-inflammatory therapy is beneficial in delaying the progression of liver fibrosis. This study aimed to investigate the effect and underlying mechanisms of adelmidrol on hepatic fibrosis induced by CCl4 and CDAA-HFD. In the CCl4 model, adelmidrol (10 mg/kg) significantly reduced the incidence of liver cirrhosis from 76.5% to 38.9%, with a reduction of ALT, AST, and extracellular matrix deposition. RNA-seq revealed adelmidrol markedly inhibited the activation of hepatic scar-associated Trem2+ macrophages and PDGFRα+ stellate cells. Adelmidrol exhibited a limited anti-fibrotic effect in CDAA-HFD-induced fibrosis. Further, inconsistencies were observed in the expression trends in liver PPARγ in both models. CCl4 injury led to the continuous decrease in hepatic PPARγ levels, adelmidrol treatment up-regulated hepatic PPARγ expression and down-regulated the expression of pro-inflammatory factor NF-κB and pro-fibrotic factor TGF-ß1. Adelmidrol also inhibited the activation of macrophages and HSCs in a PPARγ-dependent manner in vitro. GW9662, a specific PPARγ antagonist, counteracted the anti-fibrotic effect of adelmidrol. In CDAA-HFD-induced model, hepatic PPARγ expression gradually increased with the progress of modeling. Adelmidrol enhanced steatosis in hepatocytes by the activation of the PPARγ/CD36 pathway in the CDAA-HFD model and FFA-treated HepG2, showing a limited anti-fibrotic effect. GW9662 reversed the pro-steatotic effect of adelmidrol and improved fibrosis. The anti-fibrotic outcomes of adelmidrol were related to hepatic PPARγ levels, which depends on the synergistic effect of PPARγ agonism caused by adelmidrol on hepatocytes, macrophages, and HSCs in different pathological states.

Metabolic regulation of the circadian clock in classically and alternatively activated macrophages.

Macrophages exhibit a range of functional pro- and anti-inflammatory states that induce changes in their cellular metabolism. We aimed to elucidate whether these changes affect the molecular properties of their circadian clock focusing on their anti-inflammatory phenotype. Primary cell cultures of bone marrow-derived macrophages (BMDMs; nonpolarized M0 BMDM) from PER2::LUC (fusion protein of PERIOD2 and LUCIFERASE) mice were polarized into the M1 (proinflammatory) or M2 (anti-inflammatory) phenotype, and PER2-driven bioluminescence was recorded in real-time at the cell-population and single-cell levels. Viability, clock gene expression profiles, polarization plasticity and peroxisome proliferator-activated receptor γ (PPARγ) protein levels were analyzed. The effects of pharmacological activation/inhibition of PPARγ (rosiglitazone/GW9662) and inhibition of fatty acid oxidation (FAO) by etomoxir in M2 BMDM cell cultures were examined. The parameters of PER2-driven bioluminescence rhythms differed between M0, M1 and M2 BMDM cultures at cell-population and single-cell levels. Compared with M0, polarization to M2 did not change the period but increased amplitude, mean bioluminescence level and rhythm persistence. Polarization to M1 shortened the period but had no effect on the amplitude of the rhythm. The same period changes were observed after a bidirectional switch between M1- and M2-polarized states in the same culture. Both PPARγ activation/inhibition and FAO inhibition modulated the clock in M2 BMDMs, suggesting metabolic regulation of the M2 clock. Our results indicate that bidirectional changes in the properties of BMDM circadian clocks in response to their actual polarization are mediated via changes in their metabolic state. They provide new information on the interrelationship between the BMDM polarization, their circadian clock and cellular metabolism.

The Cardioprotective Effect of Corosolic Acid in the Diabetic Rats: A Possible Mechanism of the PPAR-γ Pathway.

The study was conducted to determine whether corosolic acid could protect the myocardium of diabetic rats from damage caused by isoproterenol (ISO) and, if so, how peroxisome proliferator-activated receptor gamma (PPAR-γ) activation might contribute into this protection. Diabetes in the rats was induced by streptozotocin (STZ), and it was divided into four groups: the diabetic control group, diabetic rats treated with corosolic acid, diabetic rats treated with GW9662, and diabetic rats treated with corosolic acid plus GW9662. The study was carried out for 28 days. The diabetic control and ISO control groups showed a decrease in mean arterial pressure (MAP) and diastolic arterial pressure (DAP) and an increase in systolic arterial pressure (SAP). The rat myocardium was activated by corosolic acid treatment, which elevated PPAR-γ expression. A histopathological analysis showed a significant reduction in myocardial damage by reducing myonecrosis and edema. It was found that myocardial levels of CK-MB and LDH levels were significantly increased after treatment with corosolic acid. By decreasing lipid peroxidation and increasing endogenous antioxidant levels, corosolic acid therapy showed a significant improvement over the ISO diabetic group. In conclusion, our results prove that corosolic acid can ameliorate ISO-induced acute myocardial injury in rats. Based on these results, corosolic acid seems to be a viable new target for the treatment of cardiovascular diseases and other diseases of a similar nature.

Nephropathy 1st inhibits renal fibrosis by activating the PPARγ signaling pathway.

Renal fibrosis is a manifestation of kidney injury. Nephropathy 1st is a traditional Chinese herbal medicine that has been used as a therapy for kidney disease, but the underlying mechanisms remain elusive. The aim of this study was to investigate the role and underlying mechanisms of Nephropathy 1st on the progression of kidney disease. In the present study, unilateral ureteral obstruction was performed to establish the renal fibrosis rat model. By hematoxylin-eosin staining and immunohistochemical staining analysis, the severity of renal fibrosis was evaluated in vivo. Serum creatinine (CREA) and urea nitrogen (BUN) were measured by ELISA. The expression levels of Col-I, FN, PPARγ, and Klotho were measured by Western blot in rat NRK-49F cells and in fibrotic rats. GW9662 was used to inhibit PPARγ signaling. Metabonomic analysis showed metabolic differences among groups. Nephropathy 1st administration alleviated the progression of rat renal fibrosis and reduced serum creatinine (Scr) and BUN levels. Mechanistically, Nephropathy 1st promoted the expression of PPARγ and thus activated PPARγ signaling, thereby reducing the pro-fibrotic phenotypes of fibroblasts. The therapeutic effect of Nephropathy 1st was abrogated by the PPARγ inhibitor GW9662. Moreover, Nephropathy 1st normalized the dysregulated lipid metabolism in renal fibrosis rats. In conclusion, Nephropathy 1st alleviates renal fibrosis development in a PPARγ-dependent manner.

Effects of Intra-BLA Administration of PPAR Antagonists on Formalin-Evoked Nociceptive Behaviour, Fear-Conditioned Analgesia, and Conditioned Fear in the Presence or Absence of Nociceptive Tone in Rats.

There is evidence for the involvement of peroxisome proliferator-activated receptors (PPARs) in pain, cognition, and anxiety. However, their role in pain-fear interactions is unknown. The amygdala plays a key role in pain, conditioned fear, and fear-conditioned analgesia (FCA). We investigated the effects of intra-basolateral amygdala (BLA) administration of PPARα, PPARß/δ, and PPARγ antagonists on nociceptive behaviour, FCA, and conditioned fear in the presence or absence of nociceptive tone. Male Sprague-Dawley (SD) rats received footshock (FC) or no footshock (NFC) in a conditioning arena. Twenty-three and a half hours later, rats received an intraplantar injection of formalin or saline and, 15 min later, intra-BLA microinjections of vehicle, PPARα (GW6471) PPARß/δ (GSK0660), or PPARγ (GW9662) antagonists before arena re-exposure. Pain and fear-related behaviour were assessed, and neurotransmitters/endocannabinoids measured post-mortem. Intra-BLA administration of PPARα or PPARγ antagonists potentiated freezing in the presence of nociceptive tone. Blockade of all PPAR subtypes in the BLA increased freezing and BLA dopamine levels in NFC rats in the absence of nociceptive tone. Administration of intra-BLA PPARα and PPARγ antagonists increased levels of dopamine in the BLA compared with the vehicle-treated counterparts. In conclusion, PPARα and PPARγ in the BLA play a role in the expression or extinction of conditioned fear in the presence or absence of nociceptive tone.

Antiapoptotic and anti-inflammatory effects of Pparγ agonist, pioglitazone, reversed Dox-induced cardiotoxicity through mediating of miR-130a downregulation in C57BL/6 mice.

Doxorubicin (Dox) is an antitumor agent widely used in cancer therapy, with notable side effects of cardiac toxicity. Peroxisome proliferator-activated receptor γ (PPARγ), is a transcriptional factor with antiapoptotic and anti-inflammatory properties. Recently we indicated that cardiac toxicity of Dox was due to upregulation of miR-130a and further suppressive effect on cardiac Pparγ in vitro. In this study, we extended our proposed hypothesis in vivo. To achieve this, pioglitazone (Pio) and GW9662 were used as the specific agonist and antagonist of Pparγ to treat Dox-injected mice. Heart function, apoptosis, and inflammation in heart tissue were studied. Pretreatment of Dox-injected mice with Pio resulted in elevated expression of Pparγ and suppression of miR-130a. However, GW9662 pretreatment was unable to increase miR-130a expression. Pio pretreatment led to partially cardiac toxicity limitation of Dox whereas GW9662 caused heart damage. Finally, our observation determined that activation of Pparγ was not adequate to reverse the Dox-induced toxicity completely.

Utilizing systems biology to reveal cellular responses to peroxisome proliferator-activated receptor γ ligand exposure.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that, upon activation by ligands, heterodimerizes with retinoid X receptor (RXR), binds to PPAR response elements (PPREs), and activates transcription of downstream genes. As PPARγ plays a central role in adipogenesis, fatty acid storage, and glucose metabolism, PPARγ-specific pharmaceuticals (e.g., thiazolidinediones) have been developed to treat Type II diabetes and obesity within human populations. However, to our knowledge, no prior studies have concurrently assessed the effects of PPARγ ligand exposure on genome-wide PPARγ binding as well as effects on the transcriptome and lipidome within human cells at biologically active, non-cytotoxic concentrations. In addition to quantifying concentration-dependent effects of ciglitazone (a reference PPARγ agonist) and GW 9662 (a reference PPARγ antagonist) on human hepatocarcinoma (HepG2) cell viability, PPARγ abundance in situ, and neutral lipids, HepG2 cells were exposed to either vehicle (0.1% DMSO), ciglitazone, or GW 9662 for up to 24 h, and then harvested for 1) chromatin immunoprecipitation-sequencing (ChIP-seq) to identify PPARγ-bound regions across the entire genome, 2) mRNA-sequencing (mRNA-seq) to identify potential impacts on the transcriptome, and 3) lipidomics to identify potential alterations in lipid profiles. Following exposure to ciglitazone and GW 9662, we found that PPARγ levels were not significantly different after 2-8 h of exposure. While ciglitazone and GW 9662 resulted in a concentration-dependent increase in neutral lipids, the magnitude and localization of PPARγ-bound regions across the genome (as identified by ChIP-seq) did not vary by treatment. However, mRNA-seq and lipidomics revealed that exposure of HepG2 cells to ciglitazone and GW 9662 resulted in significant, treatment-specific effects on the transcriptome and lipidome. Overall, our findings suggest that exposure of human cells to PPARγ ligands at biologically active, non-cytotoxic concentrations results in toxicity that may be driven by a combination of both PPARγ-dependent and PPARγ-independent mechanisms.

Preclinical Investigation of Alpinetin in the Treatment of Cancer-Induced Cachexia via Activating PPARγ.

The ongoing loss of skeletal muscle is a central event of cancer cachexia, and its consequences include adverse effects on patient's quality of life and survival. Alpinetin (Alp), a natural plant-derived flavonoid obtained from Alpinia katsumadai Hayata, has been reported to possess potent anti-inflammatory and antitumor activities. This study aimed to explore the therapeutic effect and underlying mechanism of Alp in the prevention of cancer cachexia. We found that Alp (25-100 µM) dose-dependently attenuated Lewis lung carcinoma-conditioned medium-induced C2C12 myotube atrophy and reduced expression of the E3 ligases Atrogin-1 and MuRF1. Moreover, Alp administration markedly improved vital features of cancer cachexia in vivo with visible reduction of the loss of tumor-free body weight and wasting of multiple tissues, including skeletal muscle, epididymal fat, and decreased expression of Atrogin-1 and MuRF1 in cachectic muscle. Alp suppressed the elevated spleen weight and serum concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6. Further, Alp treatment remained protective against cancer cachexia in the advanced stage of tumor growth. Molecular docking results suggested that Alp was docked into the active site of PPARγ with the docking score of -7.6 kcal/mol, forming a hydrogen bond interaction with PPARγ protein amino acid residue HIS449 with a bond length of 3.3 Å. Mechanism analysis revealed that Alp activated PPARγ, resulting in the downregulated phosphorylation of NF-κB and STAT3 in vitro and in vivo. PPARγ inhibition induced by GW9662 notably attenuated the improvement of Alp on the above cachexia phenomenon, indicating that PPARγ activation mediated the therapeutic effect of Alp. These findings suggested that Alp might be a potential therapeutic candidate against cancer cachexia.

Medioresinol as a novel PGC-1α activator prevents pyroptosis of endothelial cells in ischemic stroke through PPARα-GOT1 axis.

AIM: Brain microvascular endothelial cells (BMVECs), as the important structure of blood-brain barrier (BBB), play a vital role in ischemic stroke. Pyroptosis of different cells in the brain may aggravate cerebral ischemic injury, and PGC-1α plays a major role in pyroptosis. However, it is not known whether BMVECs undergo pyroptosis after ischemic stroke and whether PGC-1α activator Medioresinol (MDN) we discovered may be useful against pyroptosis of endothelial cells and ischemic brain injury. METHODS: For in vitro experiments, the bEnd.3 cells and BMVECs under oxygen and glucose-deprivation (OGD) were treated with or without MDN, and the LDH release, tight junction protein degradation, GSDMD-NT membrane location and pyroptosis-associated proteins were evaluated. For in vivo experiments, mice underwent transient middle cerebral artery occlusion (tMCAO) for ischemia model, and the neuroprotective effects of MDN were measured by infarct volume, the permeability of BBB and pyroptosis of BMVECs. For mechanistic study, effects of MDN on the accumulation of phenylalanine, mitochondrial reactive oxygen species (mtROS) were tested by untargeted metabolomics and MitoSOX Red probe, respectively. RESULTS: BMVECs underwent pyroptosis after ischemia. MDN dose-dependently activated PGC-1α, significantly reduced pyroptosis, mtROS and the expressions of pyroptosis-associated proteins (NLRP3, ASC, cleaved caspase-1, IL-1ß, GSDMD-NT), and increased ZO-1 and Occludin protein expressions in BMVECs. In tMCAO mice, MDN remarkably reduced brain infarct volume and the permeability of BBB, inhibited pyroptosis of BMVECs, and promoted long-term neurobehavioral functional recovery. Mechanistically, MDN promoted the interaction of PGC-1α with PPARα to increase PPARα nuclear translocation and transcription activity, further increased the expression of GOT1 and PAH, resulting in enhanced phenylalanine metabolism to reduce the ischemia-caused phenylalanine accumulation and mtROS and further ameliorate pyroptosis of BMVECs. CONCLUSION: In this study, we for the first time discovered that pyroptosis of BMVECs was involved in the pathogenesis of ischemic stroke and MDN as a novel PGC-1α activator could ameliorate the pyroptosis of endothelial cells and ischemic brain injury, which might attribute to reduction of mtROS through PPARα/GOT1 axis in BMVECs. Taken together, targeting endothelial pyroptosis by MDN may provide alternative therapeutics for brain ischemic stroke.

Bupleurum falcatum L. alleviates nociceptive and neuropathic pain: Potential mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: In Iranian folkloric medicine, Bupleurum falcatum L. (Chinese Thoroughwax) has been used as a selective analgesic remedy for several centuries. OBJECTIVE: The current research was conducted to explore the anti-nociceptive and anti-allodynic action of Bupleurum falcatum L. roots essential oil (BFEO) in Swiss mice. MATERIALS AND METHODS: Formalin-induced paw licking (FIPL) model was applied for exploring of BFEO antinociceptive effects (neurogenic or inflammatory pain). The involvements of L-arginine-NO-cGMP-KATP channel pathway and several receptors such as opioid, peroxisome proliferator-activated (PPA), cannabinoid, transient receptor potential vanilloid, and adrenergic receptors were assesses to detect the anti-nociceptive activity of BFEO. Cervical spinal cord contusion (CSC) paradigm was employed for induction of neuropathic pain. RESULTS: BFEO (100 mg/kg), in the FIPL model, produced significant antinociception compared to the control mice (p < 0.01). Furthermore, L-arginine, methylene blue, glibenclamide, naloxonazine, GW9662, and SR141716A pre-treatments restored the BFEO anti-nociceptive effects (p < 0.05) in the FIPL (second phase) test (p < 0.05). Intraperitoneal administration of saikosaponin A (one of the main constituents of BFEO) partially alleviated (p < 0.05) pain in FIPL test. Likewise, in CSC mice, the von Frey assay exhibited that BFEO could alter mechanical allodynia. CONCLUSION: Finally, it seems that, in male mice, BFEO has both anti-allodynic and anti-nociceptive effects. The present data also suggest activating the L-arginine-NO-cGMP-KATP channel pathway as well as interaction of opioid, PPA, and cannabinoid receptors in the BFEO anti-nociceptive activities. These results also propose that BFEO could effectively attenuate allodynia in CSC mice.

Cannabidiol selectively inhibits the contraction of rat small resistance arteries: Possible role for CGRP and voltage-gated calcium channels.

The pharmacology of cannabidiol, the non-psychoactive major component of Cannabis sativa, is of growing interest as it becomes more widely prescribed. This study aimed to examine the effects of cannabidiol on a wide range of contractile agents in rat small resistance arteries, in comparison with large arteries, and to explore its mechanism of action. The vascular actions of cannabidiol were also contrasted with effects on the contractions of bronchial, urogenital, cardiac and skeletal muscles. Isolated small or large arteries were incubated with cannabidiol (0.3-3 µM) or vehicle and concentration-contraction response curves were completed to various agents, including endothelin-1, arginine vasopressin, methoxamine, 5-HT, α-methyl 5-HT and U46619. In small arteries, the effects of cannabidiol were tested in the presence of antagonists of CB1 or CB2 receptors, calcitonin gene-related peptide (CGRP), nitric oxide synthase, cyclooxygenase, PPARγ or a combination. The role of L-type voltage-operated calcium channels was also assessed. Cannabidiol 1-3 µM significantly inhibited the contraction of small resistance arteries to all tested agents through a combination of mechanisms that include CGRP and L-type calcium channels. However, large arteries were insensitive to cannabidiol. Cannabidiol (10-100 µM) was largely without effect in bronchi, atria and hemidiaphragm, but 100 µM attenuated maximum contractions in vasa deferentia. Cannabidiol's effects in the clinical range (1-3 µM) appear to be specific to small resistance arteries. This high sensitivity of the resistance arterial circulation to cannabidiol may offer a therapeutic opportunity in peripheral vascular disease that excludes off-target sites such as the heart and non-vascular smooth muscle.

Effect of rosiglitazone on developmental competence of mouse embryos treated with lipopolysaccharide.

Lipopolysaccharide (LPS) significantly reduces pre- and post-implantation developmental competence of embryos. One of the reason of this effect could be a consequence of TLR4-mediated inflammation. In this study, we assessed the anti-inflammatory effect of peroxisome proliferator activated receptor γ (PPAR γ) agonist, rosiglitazone (RGZ), in LPS-treated mouse embryos. Initially, the optimal doses of LPS, RGZ and GW9662 (a potent and selective PPARγ antagonist) were determined by treating the mouse zygotes up to blastocyst stage and assessment of compaction and blastocyst rates. Quantitative PCR was used to assess the mRNA expression of inflammatory cytokines. Immunostaining was used to study the translocation of PPARγ in blastocysts. Finally, the blastocysts were transferred to surrogate mouse to determine the post-implantation developmental competence. 0.0625 mg/mL of LPS significantly reduced the developmental competency by around 50% compared to control group. 10 µM of RGZ significantly ameliorated the toxic effect of LPS, which was also significantly reversed by 1.25 µM GW9662. Through immunostaining, it was shown that LPS could prevent the translocation of PPARγ to nucleus; and translocation was facilitated by RGZ and this effect was reversed by GW9662. A similar effect was also observed for the mRNA expression of inflammatory cytokines (Il-1ß and Il-6). LPS significantly increased the expression of these cytokines, while RGZ significantly reduced their expression, which was also significantly reversed by GW9662. It was also shown that embryos exposed to LPS had significantly reduced post implantation developmental competence which was considerably improved by treatment with RGZ. In conclusion, these data may have clinical implications for ameliorating the adverse effects of LPS in dairy farming and infertility treatment.

Comparison of PPAR Ligands as Modulators of Resolution of Inflammation, via Their Influence on Cytokines and Oxylipins Release in Astrocytes.

Neuroinflammation is a key process of many neurodegenerative diseases and other brain disturbances, and astrocytes play an essential role in neuroinflammation. Therefore, the regulation of astrocyte responses for inflammatory stimuli, using small molecules, is a potential therapeutic strategy. We investigated the potency of peroxisome proliferator-activated receptor (PPAR) ligands to modulate the stimulating effect of lipopolysaccharide (LPS) in the primary rat astrocytes on (1) polyunsaturated fatty acid (PUFAs) derivative (oxylipins) synthesis; (2) cytokines TNFα and interleukin-10 (IL-10) release; (3) p38, JNK, ERK mitogen-activated protein kinase (MAPKs) phosphorylation. Astrocytes were exposed to LPS alone or in combination with the PPAR ligands: PPARα (fenofibrate, GW6471); PPARß (GW501516, GSK0660); PPARγ (rosiglitazone, GW9662). We detected 28 oxylipins with mass spectrometry (UPLC-MS/MS), classified according to their metabolic pathways: cyclooxygenase (COX), cytochrome P450 monooxygenases (CYP), lipoxygenase (LOX) and PUFAs: arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA). All tested PPAR ligands decrease COX-derived oxylipins; both PPARß ligands possessed the strongest effect. The PPARß agonist, GW501516 is a strong inducer of pro-resolution substances, derivatives of DHA: 4-HDoHE, 11-HDoHE, 17-HDoHE. All tested PPAR ligands decreased the release of the proinflammatory cytokine, TNFα. The PPARß agonist GW501516 and the PPARγ agonist, rosiglitazone induced the IL-10 release of the anti-inflammatory cytokine, IL-10; the cytokine index, (IL-10/TNFα) was more for GW501516. The PPARß ligands, GW501516 and GSK0660, are also the strongest inhibitors of LPS-induced phosphorylation of p38, JNK, ERK MAPKs. Overall, our data revealed that the PPARß ligands are a potential pro-resolution and anti-inflammatory drug for targeting glia-mediated neuroinflammation.

Enhancing PPARγ by HDAC inhibition reduces foam cell formation and atherosclerosis in ApoE deficient mice.

Atherosclerosis (AS) is a risky cardiovascular disease with limited treatment options. Various pan or type-selective histone deacetylase (HDAC) inhibitors are reportedly atheroprotective against atherosclerosis (AS); however, the key effectors and the main cellular processes that mediate the protective effects remain poorly defined. Here, we report that PPARγ (Peroxisome proliferator-activated receptor gamma), a transcription factor actively involved in lipid metabolism with strong tissue protective and anti-inflammation properties, is a critical mediator of the anti-AS effects by HDAC inhibition. We showed that a well-known pan-HDAC inhibitor TSA (Trichostatin A) reduced foam cell formation of macrophages that is accompanied by a marked elevation of PPARγ and its downstream cholesterol efflux transporter ABCA1 (ATP-binding membrane cassette transport protein A1) and ABCG1. In an AS model of ApoE-/- mice fed on high-fat diet, TSA treatment alleviated AS lesions, similarly increased PPARγ and the downstream cholesterol transporters and mitigated the induction of inflammatory cytokine TNFα and IL-1ß. Exploring the potential cause of PPARγ elevation revealed that TSA induced the acetylation of C/EBPα (CCAAT enhancer binding protein alpha), the upstream regulator of PPARγ, through which it increased PPARγ transactivation. More importantly, we generated a strain of PPARγ/ApoE double knockout mice and demonstrated that lack of PPARγ abrogated the protective effects of TSA on foam cell formation of peritoneal macrophages and the AS pathogenesis. Taken together, these results unravel that C/EBPα and PPARγ are the HDAC-sensitive components of an epigenetic signaling pathway mediating foam cell formation and AS development, and suggest that targeting C/EBPα/PPARγ axis by HDAC inhibitors possesses therapeutic potentials in retarding the progression of AS and the related cardiovascular diseases.

Chrysin rescues rat myocardium from ischemia-reperfusion injury via PPAR-γ/Nrf2 activation.

Pharmacological strategies aimed at co-activating peroxisome proliferator-activated receptor-gamma (PPAR-γ)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway have shown promising results in alleviating myocardial injury. The aim of the study was to evaluate the role of chrysin, a PPAR-γ agonist, in ischemia-reperfusion (IR)-induced myocardial infarction (MI) in rats and to explore the molecular mechanism driving this activity. To evaluate this hypothesis, chrysin (60 mg/kg, orally), PPAR-γ antagonist (GW9662, 1 mg/kg, intraperitoneally), or both were administered to rats for 28 days. On the 29th day, one-stage ligation of left anterior descending coronary artery for 45 min followed by 60 min of reperfusion was performed. Chrysin significantly decreased infarct size and improved cardiac functions following IR-induced MI. This improvement was corroborated by augmented PPAR-γ/Nrf2 expression as confirmed by immunohistochemistry and western blotting analysis. Chrysin exhibited strong anti-oxidant property as demonstrated by increased GSH and CAT levels and decreased 8-OHdG and TBARS levels. Our findings also imply that chrysin significantly inhibited inflammatory response as validated by decreased NF-κB, IKK-ß, CRP, TNF-α and MPO levels. In addition, chrysin decreased TUNEL/DAPI positivity, a marker of apoptotic response and normalized cardiac injury markers. The histopathological and ultrastructural analysis further supported the functional and biochemical outcomes, showing preserved myocardial architecture. Intriguingly, co-administration with GW9662 significantly diminished the cardioprotective effect of chrysin as demonstrated by depressed myocardial function, decreased PPAR-γ/Nrf2 expression and increased oxidative stress. In conclusion, the present study demonstrates that co-activation of PPAR-γ/Nrf2 by chrysin may be crucial for its cardioprotective effect.

Therapeutic potential of pharmacological agents targeting TRP channels in CNS disorders.

Central nervous system (CNS) disorders like Alzheimer's disease (AD), Parkinson disease (PD), stroke, epilepsy, depression, and bipolar disorder have a high impact on both medical and social problems due to the surge in their prevalence. All of these neuronal disorders share some common etiologies including disruption of Ca2+ homeostasis and accumulation of misfolded proteins. These misfolded proteins further disrupt the intracellular Ca2+ homeostasis by disrupting the activity of several ion channels including transient receptor potential (TRP) channels. TRP channel families include non-selective Ca2+ permeable channels, which act as cellular sensors activated by various physio-chemical stimuli, exogenous, and endogenous ligands responsible for maintaining the intracellular Ca2+ homeostasis. TRP channels are abundantly expressed in the neuronal cells and disturbance in their activity leads to various neuronal diseases. Under the pathological conditions when the activity of TRP channels is perturbed, there is a disruption of the neuronal homeostasis through increased inflammatory response, generation of reactive oxygen species, and mitochondrial dysfunction. Therefore, there is a potential of pharmacological interventions targeting TRP channels in CNS disorders. This review focuses on the role of TRP channels in neurological diseases; also, we have highlighted the current insights into the pharmacological modulators targeting TRP channels.