Kisspeptin/KISS1R Signaling Modulates Human Airway Smooth Muscle Cell Migration.

Airway remodeling is a cardinal feature of asthma, associated with increased airway smooth muscle cell (ASM) mass and up-regulation of extracellular matrix deposition. Exaggerated ASM cell migration contributes to excessive ASM mass. Previously, we demonstrated the alleviating role of kisspeptin (Kp) receptor (KISS1R) activation by Kp-10 in mitogen (PDGF)-induced human ASM cell proliferation in vitro and airway remodeling in vivo in a mouse model of asthma. Here, we examined the mechanisms by which KISS1R activation regulates mitogen-induced ASM cell migration. KISS1R activation using Kp-10 significantly inhibited PDGF-induced ASM cell migration, further confirmed using KISS1R shRNA. Furthermore, KISS1R activation modulated F/G actin dynamics and the expression of pro-migration proteins like cell division control protein 42 (CDC42) and cofilin. Mechanistically, we observed reduced ASM RhoA-GTPAse with KISS1R activation. The anti-migratory effect of KISS1R was abolished by protein kinase A (PKA)-inhibitory peptide. Conversely, KISS1R activation significantly increased cAMP and phosphorylation of cAMP-response element binding protein (CREB) in PDGF-exposed ASM cells. Overall, these results highlight the alleviating properties of Kp-10 in the context of airway remodeling.

Estrogen receptors differentially modifies lamellipodial and focal adhesion dynamics in airway smooth muscle cell migration.

Sex-steroid signaling, especially estrogen, has a paradoxical impact on regulating airway remodeling. In our previous studies, we demonstrated differential effects of 17ß-estradiol (E2) towards estrogen receptors (ERs: α and ß) in regulating airway smooth muscle (ASM) cell proliferation and extracellular matrix (ECM) production. However, the role of ERs and their signaling on ASM migration is still unexplored. In this study, we examined how ERα versus ERß affects the mitogen (Platelet-derived growth factor, PDGF)-induced human ASM cell migration as well as the underlying mechanisms involved. We used Lionheart-FX automated microscopy and transwell assays to measure cell migration and found that activating specific ERs had differential effects on PDGF-induced ASM cell migration. Pharmacological activation of ERß or shRNA mediated knockdown of ERα and specific activation of ERß blunted PDGF-induced cell migration. Furthermore, specific ERß activation showed inhibition of actin polymerization by reducing the F/G-actin ratio. Using Zeiss confocal microscopy coupled with three-dimensional algorithmic ZEN-image analysis showed an ERß-mediated reduction in PDGF-induced expressions of neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related proteins-2/3 (Arp2/3) complex, thereby inhibiting actin-branching and lamellipodia. In addition, ERß activation also reduces the clustering of actin-binding proteins (vinculin and paxillin) at the leading edge of ASM cells. However, cells treated with E2 or ERα agonists do not show significant changes in actin/lamellipodial dynamics. Overall, these findings unveil the significance of ERß activation in regulating lamellipodial and focal adhesion dynamics to regulate ASM cell migration and could be a novel target to blunt airway remodeling.

Kisspeptin regulates airway hyperresponsiveness and remodeling in a mouse model of asthma.

Asthma is a multifactorial disease of origin characterized by airway hyperresponsiveness (AHR) and airway remodeling. Several pieces of evidence from other pathologies suggest that Kisspeptins (Kp) regulate cell proliferation, migration, and invasion, mechanisms that are highly relevant to asthma. Our recent in vitro studies show Kp-10 (active peptide of Kp), via its receptor, KISS1R, inhibits human airway smooth muscle cell proliferation. Here, we hypothesize a crucial role for Kp-10 in regulating AHR and airway remodeling in vivo. Utilizing C57BL/6J mice, we assessed the effect of chronic intranasal Kp-10 exposure on mixed allergen (MA)-induced mouse model of asthma. MA-challenged mice showed significant deterioration of lung function compared to those exposed to vehicle (DPBS); Kp-10 treatment significantly improved the MA-altered lung functions. Mice treated with Kp-10 alone did not show any notable changes in lung functions. MA-exposed mice showed a significant reduction in KISS1R expression as compared to vehicle alone. MA-challenged mice showed significant alterations in immune cell infiltration in the airways and remodeling changes. Proinflammatory cytokines were significantly increased upon MA exposure, an effect abrogated by Kp-10 treatment. Furthermore, biochemical and histological studies showed Kp-10 exposure significantly reduced MA-induced smooth muscle mass and soluble collagen in the lung. Overall, our findings highlight the effect of chronic Kp-10 exposure in regulating MA-induced AHR and remodeling. © 2023 The Pathological Society of Great Britain and Ireland.

Kisspeptins inhibit human airway smooth muscle proliferation.

Sex and gender disparity in asthma is recognized and suggests a modulatory role for sex steroids, particularly estrogen. However, there is a dichotomous role for estrogen in airway remodeling, making it unclear whether sex hormones are protective or detrimental in asthma and suggesting a need to explore mechanisms upstream or independent of estrogen. We hypothesize that kisspeptin (Kp)/KISS1R signaling serves this role. Airway smooth muscle (ASM) is a key structural cell type that contributes to remodeling in asthma. We explored the role of Kp/KISS1R in regulating ASM proliferation. We report potentially novel data indicating that Kp and KISS1R are expressed in human airways, especially ASM, with lower expression in ASM from women compared with men and lower in patients with asthma compared with people without asthma. Proliferation studies showed that cleaved forms of Kp, particularly Kp-10, mitigated PDGF-induced ASM proliferation. Pharmacological inhibition and shRNA knockdown of KISS1R increased basal ASM proliferation, which was further amplified by PDGF. The antiproliferative effect of Kp-10 in ASM was mediated by inhibition of MAPK/ERK/Akt pathways, with altered expression of PCNA, C/EBP-α, Ki-67, cyclin D1, and cyclin E leading to cell cycle arrest at G0/G1 phase. Overall, we demonstrate the importance of Kp/KISS1R signaling in regulating ASM proliferation and a potential therapeutic avenue to blunt remodeling in asthma.

Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds.

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an injury leading to delayed wound healing. We selected doxycycline (DOX), as a potential drug of choice, due to its anti-bacterial properties along with its reported anti-inflammatory properties. The current study aims to formulate DOX loaded collagen-chitosan non-crosslinked (NCL) & crosslinked (CL) scaffolds and evaluate their healing ability in diabetic conditions. The characterization result of scaffolds reveals that the DOX-CL scaffold holds ideal porosity, a low swelling & degradation rate, and a sustained release of DOX compared to the DOX-NCL scaffold. The in vitro studies reveal that the DOX-CL scaffold was biocompatible and enhanced cell growth compared with CL scaffold treated and control groups. The anti-bacterial studies have shown that the DOX-CL scaffold was more effective than the CL scaffold against the most common bacteria found in DW. Using the streptozotocin and high-fat diet-induced DW model, a significantly (p≤0.05) faster rate of wound contraction in the DOX-CL scaffold treated group was observed compared to those in CL scaffold treated and control groups. The use of the DOX-CL scaffold can prove to be a promising approach for local treatment for DWs.

Network and co-expression analysis of airway smooth muscle cell transcriptome delineates potential gene signatures in asthma.

Airway smooth muscle (ASM) is known for its role in asthma exacerbations characterized by acute bronchoconstriction and remodeling. The molecular mechanisms underlying multiple gene interactions regulating gene expression in asthma remain elusive. Herein, we explored the regulatory relationship between ASM genes to uncover the putative mechanism underlying asthma in humans. To this end, the gene expression from human ASM was measured with RNA-Seq in non-asthmatic and asthmatic groups. The gene network for the asthmatic and non-asthmatic group was constructed by prioritizing differentially expressed genes (DEGs) (121) and transcription factors (TFs) (116). Furthermore, we identified differentially connected or co-expressed genes in each group. The asthmatic group showed a loss of gene connectivity due to the rewiring of major regulators. Notably, TFs such as ZNF792, SMAD1, and SMAD7 were differentially correlated in the asthmatic ASM. Additionally, the DEGs, TFs, and differentially connected genes over-represented in the pathways involved with herpes simplex virus infection, Hippo and TGF-ß signaling, adherens junctions, gap junctions, and ferroptosis. The rewiring of major regulators unveiled in this study likely modulates the expression of gene-targets as an adaptive response to asthma. These multiple gene interactions pointed out novel targets and pathways for asthma exacerbations.

Sex-Steroid Signaling in Lung Diseases and Inflammation.

Sex/gender difference exists in the physiology of multiple organs. Recent epidemiological reports suggest the influence of sex-steroids in modulating a wide variety of disease conditions. Sex-based discrepancies have been reported in pulmonary physiology and various chronic inflammatory responses associated with lung diseases like asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, and rare lung diseases. Notably, emerging clinical evidence suggests that several respiratory diseases affect women to a greater degree, with increased severity and prevalence than men. Although sex-specific differences in various lung diseases are evident, such differences are inherent to sex-steroids, which are major biological variables in men and women who play a central role to control these differences. The focus of this chapter is to comprehend the sex-steroid biology in inflammatory lung diseases and to understand the mechanistic role of sex-steroids signaling in regulating these diseases. Exploring the roles of sex-steroid signaling in the regulation of lung diseases and inflammation is crucial for the development of novel and effective therapy. Overall, we will illustrate the importance of differential sex-steroid signaling in lung diseases and their possible clinical implications for the development of complementary and alternative medicine to treat lung diseases.

Androgen receptor activation alleviates airway hyperresponsiveness, inflammation, and remodeling in a murine model of asthma.

Epidemiological studies demonstrate an apparent sex-based difference in the prevalence of asthma, with a higher risk in boys than girls, which is reversed postpuberty, where women become more prone to asthma than men, suggesting a plausible beneficial role for male hormones, especially androgens as a regulator of pathophysiology in asthmatic lungs. Using a murine model of asthma developed with mixed allergen (MA) challenge, we report a significant change in airway hyperresponsiveness (AHR), as demonstrated by increased thickness of epithelial and airway smooth muscle layers and collagen deposition, as well as Th2/Th17-biased inflammation in the airways of non-gonadectomized (non-GDX) and gonadectomized (GDX) male mice. Here, compared with non-GDX mice, MA-induced AHR and inflammatory changes were more prominent in GDX mice. Activation of androgen receptor (AR) using 5α-dihydrotestosterone (5α-DHT, AR agonist) resulted in decreased Th2/Th17 inflammation and remodeling-associated changes, resulting in improved lung function compared with MA alone challenged mice, especially in GDX mice. These changes were not observed with Flutamide (Flut, AR antagonist). Overall, we show that AR exerts a significant and beneficial role in asthma by regulating AHR and inflammation.

Cellular and Biochemical Analysis of Bronchoalveolar Lavage Fluid from Murine Lungs.

Bronchoalveolar lavage (BAL) is a technique used to collect the contents of the airways. The fluid recovered, called BAL fluid (BALF), serves as a dynamic tool to identify various disease pathologies ranging from asthma to infectious diseases to cancer in the lungs. A wide array of tests can be performed with BALF, including total and differential leukocyte counts (DLC), enzyme-linked immunosorbent assays (ELISA) or flow-cytometric quantitation of inflammatory mediators, such as cytokines, chemokines and adhesion molecules, and assessment of nitrate and nitrite content for estimation of nitric oxide synthase (NOS) activity. Here, we describe a detailed procedure for the collection of BALF for a variety of downstream usages, including DLC by cytological and flow-cytometry-based methods, multiplex cytokine analysis by flow cytometry, and NOS activity analysis by determining nitrate and nitrite levels.

Sex steroids skew ACE2 expression in human airway: a contributing factor to sex differences in COVID-19?

The incidence, severity, and mortality of ongoing coronavirus infectious disease 19 (COVID-19) is greater in men compared with women, but the underlying factors contributing to this sex difference are still being explored. In the current study, using primary isolated human airway smooth muscle (ASM) cells from normal males versus females as a model, we explored the effect of estrogen versus testosterone in modulating the expression of angiotensin converting enzyme 2 (ACE2), a cell entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using confocal imaging, we found that ACE2 is expressed in human ASM. Furthermore, Western analysis of ASM cell lysates showed significantly lower ACE2 expression in females compared with males at baseline. In addition, ASM cells exposed to estrogen and testosterone for 24 h showed that testosterone significantly upregulates ACE2 expression in both males and females, whereas estrogen downregulates ACE2, albeit not significant compared with vehicle. These intrinsic and sex steroids induced differences may help explain sex differences in COVID-19.

Differential estrogen-receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF-κB pathway.

Altered airway smooth muscle (ASM) mass and extracellular matrix (ECM) deposition in airways are characteristic features of remodeling in asthma. Increased ECM production modulates ASM cell proliferation and leads to airway remodeling. Our previous studies showed that ASM from patients with asthma exhibited increased expression of estrogen receptor (ER)-ß, which upon activation down-regulated ASM proliferation, implicating an important role for estrogen signaling in airway physiology. There is no current information on the effect of differential ER activation on ECM production. In this study, we evaluated the effect of ER-α vs. ER-ß activation on ECM production, deposition, and underlying pathways. Primary human ASM cells isolated from asthmatics and nonasthmatics were treated with E2, an ER-α agonist [propylpyrazoletriol (PPT)], and an ER-ß agonist [WAY-200070 (WAY)] with TNF-α or platelet-derived growth factor (PDGF) followed by evaluation of ECM production and deposition. Expression of proteins and genes corresponding to ECM were measured using Western blotting and quantitative RT-PCR with subsequent matrix metalloproteinase (MMP) activity. Molecular mechanisms of ER activation in regulating ECM were evaluated by luciferase reporter assays for activator protein 1 (AP-1) and NF-κB. TNF-α or PDGF significantly (P < 0.001) increased ECM deposition and MMP activity in human ASM cells, which was significantly reduced with WAY treatment but not with PPT. Furthermore, TNF-α- or PDGF-induced ECM gene expression in ASM cells was significantly reduced with WAY (P < 0.001). Moreover, WAY significantly down-regulated the activation of NF-κB (P < 0.001) and AP-1 (P < 0.01, P < 0.05) in ASM cells from asthmatics and nonasthmatics. Overall, we demonstrate differential ER signaling in controlling ECM production and deposition. Activation of ER-ß diminishes ECM deposition via suppressing the NF-κB pathway activity and might serve as a novel target to blunt airway remodeling.-Ambhore, N. S., Kalidhindi, R. S. R., Pabelick, C. M., Hawse, J. R., Prakash, Y. S., Sathish, V. Differential estrogen-receptor activation regulates extracellular matrix deposition in human airway smooth muscle remodeling via NF-κB pathway.

Role of Differential Estrogen Receptor Activation in Airway Hyperreactivity and Remodeling in a Murine Model of Asthma.

Evidence suggests that airway hyperresponsiveness (AHR) is a characteristic feature of asthma. Epidemiological studies have confirmed that the severity of asthma is greater in women, suggesting a critical role of female sex steroid hormones (especially estrogen). Very few in vivo studies have examined the role of sex steroid hormones in asthma, and the sequence of events that occur through differential activation of estrogen receptors (ERs) remains to be determined in asthmatic airways. Our recent in vitro findings indicated that ERß had increased expression in asthmatic airway smooth muscle (ASM), and that its activation by an ERß-specific agonist downregulated airway remodeling. In this study, we translated the in vitro findings to a murine asthma model and examined the differential role of ER activation in modulating lung mechanics. C57BL/6J male, female, and ovariectomized mice were exposed to mixed allergen (MA) and subcutaneously implanted with sustained-release pellets of placebo, an ERα agonist (4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol [PPT]), and/or an ERß agonist (WAY-200070). We then evaluated the effects of these treatments on airway mechanics, biochemical, molecular, and histological parameters. Mice exposed to MA showed a significant increase in airway resistance, elastance, and tissue damping, and a decrease in compliance; pronounced effects were observed in females. Compared with PPT, WAY treatment significantly reversed the MA-induced changes. The increased mRNA/protein expression of ERα, ERß, and remodeling genes observed in MA-treated mice was significantly reversed in WAY-treated mice. This novel study indicates that activation of ERß signaling downregulates AHR and airway remodeling, and is a promising target in the development of treatments for asthma.

Laser-capture microdissection of murine lung for differential cellular RNA analysis.

The lung tissue contains a heterogeneous milieu of bronchioles, epithelial, airway smooth muscle (ASM), alveolar, and immune cell types. Healthy bronchiole comprises epithelial cells surrounded by ASM cells and helps in normal respiration. In contrast, airway remodeling, or plasticity, increases surrounding of bronchial epithelium during inflammation, especially in asthmatic condition. Given the profound functional difference between ASM, epithelial, and other cell types in the lung, it is imperative to separate and isolate different cell types of lungs for genomics, proteomics, and molecular analysis, which will improve the diagnostic and therapeutic approach to treat cell-specific lung disorders. Laser capture microdissection (LCM) is the technique generally used for the isolation of specific cell populations under direct visual inspection, which plays a crucial role to evaluate cell-specific effect in clinical and preclinical setup. However, maintenance of tissue RNA quality and integrity in LCM studies are very challenging tasks. It is obvious to believe that the major factor affecting the RNA quality is tissue-fixation method. The prime focus of this study was to address the RNA quality factors within the lung tissue using the different solvent system to fix tissue sample to obtain high-quality RNA. Paraformaldehyde and Carnoy's solutions were used for fixing the lung tissue and compared RNA integrity in LCM captured lung tissue samples. To further confirm the quality of RNA, we measured cellular marker genes in collected lung tissue samples from control and mixed allergen (MA)-induced asthmatic mouse model using qRT-PCR technique. RNA integrity number showed a significantly better quality of RNA in lung tissue samples fixed with Carnoy's solution compared to paraformaldehyde solution. Isolated RNA from MA-induced asthmatic murine lung epithelium, smooth muscle, and granulomatous foci using LCM showed a significant increase in remodeling gene expression compared to control which confirm the quality and integrity of isolated RNA. Overall, the study concludes tissue fixation solvent can alter the quality of RNA in the lung and the outcome of the results.

Role of Estrogen Receptors α and ß in a Murine Model of Asthma: Exacerbated Airway Hyperresponsiveness and Remodeling in ERß Knockout Mice.

Epidemiological data suggests increased prevalence of asthma in females than males, suggesting a plausible role for sex-steroids, especially estrogen in the lungs. Estrogen primarily acts through estrogen-receptors (ERα and ERß), which play a differential role in asthma. Our previous studies demonstrated increased expression of ERß in asthmatic human airway smooth muscle (ASM) cells and its activation diminished ASM proliferation in vitro and airway hyperresponsiveness (AHR) in vivo in a mouse (wild-type, WT) model of asthma. In this study, we evaluated the receptor specific effect of circulating endogenous estrogen in regulating AHR and remodeling using ERα and ERß knockout (KO) mice. C57BL/6J WT, ERα KO, and ERß KO mice were challenged intranasally with a mixed-allergen (MA) or PBS. Lung function was measured using flexiVent followed by collection of broncho-alveolar lavage fluid for differential leukocyte count (DLC), histology using hematoxylin and eosin (H&E) and Sirius red-fast green (SRFG) and detecting αsmooth muscle actin (α-SMA), fibronectin and vimentin expression using immunofluorescence (IF). Resistance (Rrs), elastance (Ers), tissue-damping (G) and tissue-elasticity (H) were significantly increased, whereas compliance (Crs) was significantly decreased in WT, ERα KO, and ERß KO mice (males and females) challenged with MA compared to PBS. Interestingly, ERß KO mice showed declined lung function compared to ERα KO and WT mice at baseline. MA induced AHR, remodeling and immune-cell infiltration was more prominent in females compared to males across all populations, while ERß KO females showed maximum AHR and DLC, except for neutrophil count. Histology using H&E suggests increased smooth muscle mass in airways with recruitment of inflammatory cells, while SRFG staining showed increased collagen deposition in MA challenged ERß KO mice compared to ERα KO and WT mice (males and females), with pronounced effects in ERß KO females. Furthermore, IF studies showed increased expression of α-SMA, fibronectin and vimentin in MA challenged populations compared to PBS, with prominent changes in ERß KO females. This novel study indicates ERß plays a pivotal role in airway remodeling and AHR and understanding the mechanisms involved might help to surface it out as a potential target to treat asthma.

Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation.

Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERß isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERß modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17ß-estradiol, ERα-agonist and/or ERß-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERß significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERß can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways, and may point to a novel perception for blunting airway remodeling.

Brain Targeting of 1,9-Pyrazoloanthrone an c-Jun-N-terminal Kinase Inhibitor Using Liposomes for Effective Management of Parkinson's Disease.

The major challenge to treat Parkinson's disease (PD) is penetration of target molecule into the brain to improve the efficacy of drugs. To achieve better brain penetration and targeted delivery, 1,9-Pyrazoloanthrone (1,9-P) loaded liposomes were developed by solvent injection technique using ultrasonication and evaluated for particle size, morphology, entrapment efficiency, FT-IR, and in-vitro drug release studies. The potential of 1,9-Pyrazoloanthrone (1,9-P), a c-Jun-N-terminal Kinase (JNK-3) inhibitor which could stop or retard the rate of apoptosis of neuronal cells was also evaluated. In-vivo pharmacokinetic and brain uptake studies of liposomes were performed to investigate the bioavailability and brain distribution of 1,9-P. Cytotoxicity and neuroprotection were done on SH-SY5Y cell line using MTT and AO/EB apoptosis assay. The optimized batch of liposomes showed an average size of 112.33 ± 0.84 nm with a zeta potential value of -19.40 mV and 78.96 ± 0.28% drug entrapment efficiency. The in-vitro release studies demonstrated the sustained release profile of liposome up to 24 h. The pharmacokinetic data in Wistar rats over the period of 12 h demonstrated 4.82-folds greater AUC(0-12 h) for liposome in brain compared with 1,9-P suspension. Cytotoxicity assay showed no sign of toxicity, whereas apoptosis assay revealed a neuroprotective action of liposomes. The results demonstrated successful targeting of the 1,9-P, to brain as a novel strategy having significant therapeutic potential to treat PD.

Pharmacokinetic and tissue distribution studies of 1,9-pyrazoloanthrone, a c-Jun-N-terminal kinase inhibitor in Wistar rats by a simple and sensitive HPLC method.

JNK pathway activates c-Jun(s) which are responsible for cell apoptosis; as a result, inhibitors of JNK pathway have the potential to prevent dopaminergic neurons from death and decrease the loss of dopamine in substantia nigra pars compacta (SNpc). Recent in-vitro studies show that 1,9-pyrazoloanthrone (1,9-P) a potent JNK-3 inhibitor prevents the apoptosis of dopaminergic cells of brain. In the present study we formulated liposomes to increase the bioavailability of 1,9-P in the brain and developed a simple, sensitive and selective high performance liquid chromatographic method and validated for the estimation of 1,9-P in Wistar rat plasma and tissue samples. Plasma and tissue samples were extracted by protein precipitation technique using acetonitrile (ACN) and rasagiline as the internal standards. Chromatography was performed on Hibar C18 column with mobile phase of ammonium acetate (10mM, pH 8.0 adjusted with ammonia) and ACN at a flow rate of 1mL/min. The lower limit of quantification of the developed method was found to be 2.0ng/mL and 4.0ng/g in plasma and tissue samples respectively. The liposomes of 1,9-P administered to animals at the dose equivalent to 15mg/kg orally demonstrated remarkable absorption into the systemic circulation with maximum concentration (∼7500ng/mL) within 2.0h. The order of the area under curve was found to be kidney>liver>brain>lungs>spleen>heart. The liposomes of 1,9-P were rapidly taken up into brain and showed a good brain concentration after 2.0h; sustenance up to 4.0h was achieved which is better than 1,9-P solution.