Inhibition of CK2 Diminishes Fibrotic Scar Formation and Improves Outcomes After Ischemic Stroke via Reducing BRD4 Phosphorylation.

Fibrotic scars play important roles in tissue reconstruction and functional recovery in the late stage of nervous system injury. However, the mechanisms underlying fibrotic scar formation and regulation remain unclear. Casein kinase II (CK2) is a protein kinase that regulates a variety of cellular functions through the phosphorylation of proteins, including bromodomain-containing protein 4 (BRD4). CK2 and BRD4 participate in fibrosis formation in a variety of tissues. However, whether CK2 affects fibrotic scar formation remains unclear, as do the mechanisms of signal regulation after cerebral ischemic injury. In this study, we assessed whether CK2 could modulate fibrotic scar formation after cerebral ischemic injury through BRD4. Primary meningeal fibroblasts were isolated from neonatal rats and treated with transforming growth factor-ß1 (TGF-ß1), SB431542 (a TGF-ß1 receptor kinase inhibitor) or TBB (a highly potent CK2 inhibitor). Adult SD rats were intraperitoneally injected with TBB to inhibit CK2 after MCAO/R. We found that CK2 expression was increased in vitro in the TGF-ß1-induced fibrosis model and in vivo in the MCAO/R injury model. The TGF-ß1 receptor kinase inhibitor SB431542 decreased CK2 expression in fibroblasts. The CK2 inhibitor TBB reduced the increases in proliferation, migration and activation of fibroblasts caused by TGF-ß1 in vitro, and it inhibited fibrotic scar formation, ameliorated histopathological damage, protected Nissl bodies, decreased infarct volume and alleviated neurological deficits after MCAO/R injury in vivo. Furthermore, CK2 inhibition decreased BRD4 phosphorylation both in vitro and in vivo. The findings of the present study suggested that CK2 may control BRD4 phosphorylation to regulate fibrotic scar formation, to affecting outcomes after ischemic stroke.

M2a macrophages regulate fibrosis and affect the outcome after stroke via PU.1/mTOR pathway in fibroblasts.

The moderate formation of the fibrotic scar plays an important role in functional recovery after stroke. M2a macrophages have been identified as an important source of early fibrosis after cerebral ischemia. However, the underlying mechanisms by which macrophages interact with fibroblasts in this context remain largely unknown. Therefore, our study aimed to further investigate the potential mechanisms underlying the effects of macrophages on fibroblasts following ischemic stroke. In vitro and in vivo, recombinant rat interleukin 4 (IL4) was used to induce macrophages to polarize into M2a macrophages. In vitro, primary Sprague-Dawley newborn rat meningeal-derived fibroblasts were treated with PU.1 knockdown, the PU.1 inhibitor DB1976 or the mTOR inhibitor rapamycin, which were then co-cultured with M2a macrophage conditioned medium (MCM). In vivo, Sprague-Dawley adult rats were infected with negative control adenoviruses or PU.1-shRNA adenoviruses. Ten days after infection, an injury model of middle cerebral artery occlusion/reperfusion (MCAO/R) was constructed. Subsequently, IL4 was injected intracerebroventricularly to induce M2a macrophages polarization. In vitro, M2a MCM upregulated PU.1 expression and promoted the differentiation, proliferation, migration and extracellular matrix generation of fibroblasts, which could be reversed by treatment with the PU.1 inhibitor DB1976 or PU.1 knockdown. In vivo, PU.1 expression in fibroblasts was increased within ischemic core following MCAO/R, and this upregulation was further enhanced by exposure to IL4. Treatment with IL4 promoted fibrosis, increased angiogenesis, reduced apoptosis and infarct volume, as well as mitigated neurological deficits after MCAO/R, and these effects could be reversed by PU.1 knockdown. Furthermore, both in vivo and in vitro studies showed that IL4 treatment increased the levels of phosphorylated Akt and mTOR proteins, which were markedly decreased by PU.1 knockdown. Additionally, the use of an mTOR inhibitor rapamycin obviously suppressed the migration and differentiation of fibroblasts, and Col1 synthesis. In conclusion, our findings suggest for the first time that M2a macrophages, at least in part, regulate fibrosis and affect the outcome after cerebral ischemic stroke via the PU.1/mTOR signaling pathway in fibroblasts.

Sirt1 Overexpression Inhibits Fibrous Scar Formation and Improves Functional Recovery After Cerebral Ischemic Injury Through the Deacetylation of 14-3-3ζ.

Cerebral ischemic stroke is one of the leading causes of human death. The fibrous scar is one of major factors influencing repair in central nervous system (CNS) injury. Silencing information regulator 2-related enzyme 1 (Sirt1) can regulate peripheral tissue and organ fibrosis. However, it is unclear how the fibrous scar forms and is regulated and it is unknown whether and how Sirt1 regulates the formation of the fibrous scar after cerebral ischemic stroke. Therefore, in the present study, we examined the effects of Sirt1 on the formation of the fibrotic scar after middle cerebral artery occlusion/reperfusion (MCAO/R) injury in vivo and on the transforming growth factor ß1 (TGF-ß1)-induced meningeal fibroblast fibrotic response in vitro, and we explored the molecular mechanisms underlying the Sirt1-regulated fibrosis process in vitro. We found that MCAO/R injury induced fibrotic scar formation in the ischemic area, which was accompanied by the downregulation of Sirt1 expression. The overexpression of Sirt1 reduced the infarct volume, improved Nissl body structure and reduced neurons injury, attenuated formation of fibrotic scar, upregulated growth associated protein43 (GAP43) and synaptophysin (SYP) expression, and promoted neurological function recovery. Similarly, Sirt1 expression was also downregulated in the TGF-ß1-induced fibrosis model. Sirt1 overexpression inhibited fibroblast migration, proliferation, transdifferentiation into myofibroblasts, and secretion of extracellular matrix(ECM) by regulating the deacetylation of lysine at K49 and K120 sites of 14-3-3ζ in vitro. Therefore, we believe that Sirt1 could regulate fibrous scar formation and improve neurological function after cerebral ischemic stroke through regulating deacetylation of 14-3-3ζ.

M2 macrophages mediate fibrotic scar formation in the early stages after cerebral ischemia in rats.

In the central nervous system, the formation of fibrotic scar after injury inhibits axon regeneration and promotes repair. However, the mechanism underlying fibrotic scar formation and regulation remains poorly understood. M2 macrophages regulate fibrotic scar formation after injury to the heart, lung, kidney, and central nervous system. However, it remains to be clarified whether and how M2 macrophages regulate fibrotic scar formation after cerebral ischemia injury. In this study, we found that, in a rat model of cerebral ischemia induced by middle cerebral artery occlusion/reperfusion, fibrosis and macrophage infiltration were apparent in the ischemic core in the early stage of injury (within 14 days of injury). The number of infiltrated macrophages was positively correlated with fibronectin expression. Depletion of circulating monocyte-derived macrophages attenuated fibrotic scar formation. Interleukin 4 (IL4) expression was strongly enhanced in the ischemic cerebral tissues, and IL4-induced M2 macrophage polarization promoted fibrotic scar formation in the ischemic core. In addition, macrophage-conditioned medium directly promoted fibroblast proliferation and the production of extracellular matrix proteins in vitro. Further pharmacological and genetic analyses showed that sonic hedgehog secreted by M2 macrophages promoted fibrogenesis in vitro and in vivo, and that this process was mediated by secretion of the key fibrosis-associated regulatory proteins transforming growth factor beta 1 and matrix metalloproteinase 9. Furthermore, IL4-afforded functional restoration on angiogenesis, cell apoptosis, and infarct volume in the ischemic core of cerebral ischemia rats were markedly impaired by treatment with an sonic hedgehog signaling inhibitor, paralleling the extent of fibrosis. Taken together, our findings show that IL4/sonic hedgehog/transforming growth factor beta 1 signaling targeting macrophages regulates the formation of fibrotic scar and is a potential therapeutic target for ischemic stroke.

Inhibition of BRD4 decreases fibrous scarring after ischemic stroke in rats by inhibiting the phosphorylation of Smad2/3.

AIMS: Fibrous scarring may play a much more important role in preventing secondary expansion of tissue damage and hindering repair and regeneration than glial scarring after central nervous system (CNS) injury. However, relatively little is known about how fibrous scars form and how fibrous scar formation is regulated after CNS injury. Bromodomain-containing protein 4 (BRD4) is involved in fibrosis in many tissues, and transforming growth factor-ß1 (TGF-ß1)/Smad2/3 signaling is one of the critical pathways of fibrosis. However, it is unclear whether and how BRD4 affects fibrous scar formation after ischemicbraininjury. In the present study, whether BRD4 can regulate the formation of fibrous scars after ischemic stroke via TGF-ß1/Smad2/3 signaling was assessed. MATERIALS AND METHODS: Primary meningeal fibroblasts isolated from neonatal SD rats were treated with TGF-ß1, SB431542 (a TGF-ß1 receptor inhibitor) and JQ1 (a small-molecule BET inhibitor that can also inhibit BRD4). BRD4 was knocked down in adult Sprague-Dawley (SD) rats by using adenovirus before middle cerebral artery occlusion/reperfusion (MCAO/R) injury. The proliferation and migration of meningeal fibroblasts in vitro were evaluated with the Cell Counting Kit-8 (CCK-8) assay and scratch test, respectively. Neurological function was assessed with Longa scores, modified Bederson Scores and modified neurological severity scores (mNSSs). The infarct volume was assessed with TTC staining. The protein expression of synaptophysin (SY), BRD4, Smad2/3, p-Smad2/3, α-smooth muscle actin (α-SMA), collagen-1 (COL1) and fibronectin (FN) in vivo and in vitro was examined with immunocytochemistry, immunofluorescence, and Western blotting. KEY FINDINGS: BRD4 expression was upregulated in a TGF-ß1-induced meningeal fibroblast fibrosis model and was downregulated by the TGF-ß1 receptor inhibitor SB431542 in vitro. JQ1, a small-molecule BET inhibitor, inhibited BRD4 and decreased TGF-ß1-induced meningeal fibroblast proliferation, migration and activation. Furthermore, MCAO/R injury induced fibrosis and upregulated BRD4 expression in the cerebral infarct center. BRD4 knockdown by adenovirus inhibited fibrous scarring, promoted synaptic survival, decreased the infarct volume, and improved neurological function after MCAO/R injury. Moreover, inhibition of BRD4, either by JQ1 in vitro or adenovirus in vivo, decreased the phosphorylation of Smad2/3. CONCLUSIONS: This study is the first to indicate that inhibition of BRD4 delays fibrous scarring after ischemic stroke through mechanisms involving the phosphorylation of Smad2/3.

Potential mechanisms and therapeutic targets of mesenchymal stem cell transplantation for ischemic stroke.

Ischemic stroke is one of the major causes of death and disability in the world. Currently, most patients cannot choose intravenous thrombolysis or intravascular mechanical thrombectomy because of narrow therapeutic windows and severe complications. Stem cell transplantation is an emerging treatment and has been studied in various central nervous system diseases. Animal and clinical studies showed that transplantation of mesenchymal stem cells (MSCs) could alleviate neurological deficits and bring hope for ischemic stroke treatment. This article reviewed biological characteristics, safety, feasibility and efficacy of MSCs therapy, potential therapeutic targets of MSCs, and production process of Good Manufacturing Practices-grade MSCs, to explore the potential therapeutic targets of MSCs in the process of production and use and provide new therapeutic directions for ischemic stroke.

Resveratrol pretreatment protects neurons from oxygen-glucose deprivation/reoxygenation and ischemic injury through inhibiting ferroptosis.

Ferroptosis, a newly discovered iron-dependent cell death, is involved in brain ischemia-reperfusion injury. Iron scavengers or ferroptosis inhibitors could reduce infarct volume and improve neurological function in mice. Resveratrol has neuroprotective and neurorestorative effects. However, it is unclear whether resveratrol can play a neuroprotective role via inhibiting ferroptosis. Our study showed that resveratrol pretreatment had a similar effect with ferrostatin­1, which inhibited neuronal ferroptosis-related changes, such as iron overload, damages of oxidation-reduction system, and destruction of mitochondrial structure, after oxygen-glucose deprivation/reoxygenation (OGD/R) and application of ferroptosis inducers. In addition, middle cerebral artery occlusion/reperfusion (MCAO/R) injury in vivo also induced ferroptosis, and resveratrol pretreatment could inhibit ferroptosis and reduce degenerative neurons, cerebral ischemic damage and infarction volume. Our results are the first to indicate that resveratrol pretreatment might inhibit ferroptosis induced by OGD/R and ferroptosis inducers in neurons, and MCAO/R in rats.

Effect of Yin-Zhi-Huang on up-regulation of Oatp2, Ntcp, and Mrp2 proteins in estrogen-induced rat cholestasis.

CONTEXT: Yin-Zhi-Huang (YZH), a prescription of traditional Chinese medicine, is widely used to treat neonatal jaundice or cholestasis. OBJECTIVE: This study investigates the regulatory effect of YZH on the localization and expression of organic anion transporting polypeptides 2 (Oatp2), Na(+)-taurocholate co-transporting polypeptide (Ntcp), multidrug-resistance-associated protein 2 (Mrp2), and bile salt export pump (Bsep) in estrogen-induced cholestasis rats. MATERIAL AND METHODS: Cholestasis model rats were induced via subcutaneous injection of estradiol benzoate (EB, 5 mg/kg/d) for 5 d. Other EB-induced rats were treated with saline (2 ml) or YZH (1.5 g/kg, two times a day) for 7, 14, and 21 d. The biochemical and pathologic examinations were performed. Moreover, the localization and expression of Oatp2, Ntcp, Mrp2, and Bsep were determined by immunohistochemisty and Western blotting, respectively. RESULTS: YZH treatment could significantly decrease the serum total bile acids (TBA) (4.9 ± 0.6-2.8 ± 0.8) and direct bilirubin (DBIL) (2.6 ± 0.7-1.0 ± 0.1) levels, improve the histological disorganization, and, respectively, increase the expression of Oatp2 and Ntcp by 46% and 28% compared with saline-treated (p < 0.05) rats at 14 d. The expression of Mrp2 increased by 45% was observed in YZH treated compared with saline-treated (p < 0.05) rats at 7 d. However, there was a little change in the expression of Bsep (p > 0.05) after YZH treatment for 7, 14, and 21 d. DISCUSSION AND CONCLUSION: In conclusion, the therapeutic effect of YZH to cholestasis could be attributed to the regulation of Oatp2, Ntcp, Mrp2, and Bsep.

Role of age-related decrease of renal organic cation transporter 2 in the effect of atenolol on renal excretion of metformin in rats.

Many diabetes patients, especially the elder ones, suffered from hypertension simultaneously. Therefore, it is very likely that a large number of diabetes patients receiving metformin hydrochloride may simultaneously be given beta-blockers. Knowing that both metformin and atenolol are eliminated by organic cation transporter 2 (OCT2/SLC22A2) expressed in the renal basolateral membrane, it is not clear whether there is a competitive effect on the renal excretion of metformin and/or atenolol when metformin and atenolol were co-administered, and whether age was involved in this drug-drug interaction. In this present study, both young rats (aged 3 months) and aged rats (aged 12 months) were used, rats were divided into metformin-treated group and metformin and atenolol co-administrated group, respectively. Either metformin (2.5 mg/kg) alone or metformin (2.5 mg/kg) in combination with atenolol (8 mg/kg) was administered to rats by tail vein injection. Then, urine was collected and the metformin concentration in urine was determined by HPLC. The localization and expression of rOCT2 in kidney were also investigated by Western blotting and immunohistochemistry. Significant differences of t 1/2, K e, CLtot and the accumulated metformin excretion in urine were founded in aged rats, but not in young rats, between metformin-treated group (2.002 ± 0.51 h, 0.346 ± 0.07/h, 57.161 ± 18.59 %, 4,287.087 ± 458.08 µg) and metformin plus atenolol-treated group (3.03 ± 0.67 h, 0.228 ± 0.05/h, 43.199 ± 10.28 %, 3,239.972 ± 446.61 µg). Moreover, a significant age-related decrease in rOCT2 protein expression was observed in the aged rats (P < 0.01), which may be responsible for the effect of atenolol on the renal excretion of metformin in the aged rats. In conclusion, there is a drug-drug interaction between atenolol and metformin, and more attention should be paid when atenolol and metformin were co-administered to the aged people inclinical.

The impact of drug transporters on adverse drug reaction.

In this review, we have highlighted the adverse drug reaction mediated by transporters from two aspects: (1) competitive interactions between drug and drug/metabolite/endogenous substance mediated by transporters; (2) the expression/function change of transporter due to physiologic factors, disease, and drugs induction. It indicated that transporters exhibited a broad substrate specificity with a degree of overlap, which could change the pharmacokinetics of drugs and cause toxicity due to competition interactions among substrates. In addition, the expression and function of transporters were regulated by physiological conditions, pathological conditions, and drugs induction, which could cause adverse drug reaction and interindividual differences. Furthermore, one substrate was always medicated by several transporters and often subjected to metabolism by CYP enzymes, so we should be more aware of the increased plasma concentration of drugs caused by drug transporters as well as drug metabolizing enzymes synergistically, especially for drugs with narrow therapeutic window. In addition, the weightiness for one transporter to induce drugs plasma/tissue concentration change could be different in different condition. On the whole, transporters were corresponding with systemic/organs exposure of drug/metabolites/endogenous compounds. So understanding the expression and function in drug transporters will result in better strategies for optimal dosage regimen and reduce the risk for drug adverse reaction as well as adverse drug-drug interactions.

Effects of pyridoxine on the intestinal absorption and pharmacokinetics of isoniazid in rats.

Pyridoxine is always simultaneously administered orally with isoniazid for tuberculosis patients in the clinic to prevent or treat the nervous system side effects induced by isoniazid. So the aim of this research was to investigate the effects of pyridoxine on the intestinal absorption and pharmacokinetics of isoniazid. The intestinal absorption of isoniazid with or without pyridoxine was investigated by the rat single-pass intestinal perfusion model in situ, and a high-performance liquid chromatographic method was applied to study the pharmacokinetics of isoniazid with or without pyridoxine. The results suggested that the intestinal apparent permeability (P app) and intestinal absorption rate constant (K a) for isoniazid (30 µg/ml) were decreased by 43.7 and 36.4 %, respectively, by co-perfused pyridoxine (40 µg/ml). In vivo, the effect of pyridoxine on isoniazid pharmacokinetic correlated with the doses of pyridoxine. The blood concentrations of isoniazid at the absorption phase were affected by co-administered pyridoxine, but the AUC and C max of isoniazid were not greatly affected by pyridoxine as expected from the inhibition by pyridoxine of the intestinal absorption of isoniazid, which could be caused by its rapid absorption phase. Therefore, although the intestinal absorption of isoniazid could be significantly inhibited by pyridoxine, the pharmacokinetics of isoniazid oral administration was not greatly affected by the decreased intestinal absorption of isoniazid due to its rapid absorption.