The efficacy of a paeoniflorin-sodium alginate-gelatin skin scaffold for the treatment of diabetic wound: An in vivo study in a rat model.

OBJECTIVE: To investigate the efficacy of a paeoniflorin-sodium alginate (SA)-gelatin skin scaffold for treating diabetic wound in a rat model. METHODS: Bioinks were prepared using various percentages of paeoniflorin in the total weight of a solution containing SA and gelatin. Skin scaffolds containing 0%, 1%, 3%, 5%, and 10% paeoniflorin were printed using 3D bioprinting technology, and scaffold microstructure was observed with scanning electron microscopy. Skin scaffolds were then used in rats with diabetic wounds. H&E staining, Masson staining, and immunohistochemical staining for IL-1ß and CD31 were performed on days 7 and 14. RESULTS: All skin scaffolds had a mesh-like structure with uniform pore distribution. Wounds healed well in each group, with the 1% and 3% groups demonstrating the most complete healing. H&E staining showed that skin accessory organs had appeared in each group. On day 7, collagen deposition in the 3% group was higher than in the other groups (P＜0.05), and IL-1ß infiltration was lower in the 10% group than in the 3% group (P = 0.002). On day 14, IL-1ß infiltration was not significantly different between the 10% and 3% groups (P = 0.078). The CD31 level was higher in the 3% group than in the other groups on days 7 and 14 (P＜0.05). CONCLUSION: A 3% paeoniflorin-SA-gelatin skin scaffold promoted the healing of diabetic wounds in rats. This scaffold promoted collagen deposition and microvascular regeneration and demonstrated anti-inflammatory properties, suggesting that this scaffold type could be used to treat diabetic wounds.

Celecoxib and Myrtol: A Novel Therapy for Recurrent Appendiceal Mucinous Neoplasms With Extensive Peritoneal Dissemination.

BACKGROUND: Unresectable appendiceal mucinous neoplasms (AMNs) with extensive peritoneal dissemination cause significant morbidity and have limited treatment options. We evaluated a novel combination of Celecoxib and Myrtol in treating such AMNs. METHODS: Patients with recurrent AMNs with extensive peritoneal disease treated with a daily regimen of 200 mg Celecoxib and 1200 mg Myrtol Standardized were included. Progression-free survival (PFS) and overall survival (OS) were calculated, and carcinoembryonic antigen (CEA) trends were compared pretreatment and post-treatment in terms of percentage change. RESULTS: Thirteen patients with extensive, recurrent disease (median peritoneal carcinomatosis index of 36) were included between 2017 and 2020. The median age was 63 years (interquartile range: 55 to 67) and 7 (54%) were male. A total of 85% had undergone prior cytoreductive surgery while 15% underwent cytoreductive surgery >2 times. 54% had received multiple cycles of systemic chemotherapy before starting Celecoxib-Myrtol. After a median follow-up of 8 months, median PFS and OS were 16 months (interquartile range: 5 to 17) and 27 months, respectively. Nine (69.2%) showed improvement in CEA values 3 months after treatment compared with 3-month pretreatment CEA trends. None had adverse events attributable to Celecoxib-Myrtol. CONCLUSIONS: Our feasibility study suggests that a regimen of Celecoxib-Myrtol is well tolerated and may prolong PFS and OS in patients with recurrent AMNs with peritoneal spread.

Paeoniflorin-loaded pH-sensitive liposomes alleviate synovial inflammation by altering macrophage polarity via STAT signaling.

Macrophage polarization plays a prominent role in the pathogenesis of rheumatoid arthritis (RA) and could be regulated by natural extracts paeoniflorin (Pae) but with low bioavailability. In the present study, Pae-loaded liposomes (Pae-LS) with co-conjugation of folate and PEG were prepared for the improvement of therapeutic benefits. We evaluated biophysical characterizations of Pae-LS and macrophage uptake of liposomes, as well as gain insight into whether Pae-LS can improve synovial inflammation in CIA rats and how Pae-LS promoted RAW 264.7 macrophages phenotype switch. We found that Pae-LS showed physical stability, sustained release, long circulation, pH-responsive properties, and higher uptake by active macrophages than free Pae. Furthermore, Pae-LS could repress STAT1 phosphorylation to reduce the levels of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and iNOS expression, as well as lead to a marked increase in anti-inflammatory cytokine (IL-10) and CD206 levels via elevated p-STAT6. In contrast to free Pae, Pae-LS treatment was more effective in alleviating synovial inflammation and hyperplasia in the ankle joint of CIA rats. Our study revealed Pae-LS could effectively suppress synovial inflammation of CIA rats by regulating macrophage polarization via STAT signaling and had the potential for RA treatment as liposome delivery carriers systems.

Linalyl acetate as a potential preventive agent against muscle wasting in rheumatoid arthritis rats chronically exposed to nicotine.

Cigarette smoking has detrimental effects on rheumatoid arthritis (RA), characterized by muscle wasting. Linalyl acetate (LA), the main component of Lavandula angustifolia Mill (lavender) oil, has anti-inflammatory properties. We investigated the detrimental effects of chronic nicotine exposure in rats with RA, as well as the abilities of lavender oil and LA to prevent muscle wasting. Rats with RA induced by type II collagen were exposed to nicotine for 22 days from day 1. Lavender oil or LA was administered twice a week during the experiment. Compared with control, collagen-induced arthritis (CIA) and chronic nicotine exposure plus CIA (NicoCIA) showed increases in hind paw thickness and serum interleukin (IL)-6 and decreases in body weight and serum insulin-like growth factor (IGF)-1 levels. Moreover, weight and fiber cross-sectional area of the gastrocnemius muscle were much lower, and mitochondrial membrane potential of the gastrocnemius muscle was higher, in the NicoCIA than in the CIA. These alterations in the NicoCIA were prevented by lavender oil and LA. Importantly, LA showed greater activity than lavender oil in preventing IGF-1 reduction in the NicoCIA. These findings suggest that lavender oil and LA may have preventive benefit in RA by counteracting muscle wasting associated with chronic nicotine exposure.

New Broth Macrodilution Volatilization Method for Antibacterial Susceptibility Testing of Volatile Agents and Evaluation of Their Toxicity Using Modified MTT Assay In Vitro.

In this study, a new broth macrodilution volatilization method for the simple and rapid determination of the antibacterial effect of volatile agents simultaneously in the liquid and vapor phase was designed with the aim to assess their therapeutic potential for the development of new inhalation preparations. The antibacterial activity of plant volatiles (ß-thujaplicin, thymohydroquinone, thymoquinone) was evaluated against bacteria associated with respiratory infections (Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) and their cytotoxicity was determined using a modified thiazolyl blue tetrazolium bromide assay against normal lung fibroblasts. Thymohydroquinone and thymoquinone possessed the highest antibacterial activity against H. influenzae, with minimum inhibitory concentrations of 4 and 8 µg/mL in the liquid and vapor phases, respectively. Although all compounds exhibited cytotoxic effects on lung cells, therapeutic indices (TIs) suggested their potential use in the treatment of respiratory infections, which was especially evident for thymohydroquinone (TI > 34.13). The results demonstrate the applicability of the broth macrodilution volatilization assay, which combines the principles of broth microdilution volatilization and standard broth macrodilution methods. This assay enables rapid, simple, cost- and labor-effective screening of volatile compounds and overcomes the limitations of assays currently used for screening of antimicrobial activity in the vapor phase.

Optical Coherence Tomography and Microdialysis for Microneedle-Mediated Penetration Enhancement Study of Paeoniflorin-Loaded Ethosomes.

BACKGROUND: To understand the cumulative effect of topical formulations after medication, evaluate the therapeutic effect of microneedle-assisted (MN-assisted) paeoniflorin-loaded ethosomes (TGP-E), and explore the potential for deep penetration of drugs, this paper uses microdialysis to systematically study the percutaneous pharmacokinetics of TGP-E. METHODS: First, optical coherence tomography (OCT) was used to study the effectiveness of microneedle puncture. Second, a microdialysis method and a UPLC-MS method for determining the amount of paeoniflorin (Pae) in dialysate were established. Finally, the transdermal pharmacokinetics of TGP-E was studied using in vivo microdialysis in rats under the above MN-assisted conditions. RESULTS: The optimal MN-assisted conditions were obtained at a microneedle length of 500 µm, a pressure of 3 N, and an action time of 3 min. The pharmacokinetic results demonstrated that the maximum drug concentration (Cmax) and the area under the curve (AUC) of the TGP-E gel were higher than the TGP-saline solution gel, and the mean retention time was lower. These indicated that microneedle can promote the entry of the ethosomes into the skin for in vivo experiments and greatly improve the possibility of deep penetration of the water-soluble Pae. CONCLUSION: Therefore, the microneedle-ethosomes delivery system is a more ideal means for promoting the deep penetration of Pae. These findings may provide a reference for the combination of multiple penetration-enhancement ways to promote drug absorption, and also provide a new insight to realize the development of novel, safe, and more effective dosage forms and administration routes of drugs.

Pharmacokinetic interaction between peimine and paeoniflorin in rats and its potential mechanism.

CONTEXT: Peimine and paeoniflorin can be combined for the treatment of cough in paediatrics. The interaction during the co-administration could dramatically affect the bioavailability of drugs. OBJECTIVE: The interaction between peimine and paeoniflorin was investigated in this study. MATERIALS AND METHODS: The pharmacokinetics of paeoniflorin (20 mg/kg) with or without the coadministration of peimine (5 mg/kg for 10 days before paeoniflorin) was orally investigated in Sprague-Dawley rats (n = 6). The group without the peimine was set as the control group. The metabolic stability of paeoniflorin was studied in rat liver with microsomes. The effect of peimine on the absorption of paeoniflorin was investigated with Caco-2 cell monolayers. RESULTS: The Cmax (244.98 ± 10.95 vs. 139.18 ± 15.14 µg/L) and AUC(0-t) (3295.92 ± 263.02 vs. 139.18 ± 15.14 h·µg/L) of paeoniflorin was increased by peimine. The t1/2 was prolonged from 5.33 ± 1.65 to 14.21 ± 4.97 h and the clearance was decreased from 15.43 ± 1.75 to 4.12 ± 0.57 L/h/kg. Consistently, peimine increased the metabolic stability of paeoniflorin with rat liver microsomes with the increased t1/2 (56.78 ± 2.62 vs. 26.33 ± 3.15 min) and the decreased intrinsic clearance (24.42 ± 3.78 vs. 52.64 ± 4.47 µL/min/mg protein). Moreover, the transportation of paeoniflorin was also inhibited by peimine as the efflux ratio decreased from 3.06 to 1.63. DISCUSSION AND CONCLUSIONS: Peimine increased the systemic exposure of paeoniflorin through inhibiting the activity of CYP3A4 and P-gp. These results provide a reference for further in vivo studies in a broader population.

Ferroportin-Dependent Iron Homeostasis Protects against Oxidative Stress-Induced Nucleus Pulposus Cell Ferroptosis and Ameliorates Intervertebral Disc Degeneration In Vivo.

Ferroptosis is a specialized form of regulated cell death that is charactered by iron-dependent lethal lipid peroxidation, a process associated with multiple diseases. However, its role in the pathogenesis of intervertebral disc degeneration (IVDD) is rarely investigated. This study is aimed at investigating the role of ferroptosis in oxidative stress- (OS-) induced nucleus pulposus cell (NPC) decline and the pathogenesis of IVDD and determine the underlying regulatory mechanisms. We used tert-butyl hydroperoxide (TBHP) to simulate OS conditions around human NPCs. Flow cytometry and transmission electron microscopy were used to identify ferroptosis, while iron assay kit, Perl's staining, and western blotting were performed to assay the intracellular iron levels. A ferroportin- (FPN-) lentivirus and FPN-siRNA were constructed and used to explore the relationship between FPN, intracellular iron homeostasis, and ferroptosis. Furthermore, hinokitiol, a bioactive compound known to specifically resist OS and restore FPN function, was evaluated for its therapeutic role in IVDD both in vitro and in vivo. The results indicated that intercellular iron overload plays an essential role in TBHP-induced ferroptosis of human NPCs. Mechanistically, FPN dysregulation is responsible for intercellular iron overload under OS. The increase in nuclear translocation of metal-regulatory transcription factor 1 (MTF1) restored the function of FPN, abolished the intercellular iron overload, and protected cells against ferroptosis. Additionally, hinokitiol enhanced the nuclear translocation of MTF1 by suppressing the JNK pathway and ameliorated the progression of IVDD in vivo. Taken together, our results demonstrate that ferroptosis and FPN dysfunction are involved in the NPC depletion and the pathogenesis of IVDD under OS. To the best of our knowledge, this is the first study to demonstrate the protective role of FPN in ferroptosis of NPCs, suggesting its potential used as a novel therapeutic target against IVDD.

Perillyl alcohol decreases the frequency and severity of convulsive-like behavior in the adult zebrafish model of acute seizures.

This research aimed to assess the effect of perillyl alcohol (PA) on convulsive behavior in vivo using adult zebrafish (Danio rerio, both sexes). The seizures were induced with pentylenetetrazole (PTZ) intraperitoneally at 170 mg/kg, and diazepam (DZP) was used as the control anticonvulsant (2 mg/kg, oral); PA was tested at 10, 50, and 100 mg/kg orally. The groups had ten animals per group (total n = 60), observed for 10 minutes after seizure induction. We manually appraised typical seizure phenotypes for quantification and used an animal tracking software (Toxtrac) to assess the motor parameters. Next, we sought to find a mechanism of action for PA anticonvulsant activity in silico using a structure-based activity prediction server and molecular docking. The results show that PTZ induced seizure-like behavior in all untreated animals with hyperlocomotion episodes, seizure itself, posture loss, and immobility. DZP inhibited the seizures in all animals of the positive control group. PA, in turn, inhibited the occurrence of seizures in a dose-dependent manner, with frequencies of 90%, 70%, and 40% (for 10, 50, and 100 mg/kg, respectively). The PA treatments also decreased several seizure endpoints in a dose-dependent manner. Also, the difference of the group treated with highest dose of PA was statistically significant compared with the negative control group for all the endpoints assessed (p < 0.05, Kruskal-Wallis). The in silico analyses suggested that PA can affect the GABAergic system, which might be involved in its anticonvulsant activity, but other mechanisms cannot be ruled out. Overall, our results suggest an anticonvulsant potential in perillyl alcohol.

Antidepressant Effect of Paeoniflorin Is Through Inhibiting Pyroptosis CASP-11/GSDMD Pathway.

Nod-like receptor protein 3 (NLRP3)-associated neuroinflammation mediated by activated microglia is involved in the pathogenesis of depression. The role of the pore-forming protein gasdermin D (GSDMD), a newly identified pyroptosis executioner downstream of NLRP3 inflammasome mediating inflammatory programmed cell death, in depression has not been well defined. Here, we provide evidence that paeoniflorin (PF), a monoterpene glycoside compound derived from Paeonia lactiflora, ameliorated reserpine-induced mouse depression-like behaviors, characterized as increased mobility time in tail suspension test and forced swimming test, as well as the abnormal alteration of synaptic plasticity in the depressive hippocampus. The molecular docking simulation predicted that PF would interact with C-terminus of GSDMD. We further demonstrated that PF administration inhibited the enhanced expression of GSDMD which mainly distributed in microglia, along with the proteins involved in pyroptosis signaling transduction including caspase (CASP)-11, CASP-1, NLRP3, and interleukin (IL)-1ß in the hippocampus of mice treated with reserpine. And also, PF prevented lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced pyroptosis in murine N9 microglia in vitro, evidenced by inhibiting the expression of CASP-11, NLRP3, CASP-1 cleavage, as well as IL-1ß. Furthermore, VX-765, an effective and selective inhibitor for CASP-1 activation, reduced the expression of inflammasome and pyroptosis-associated proteins in over-activated N9 and also facilitated PF-mediated inhibition of pyroptosis synergistically. Collectively, the data indicated that PF exerted antidepressant effects, alleviating neuroinflammation through inhibiting CASP-11-dependent pyroptosis signaling transduction induced by over-activated microglia in the hippocampus of mice treated with reserpine. Thus, GSDMD-mediated pyroptosis in activated microglia is a previously unrecognized inflammatory mechanism of depression and represents a unique therapeutic opportunity for mitigating depression given PF administration.

Glycyrrhizic acid-based self-assembled micelles for improving oral bioavailability of paeoniflorin.

BACKGROUND: Paeoniflorin (Pae), a water-soluble monoterpene glucoside, has high potential clinical value in autoimmune and inflammatory diseases. However, the extremely low oral bioavailability of Pae (approximately 3%-4%) limits its formulation development and clinical application. This study aimed to develop micelles using the glycyrrhizic acid (GL) as the carrier to improve the oral absorption of Pae. METHODS: Pae-loaded GL micelles were prepared by the ultrasonic dispersion method and its formulation was optimized by single-factor tests. Characterizations of Pae-loaded GL micelles including particle size, zeta potential, entrapment efficiency (EE), drug loading (DL), morphology, and drug release in vitro were carried out. The single-pass intestinal perfusion and pharmacokinetic studies of Pae-loaded GL micelles were also evaluated in rats and compared with Pae solution and the mixed solution of Pae and GL. RESULTS: The optimized Pae-loaded GL micelles had EE of (42.21 ± 0.89)%, particle size of (58.89 ± 4.24) nm with PDI of (0.194 ± 0.010), zeta potential of (-24.40 ± 1.90) mV. Pae-loaded GL micelles showed a nearly spherical shape under TEM. Drug release of micelles demonstrated a delayed drug release compared to Pae solution. The single-pass intestinal perfusion study showed a significantly higher permeability of Pae in duodenum (p < 0.05), jejunum (p < 0.05), ileum (p < 0.01) and colon (p < 0.01) intestine after perfusion of Pae-loaded GL micelles as compared to Pae solution. The in vivo pharmacokinetics demonstrated that the Cmax and AUC0-t values of Pae-loaded GL micelles were approximately 2.18- and 3.64-fold superior than the Pae solution. CONCLUSION: These results suggested GL could be a potential carrier for the oral delivery of Pae.

Intranasal administration of perillyl alcohol-loaded nanoemulsion and pharmacokinetic study of its metabolite perillic acid in plasma and brain of rats using ultra-performance liquid chromatography/tandem mass spectrometry.

Perillyl alcohol (POH) is a monocyclic terpene that has strong antitumor activity. Brain tumors are particularly difficult to treat with therapeutic agents, and clinical trials have shown their low tolerance through oral administration. We proposed the entrapment of POH into an oil-in-water chitosan nanoemulsion aiming its intranasal administration for brain targeting. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the quantitation of total metabolite perillic acid (PA) in plasma and brain of rats. The rat samples containing the metabolite were treated by liquid-liquid extraction with acetonitrile. The mobile phase was 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B), at a flow rate of 0.3 mL min-1 in gradient elution. The chromatography was run for 10 min, and analytical curves were built in acetonitrile, plasma, and brain. The PA was detected in positive ion mode with multiple reaction monitoring. The method has shown high selectivity, sensitivity, and throughput. The low quantification limits of 162, 178, and 121 ng mL-1 for acetonitrile, brain, and plasma, respectively, indicate a good detectability of the method. The repeatability and precision observed were within the limits recommended in the literature. The accuracy of the method was verified through high recovery rates (106-118%). The validated method was successfully applied to the pharmacokinetic study of the metabolite PA after the intranasal administration of free or POH-loaded nanoemulsion in rats. The results showed that chitosan nanoemulsion improved the plasma and brain bioavailability of POH, representing a promising alternative to free POH treatment.

miR-18a Inhibits BMP4 and HIF-1α Normalizing Brain Arteriovenous Malformations.

RATIONALE: Brain arteriovenous malformations (AVMs) are abnormal tangles of vessels where arteries and veins directly connect without intervening capillary nets, increasing the risk of intracerebral hemorrhage and stroke. Current treatments are highly invasive and often not feasible. Thus, effective noninvasive treatments are needed. We previously showed that AVM-brain endothelial cells (BECs) secreted higher VEGF (vascular endothelial growth factor) and lower TSP-1 (thrombospondin-1) levels than control BEC; and that microRNA-18a (miR-18a) normalized AVM-BEC function and phenotype, although its mechanism remained unclear. OBJECTIVE: To elucidate the mechanism of action and potential clinical application of miR-18a as an effective noninvasive treatment to selectively restore the phenotype and functionality of AVM vasculature. METHODS AND RESULTS: The molecular pathways affected by miR-18a in patient-derived BECs and AVM-BECs were determined by Western blot, RT-qPCR (quantitative reverse transcription polymerase chain reaction), ELISA, co-IP, immunostaining, knockdown and overexpression studies, flow cytometry, and luciferase reporter assays. miR-18a was shown to increase TSP-1 and decrease VEGF by reducing PAI-1 (plasminogen activator inhibitor-1/SERPINE1) levels. Furthermore, miR-18a decreased the expression of BMP4 (bone morphogenetic protein 4) and HIF-1α (hypoxia-inducible factor 1α), blocking the BMP4/ALK (activin-like kinase) 2/ALK1/ALK5 and Notch signaling pathways. As determined by Boyden chamber assays, miR-18a also reduced the abnormal AVM-BEC invasiveness, which correlated with a decrease in MMP2 (matrix metalloproteinase 2), MMP9, and ADAM10 (ADAM metallopeptidase domain 10) levels. In vivo pharmacokinetic studies showed that miR-18a reaches the brain following intravenous and intranasal administration. Intranasal co-delivery of miR-18a and NEO100, a good manufacturing practices-quality form of perillyl alcohol, improved the pharmacokinetic profile of miR-18a in the brain without affecting its pharmacological properties. Ultra-high-resolution computed tomography angiography and immunostaining studies in an Mgp-/- AVM mouse model showed that miR-18a decreased abnormal cerebral vasculature and restored the functionality of the bone marrow, lungs, spleen, and liver. CONCLUSIONS: miR-18a may have significant clinical value in preventing, reducing, and potentially reversing AVM.

Anti-psoriatic effect of Lavandula angustifolia essential oil and its major components linalool and linalyl acetate.

ETHNO-PHARMACOLOGICAL RELEVANCE: Lavender oil (LO) is an aromatic/essential oil extracted from Lavandula angustifolia and traditionally used as an aromatherapy massage oil due to its anti-inflammatory and wound healing property and also for providing the relief in other skin conditions such as psoriasis, dermatitis and eczema. However, LO has not been evaluated scientifically for psoriasis like skin inflammation. AIM OF THE STUDY: This study was aimed to investigate the LO and its major components linalool (L) and linalyl acetate (LA) against psoriasis like skin inflammation. MATERIALS AND METHODS: Anti-psoriatic activity was done using Imiquimod (IMQ) induced psoriasis like skin inflammation in BALB/c mice. Assessment of anti-psoriatic effect of LO, L and LA was done on the basis of change in ear thickness, psoriasis area severity index (PASI) scoring at alternative day, CosCam scoring using skin analyzer equipped with SkinSys software, biochemical, immunohistochemical and histological investigations. Level of effectiveness against psoriasis was investigated by percent reduction in PASI scores, CosCam scores and level of Th-1 and Th-17 cell expressing cytokines, as compared to the diseased mice. RESULTS: Topical application of LO 10% showed 73.67% recovery in PASI and 87% in Th-17 cell-specific cytokines towards normal as compared to disease group. L and LA were identified as the major components of LO and favoured ligands for selected psoriasis targets. At 2% topical dose, L and LA showed 64% and 47.61% recovery in PASI scores, respectively. Both, L and LA showed significant recovery in Th-1 specific TNF-α and IL-1ß however, only L showed significant recovery of Th-17 cytokines (IL-17 and IL-22). In contrast to LA (which restored granulosis), L restored epidermal hyperplasia and parakeratosis toward the normal condition. On the other hand, L also reduced the expression of NF-κß, ccr6 and IL-17, while LA reduced the expression of NF-κß only. At 10% topical dose, LO was observed to be slight irritant while at 2% topical dose, L and LA were found non-irritant to the skin. CONCLUSION: This study proves the effectiveness of LO and its major phytoconstituents linalool and linalyl acetate against IMQ induced psoriasis like skin inflammation and provides the scientific evidence for topical use of lavender oil.

Paeoniflorin attenuates early brain injury through reducing oxidative stress and neuronal apoptosis after subarachnoid hemorrhage in rats.

Paeoniflorin is a natural monoterpene glucoside from Paeoniae Radix with neuroprotective properties. However, it is still unclear whether paeoniflorin has neuroprotective effects on subarachnoid hemorrhage (SAH). This study explores the effect of paeoniflorin on early brain injury (EBI) using rat SAH model. We found that paeoniflorin significantly improves neurological deficits, attenuates brain water content and Evans blue extravasation at 72 h after SAH. Paeoniflorin attenuates the oxidative stress following SAH as evidenced by decrease of reactive oxygen species (ROS), malondialdehyde (MDA), 3-Nitrotyrosine, and 8-Hydroxy-2-deoxy guanosine (8-OHDG) level, increase of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase activity, and up-regulates the nuclear factor erythroid­related factor 2 (Nrf2)/heme oxygenase­1 (HO-1) pathway. Inhibition of microglia activation and neuro-inflammatory response both contributed to paeoniflorin's protective effects. Moreover, paeoniflorin treatment significantly reduces the ratio of Bax/Bcl-2, active caspase-3/ neuronal nuclei (NeuN) and TUNEL/DAPI positive cells at 72 h following SAH. Our results indicate that paeoniflorin may attenuate early brain injury after experimental SAH.

Paeoniflorin Ameliorates Chronic Hypoxia/SU5416-Induced Pulmonary Arterial Hypertension by Inhibiting Endothelial-to-Mesenchymal Transition.

BACKGROUND: Endothelial cells dysfunction is one of the hallmark pathogenic features of pulmonary arterial hypertension (PAH). Paeoniflorin (PF) is a monoterpene glycoside with endothelial protection, vasodilation, antifibrotic, anti-inflammatory and antioxidative properties. However, the effects of PF on PAH remain unknown. METHODS: Here, we investigated the efficacy of PF in the SU5416/hypoxia (SuHx) rat model of PAH. Human pulmonary arterial endothelial cells (HPAECs) were exposed to 1% O2 with or without PF treatment. RESULTS: Hemodynamics analysis showed that prophylactic treatment with PF (300 mg/kg i.g. daily for 21 days) significantly inhibited chronic hypoxia/SU5416-induced elevations of right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index in rats. Meanwhile, PF significantly reduced pulmonary vascular remodeling, as well as alleviated collagen deposition in lungs and right ventricles in SuHx rats. Additionally, PF inhibited SuHx-induced down-regulation of endothelial marker (vascular endothelial cadherin) and up-regulation of mesenchymal markers (fibronectin and vimentin) in lung, suggesting that PF could inhibit SuHx-induced endothelial-to-mesenchymal transition (EndMT) in lung. Further in vitro studies confirmed that PF treatment suppressed hypoxia-induced EndMT in HPAECs, which was abolished by the knockdown of bone morphogenetic protein receptor type 2 (BMPR2) in HPAECs. CONCLUSION: Taken together, our findings suggest that PF ameliorates BMPR2 down-regulation-mediated EndMT and thereafter alleviates SuHx-induced PAH in rats.

Paeoniflorin Attenuates Myocardial Fibrosis in Isoprenaline-induced Chronic Heart Failure Rats via Inhibiting P38 MAPK Pathway.

Paeoniforin (Pae) is a monoterpenoid glycoside compound and has many biological activities, such as immunosuppression, anti-inflammation and anti-cell proliferation. However, the effects and mechanisms of Pae on chronic heart failure (CHF) remain unclear. This study was conducted to assess the effects and mechanisms of Pae on myocardial fbrosis in isoprenaline (Iso)-induced CHF rats. Pae (20 mg/kg) was intragastrically administrated to CHF rats for 6 weeks. Cardiac structure and function were assessed. The protein and mRNA levels of transforming growth factor ß1 (TGF-ß1) and p38 were detected. Compared to Iso group, Pae could alleviate myocardial fibrosis and improve cardiac function in CHF rats. The levels of collagen volume fraction (13.75%±3.77% vs. 30.97%±4.22%, P<0.001) and perivascular collagen volume area (14.32%±2.50% vs. 28.31%±3.16%, P<0.001) were signifcantly reduced in Pae group as compared with those in Iso group. The expression of TGF-ß1 protein (0.30±0.07 vs. 0.66±0.07, P<0.05) and mRNA (3.51±0.44 vs. 7.58±0.58, P<0.05) decreased signifcantly in Pae group as compared with that in Iso group. The expression of p38 protein (0.36±0.12 vs. 0.81±0.38, P<0.05) and mRNA (3.84±0.05 vs. 4.40±0.17, P<0.05) also decreased markedly in Pae group as compared with that in Iso group. Pae could attenuate myocardial fbrosis and improve cardiac function in CHF rats by down-regulating the p38 MAPK signaling pathway.

Intranasal perillyl alcohol therapy improves survival of patients with recurrent glioblastoma harboring mutant variant for MTHFR rs1801133 polymorphism.

BACKGROUND: Polymorphisms in MTHFR gene influence risk and overall survival of patients with brain tumor. Global genomic DNA (gDNA) methylation profile from tumor tissues is replicated in peripheral leukocytes. This study aimed to draw a correlation between rs1801133 MTHFR variants, gDNA methylation and overall survival of patients with recurrent glioblastoma (rGBM) under perillyl alcohol (POH) treatment. METHODS: gDNA from whole blood was extracted using a commercially available kit (Axygen) and quantified by spectrophotometry. Global gDNA methylation was determined by ELISA and rs1801133 polymorphism by PCR-RFLP. Statistical analysis of gDNA methylation profile and rs1801133 variants included Mann-Whitney, Kruskal-Wallis, Spearman point-biserial correlation tests (SPSS and Graphpad Prism packages; significant results for effect size higher than 0.4). Prognostic value of gDNA methylation and rs1801133 variants considered survival profiles at 25 weeks of POH treatment, having the date of protocol adhesion as starting count and death as the final event. RESULTS: Most rGBM patients showed global gDNA hypomethylation (median = 31.7%) and a significant, moderate and negative correlation between TT genotype and gDNA hypomethylation (median = 13.35%; rho = - 0.520; p = 0.003) compared to CC variant (median = 32.10%), which was not observed for CT variant (median = 33.34%; rho = - 0.289; p = 0.06). gDNA hypermethylated phenotype (median = 131.90%) exhibited significant, moderate and negative correlations between TT genotype (median = 112.02%) and gDNA hypermethylation levels when compared to CC (median = 132.45%; rho = - 0,450; p = 0.04) or CT (median = 137.80%; rho = - 0.518; p = 0.023) variants. TT variant of rs1801133 significantly decreased gDNA methylation levels for both patient groups, when compared to CC (d values: hypomethylated = 1.189; hypermethylated = 0.979) or CT (d values: hypomethylated = 0.597; hypermethylated = 1.167) variants. Positive prognostic for rGBM patients may be assigned to gDNA hypermethylation for survivors above 25 weeks of treatment (median = 88 weeks); and TT variant of rs1801133 regardless POH treatment length. CONCLUSION: rGBM patients under POH-based therapy harboring hypermethylated phenotype and TT variant for rs1801133 had longer survival. Intranasal POH therapy mitigates detrimental effects of gDNA hypomethylation and improved survival of patients with rGBM harboring TT mutant variant for MTHFR rs1801133 polymorphism. TRIAL REGISTRATION: CONEP -9681- 25,000.009267 / 2004. Registered 12th July, 2004.

Exploring and characterizing a novel combination of paeoniflorin and talatizidine for the treatment of rheumatoid arthritis.

Wutou Decoction (WTD) achieves favorable therapeutic response in treating rheumatoid arthritis (RA), especially for wind-cold-dampness stimulating RA. However, its material basis and molecular mechanisms remain unclear. To address this problem, the main bioactive compounds (BACs) of WTD against RA and the candidate targets were identified in the current study via transcriptional regulatory network analysis, computational structure-based methods, as well as in vivo and in vitro experimental validations. As a result, we successfully established a RA rat model named AIA-S, which simulated the clinical manifestations and pathological changes of wind-cold-dampness stimulating RA, and also displayed the distinctive characteristics and biological basis of inflammatory-immune system imbalance and abnormal energy metabolism changes. In addition, ALOX15B-PPAR-γ-PTGS2-FGF2-IL-1ß-c-JUN-MMP13-TGF-ß1 signal axis, involved into thermogenesis and energy metabolism, as well as maintaining the balance of inflammation-immune system, was identified as a candidate target of WTD against RA, according to the transcriptional regulatory network analysis on "RA-related gene-WTD-effective gene interaction network". Moreover, Paeoniflorin (PAE) and Talatizidine (TLT) were demonstrated to be the main BACs of WTD against RA for the following reasons: firstly, both PAE and TLT were the BACs of WTD according to ADME analysis in silico and the pharmacokinetics analysis in vivo. Secondly, both PAE and TLT were able to bind with PPAR-γ, c-JUN, MMP13 and TGF-ß1, which were the candidate targets of WTD against RA, with the strong binding affinity. Thirdly, the PAE and TLT combination exerted significant therapeutic effects on AIA-S rats through reversing the imbalance of inflammatory-immune system, and the disturbance of thermogenesis and energy metabolism, which were similar to WTD. More importantly, the administration of TLT or PAE alone didn't exert as prominently therapeutic effects as that of the two-BAC-combination did. Fourthly, the PAE and TLT combination promoted adipogenesis and lipogenesis by upregulating the PPAR-γ-induced lipogenic proteins. In conclusion, this study identified PAE and TLT as the main BACs of WTD in alleviating the severity of RA, and also developed a novel combination of PAE and TLT as a promising candidate drug for RA therapy.

Anthelmintic activity of nanoencapsulated carvacryl acetate against gastrointestinal nematodes of sheep and its toxicity in rodents.

The objective of this study was to evaluate the efficacy of carvacryl acetate (CVA) and nanoencapsulated CVA (nCVA) on gastrointestinal nematodes of sheep. The CVA was nanoencapsulated with chitosan/gum arabic and the efficacy of nanoencapsulation (EE), yield, zeta potential, nanoparticle morphology and release kinetics at pH 3 and 8 were analyzed. Acute and subchronic toxicity were evaluated in rodents and reduction of egg counts in the faeces (FECRT) of sheep. The sheep were divided into four groups (n = 10): G1, 250 mg/kg CVA; G2, 250 mg/kg nCVA; G3, polymer matrix and G4: 2.5 mg/kg monepantel. EE and nCVA yield were 65% and 57%, respectively. The morphology of the nanoparticles was spherical, size (810.6±286.7 nm), zeta potential in pH 3.2 (+18.3 mV) and the 50% release of CVA at pHs 3 and 8 occurred at 200 and 10 h, respectively. nCVA showed LD50 of 2,609 mg/kg. CVA, nCVA and monepantel reduced the number of eggs per gram of faeces (epg) by 57.7%, 51.1% and 97.7%, respectively. The epg of sheep treated with CVA and nCVA did not differ from the negative control (P>0.05). Nanoencapsulation reduced the toxicity of CVA; however, nCVA and CVA presented similar results in the FECRT.