Background
Neuroinflammation accompanies all neurological afflictions from traumatic brain injuries and nervous system infections to chronic neurodegenerative diseases. Microglia, resident immune cells in the brain, are the key contributors of this neuroinflammation resulting in neurodegeneration. The microglial-mediated neuroinflammation has been the characteristic diagnostic feature of various neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), trauma, multiple sclerosis (MS), and cerebral ischemia [
1]. Under normal physiological conditions, these special brain macrophages serve the role of immune surveillance displaying M2 phenotype by secreting various neurotrophic and anti-inflammatory factors creating protective microenvironment for the neurons. However, in response to infection or injury, they readily become activated and converted in the form of M1 phenotype displaying a variety of surface receptors, including the MHCs and complement receptors [
2]. They also undergo dramatic morphological changes from resting ramified cells to activated amoeboid microglia [
3]. Chronic activation of microglia results in neuroinflammation by secreting various neurotoxic and pro-inflammatory mediators causing demyelination and neuronal death [
4]. These mediators include PGE
2, monocyte chemoattractant protein-1 (MCP-1), inflammatory cytokines such as interleukin-1 β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) and free radicals such as nitric oxide (NO) and superoxide, fatty acid metabolites such as eicosanoids, and quinolinic acid [
5].
Conventionally, various nonsteroidal anti-inflammatory drugs (NSAIDs) like rofecoxib, COX 2 inhibitors, aspirin, mefenamic acid, indomethacin, and ketoprofen are used to treat neurodegenerative diseases [
6,
7]. However, treatment with NSAIDs is generally accompanied by various side effects like gastrointestinal problems, dizziness, headache, and dyspepsia as their major drawback. Recently, many medicinal herbs and their bioactive components like epigallocatechin-3-gallate (EGCG) from
Camellia sinensis, curcumin from
Cucurma longa, and resveratrol from grapes have been reported to possess anti-inflammatory activity and are more safe, effective, and easy to use than the NSAIDs with negligible potential risks [
8‐
11].
Withania somnifera is the most popular medicinal plant in Ayurveda for its nerve tonic and memory enhancing properties. It is also well acknowledged for its longevity and vitality increasing properties [
12]. Ashwagandha has been reported to possess a number of therapeutic properties including anti-inflammatory, sedative, hypnotic, narcotic, and general tonic [
13]. The root extract of
Withania has been found to be effective in protecting against hydrogen peroxide- and β-amyloid (1–42)-induced cytotoxicity in NGF-differentiated PC12 cells [
14]. Withania contains active ingredients like steroidal alkaloids and lactones known as “withanolides.” Among the bioactive components of
Withania, Withaferin A (Wit A) and withanolide D are well characterized which contribute to the most of the pharmaceutical properties of this plant [
15]. Wit A has been shown to exhibit anti-inflammatory acitivity via NF-kB activation inhibition by blocking IkB phosphorylation and inhibiting IkB kinase activation [
16,
17]. It also inhibited lipopolysaccharide (LPS)-induced PGE
2 production and COX-2 expression in BV-2 cells and primary microglia, and these effects are mediated, at least in part, by reduced phosphorylation and nuclear translocation of STAT1 [
18].
The anticancerous activity of Ashwagandha has already been well documented by our lab using both in vitro and in vivo model systems [
19‐
21]. Further, it has also been shown to have neuroprotective potential against glutamate cytotoxicity [
22].
In view of the previous reports, the present study was planned to further elucidate anti-neuroinflammatory potential of Ashwagandha leaf water extract (ASH-WEX) and one of its active chloroform fraction (fraction IV (FIV)) in LPS-induced primary microglial cells and BV-2 murine microglial cell line as an in vitro model system. LPS-induced activation of the microglial cells is a useful model for the study of neuroinflammation and nerve cell injury as they release various pro-inflammatory and neurotoxic factors [
23]. We have also done preliminary study using β-amyloid-activated BV-2 and primary microglial cells as the inflammatory model to confirm the anti-neuroinflammatory potential of ASH-WEX and FIV in alternative condition. β-amyloid both recruits and activates the microglia, thereby initiating vicious cycle of the inflammation between β-amyloid accumulation, activated microglia and microglial inflammatory mediators, which enhance β-amyloid deposition and neuroinflammation thus considered as the unifying factor in the development of AD [
24]. Further, we examined the signaling cascades associated with ASH-WEX and FIV on the proliferation and migration of LPS-induced microglial cells and their phenotypic changes which have been found to be responsible for neuronal damage in various neurological disorders. We further studied the effect of ASH-WEX and FIV on various inflammatory pathways like nuclear factor-kB (NFkB), activator protein 1 (AP1), and pAkt
SER-473 which are known to mediate the production of various pro-inflammatory mediators like reactive nitrogen species (RNS), reactive oxygen species (ROS), inflammatory cytokines, and matrix metalloproteinases (MMPs) as a result of microglial cell activation.
Methods
Ashwagandha leaf extract
The ASH-WEX was prepared by suspending 10 g of dry leaf powder of Ashwagandha in 100 ml of water and stirring it overnight at 45 °C in shaker incubator. The suspension was then filtered under sterile conditions to obtain the aqueous leaf extract. The filtrate obtained was treated as 100 % aqueous extract and diluted in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % fetal bovine serum (FBS) according to experimental requirement.
One of its fractions was prepared by solvent extraction in the following order: hexane → chloroform → ethylacetate → butanol. Then, the active chloroform fraction was further fractionated by thin-layer chromatography using hexane: ethylacetate as the mobile phase and four fractions were obtained. Out of these, one fraction was active in suppressing LPS-induced microglial activation and was labeled as the FIV. It was then dried and dissolved in DMSO at the concentration of the 50 mg/ml and diluted in DMEM according to the experimental requirement. ASH-WEX and FIV were also analyzed for the presence of the possible bioactive components Wit A and Withanone by reverse-phase chromatography. For HPLC, the samples were prepared by dissolving dried ASH-WEX and FIV in methanol at 10 and 2 mg/ml concentration, respectively. Purified Wit A and Withanone dissolved in methanol were used as the standards to determine their concentration in ASH-WEX and FIV.
Primary microglial cultures
Primary microglia-enriched cultures were obtained from primary mixed glial cultures from 1-day-old Wistar rat pups. Animal care and procedures were followed in accordance with the guidelines of the Animal Ethical Committee, Guru Nanak Dev University, Amritsar, India. To obtain mixed glial cultures, the brain was dissected; their meninges were carefully removed and digested with 0.05 % trypsin-EDTA solution for 10 min at 37 °C. Trypsinization was stopped by adding an equal volume of DMEM culture medium supplemented with 10 % FBS. Cells were pelleted (5 min, 200 g), resuspended in culture medium, and brought to a single-cell suspension by repeated pipetting. Cells were seeded and cultured at 37 °C in a 5 % CO
2 humidified atmosphere. Medium was replaced after every 3 days. Microglial cultures were prepared by the mild trypsinization method [
25]. Briefly, after 18–20 days, in vitro (DIV) mixed glial cultures were treated for 30 min with 0.25 % trypsin containing 1 mM EDTA diluted with DMEM containing 1 mM Ca
2+ in 1:3 ratio. This resulted in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving a population of firmly attached cells identified as 98 % microglia. Isolated microglial cells were seeded in multiwell plates by trypsinization with 0.25 % trypsin-EDTA for 10 min and treated after 24 h of seeding.
BV-2 cell culture and treatments
BV-2 cell line was obtained from National Brain Research Centre (NBRC), Manesar, Haryana, India. BV-2 cells are murine-cultured microglial cells immortalized after transfection with a v-raf/v-myc recombinant retrovirus. The cells were maintained in DMEM medium with 10 % FBS (Biological Industries) and 1× PSN mix (Invitrogen, Carlsbad, CA, USA) at 37 °C in 5 % CO2 and humid environment.
Both primary and BV-2 microglial cultures were pretreated with ASH-WEX and FIV for 2 h before adding 100 ng/ml LPS to cultures (LPS (rough strains) from Escherichia coli F583 (Rd mutant) and 5 nM β-amyloid (Sigma-Aldrich, Saint Louis, Missouri, 63103 USA). Treatment was done for 36 h before harvesting for different assays.
Cytotoxicity and cell viability assays
ASH-WEX and one of its active fraction (FIV) were tested for cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The cell viability was quantified by the conversion of yellow MTT by mitochondrial dehydrogenases of living cells to purple MTT formazan at 595-nm wavelength [
26].
Cell morphology studies
Morphological changes in both primary and BV-2 microglial cells treated with different concentrations of ASH-WEX and FIV with or without activation with LPS and β-amyloid were studied by phase contrast microscopy. These experiments were performed in 24-well plates containing poly-l-lysine-coated coverslips (20,000 cells/ml). The phase contrast images of culture were taken using Phase Contrast Inverted Microscope (Nikon TE2000). Nuclear morphology was studied by staining the nucleus with an AT-rich region-specific fluorescent stain, i.e., 4′,6-diamidino-2-phenylindole (DAPI).
Immunostaining
Both primary and BV-2 microglial cells, control and treated, were washed with 1× phosphate-buffered saline (PBS) thrice and were fixed with acetone to methanol followed by permeabilization with 0.3 % Triton X-100 in PBS (PBST). Cells were then incubated with anti-α-tubulin (1:500) or anti-NF-kB (1:300) or anti-AP1 (1:300) or anti-HSP-70 (1:300) (from Sigma-Aldrich) diluted in 2 % BSA, for 24 h at 4 °C in humid chamber. After two to three washings with 0.1 % PBST, the cells were incubated with the secondary antibody anti-mouse IgG 488 and anti-rabbit IgG 488 (prepared in 2 % BSA (1:500)) for 2 h at room temperature. Cells were incubated with (DAPI, 1:5000 in 1× PBS) for 10 min for nuclear staining and then mounted with anti-fading reagent (Fluoromount, Sigma). For CellRox and Mitotracker staining, after treatment period, the dye was added in culture according to the manufacturer’s instructions; cells were then fixed and mounted. Images were captured using Nikon AIR Confocal Laser Microscope and analyzed using NIS elements AR analysis software version 4.11.00. Experiment was performed in triplicate.
Protein assay and Western blotting
For total protein extraction, BV-2 microglial cells were grown and treated in 100-mm petri dishes followed by harvesting with PBS-EDTA (1 mM). Nuclear and cytoplasmic protein fractionation was performed using a CelLytic NuCLEAR extraction kit from Sigma-Aldrich. Briefly, the cell pellet obtained was allowed to swell with hypotonic buffer (100 mM HEPES, pH 7.9, 15 mM MgCl2, and 100 mM KCl). To the swollen cells in lysis buffer, 10 % IGEPAL CA-630 solution was added to a final concentration of 0.6 % and vortexed vigorously for 10 s. The cells are then disrupted, the cytoplasmic fraction was removed, and the nuclear proteins were released from the nuclei by a high-salt buffer (20 mM HEPES, pH 7.9, with 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, and 25 % (v/v) glycerol). For total protein extraction, the cell pellet was homogenized in RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 1.0 % NP-40). Protein concentration was determined by the Bradford method. Protein lysate (30–50 μg) was resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto a PVDF membrane (Hybond-P) using the semi-dry Novablot system (Amersham Pharmacia). Further, membranes were probed with anti-mortalin (1:1000), anti-HSP70 (1:2500), anti-NFkB (1:2500), anti-OX-42 (1:1000), anti-OX-6 (1:1000), anti-PARP (1:1500), anti-Bcl-xl (1:1000), anti-AP1 (1:2500), anti-Akt (1:2500), and anti-Cyclin D1 (1:2000) (Sigma-Aldrich) and anti-proliferating cell nuclear antigen (PCNA) (1:2000) (Chemicon International, Temecula, CA, USA), for overnight at 4 °C. This was followed by washing with 0.1 % TBST and incubation with HRP-labeled secondary antibodies for 2 h at room temperature. Immunoreactive bands were detected by ECL Plus Western blot detection system (Amersham Biosciences) using LAS 4000 (GE Biosciences). α-Tubulin has been used as an endogenous control for normalizing the expression of the protein of interest. α-Tubulin and histone 3 were used as internal control for cytoplasmic and nuclear expression of proteins of interest, respectively. The change in expression of gene of interests was taken on the average of IDV values obtained from at least three independent experiments.
Cell cycle analysis
BV-2 microglial cells were seeded at the cell density 2.5 × 105 per ml in 100-mm-diameter petri plates and then treated according to the regime for 36 h. Cells from four petri dishes were pooled together. After 36 h of treatment, cells were trypsinized and then centrifuged at 1000 rpm. The cell pellet was resuspended in 1 ml of ice-cold PBS and then fixed with ice-cold 70 % ethanol for 2 h at 4 °C. Cells were then centrifuged and resuspended in 1 ml of PBS and incubated for 15 min and again centrifuged and resuspended in propidium iodide (PI) staining solution (100 mM Tris pH 7.4, 150 mM CaCl2, 0.5 mM MgCl2, 0.1 % NP-40, and 3 μM PI) and scanned with BD Accuri C6 Flow cytometer (BD Biosciences). DNA content histograms and cell cycle phase distribution were analyzed from at least 50,000 single events by excluding cell aggregates based on scatter plots of fluorescence pulse area versus fluorescence pulse width using FCS Express 4 flow research edition software (De Novo software).
Annexin V-FITC (apoptosis) assay
To further explore whether ASH-WEX and FIV caused the apoptosis of the activated BV-2 microglia, cell suspension was harvested from cells grown and treated in 100 mm petris. Cells from the four petri dishes were pooled together. Cells were stained with Annexin V conjugated with FITC and PI using Annexin V-FITC Apoptosis Detection Kit (Miltenyi Biotech, San Diego, CA, USA). Briefly, 1 ml of 1× binding buffer was added to cells, and pellet was obtained after centrifugation at 5000 rpm. One hundred microliters of 1× binding buffer and 10 μl Annexin V-FITC was added to it and incubated for 15 min in the dark. Pellet was obtained by centrifugation at 5000 rpm after adding 1 ml 1× binding buffer and resuspended in 500 μl of 1× binding buffer. Five microliters of propidine iodide solution was added immediately prior to analysis by BD C6 Accuri flow cytometer.
Wound scratch assay
In order to test anti-migration potential of ASH-WEX and FIV, BV-2 microglial cells were grown to confluent monolayer and then wounded by scratching the surface with a pipette tip. Cells were firstly pretreated with the ASH-WEX and FIV followed by activation with LPS. The initial wounding and the migration of cells in the scratched area were photographically monitored for 12–24 h after the treatment. Images were analyzed by Image Pro Plus software version 4.5.1 from the media cybernetics for the migration of cells in the scratched area by calculating the distance covered by the invading cells after incubation. Experiment was repeated for at least three times in triplicate.
Gelatin zymogram study
In order to study the effect of ASH-WEX and FIV on matrix metalloproteinase (MMP 2, MMP 9)-conditioned media from both primary and BV-2 microglial cell cultures after treatment were separated on a 10 % SDS-PAGE containing 0.1 % gelatin. After electrophoresis, gels were washed with renaturation buffer (Invitrogen) for 1 h to remove SDS. The gel was then incubated in zymogram developing buffer (Invitrogen) at 37 °C for 48 h followed by staining with Coomassie brilliant blue and destaining in buffer containing 50 % methanol and 10 % acetic acid (v/v). The location of gelatinolytic activity was detected as clear white bands.
Nitrite determination
Both primary and BV-2 microglial cells were seeded and treated as per the regimen. Nitrite determination was done using the Griess reagent nitrite determination kit (Molecular Probes, Invitrogen) as per the manufacturer’s protocol. Briefly, the culture conditioned media was collected and mixed with the Griess reagent, and the readings were obtained at the 548-nm wavelength. The nitrite concentrations were calculated using the sodium nitrite standard curve.
Pro-inflammatory cytokine assay
Cell culture conditioned media was collected from the different treatment conditions and analyzed for the presence of the pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) using the pro-inflammatory cytokine sandwich ELISA-based kits (from Sigma-Aldrich).
Statistical analysis
Values are expressed as mean ± standard error of the mean (SEM) obtained from at least three independent experiments. The Sigma Stat for Windows (version 3.5) was used to analyze the results by one-way ANOVA (Holm-Sidak post hoc method), in order to determine the significance of the mean values. Values of p < 0.05 were considered as statistically significant.
Discussion
Suppression of microglial-mediated neuroinflammation is considered as an important target in developing the therapeutics for the treatment of neurodegenerative diseases. The current study was aimed to study the anti-neuroinflammatory potential of the ASH-WEX and its active chloroform fraction (FIV). MTT assay revealed that treatment of the BV-2 microglial cells with ASH-WEX and FIV for 48 h reduced their proliferation rate, since ASH-WEX and FIV treatment decreased the cell viability beyond the concentration ≥0.5 % and ≥15 μg/ml, respectively, in dose dependent manner. So 0.2 % of ASH-WEX and 10 μg/ml of FIV were chosen as the effective dose for further experiments. BV-2 and primary microglia activated with both 100 ng/ml of bacterial endotoxin LPS and 5 nm of β-amyloid were used as in vitro model system for preliminary experiments.
In the adult brain, microglia exhibit ramified morphology and are involved in immune surveillance. In response to the immunological insult, these quiescent microglial cells retract their ramifications, transforming into an amoeboid-like cells with motile protrusions responsible for its inflammatory phenotype [
27]. α-Tubulin immunostaining and phase contrast images showed that ASH-WEX and FIV inhibited both LPS- and β-amyloid-induced amoeboid morphology of these cells and restored their ramified morphology. At the molecular level, ASH-WEX and FIV pretreatment significantly suppressed the expression of the microglial-specific marker Iba-1 which was upregulated in both LPS- and β-amyloid-treated BV-2 and primary microglial cells. Iba-1, 17 kDa calcium-binding adaptor protein, has been reported to be upregulated during inflammation which helps to discriminate between the surveilling and activated microglia [
28,
29]. These observations provided the first line of evidence that ASH-WEX and FIV inhibited LPS- and β-amyloid-induced microglial activation xand suppressed neuroinflammation. Further, ASH-WEX- and FIV-pretreated BV-2 microglial cells also showed the downregulated expression of the MHC II compared to LPS-treated BV-2 microglial cells as detected by OX-6 immunostaining.
LPS-induced activation of both primary and BV-2 microglial cells was seen to increase the production of ROS (as detected by CellROX staining) which was associated with higher mitochondrial activity as evident from increased fluorescence of Green FM dye. The mitochondrial dysfunction plays important role in the ROS production in various models of chronic inflammation [
30,
31]. LPS-treated primary and BV-2 microglial cells also showed higher level of nitrite released in the media which is already reported to cause upregulation of iNOS and results in the synthesis of high levels of NO [
32]. Interestingly, pretreatment with ASH-WEX and FIV suppressed the effects of LPS as both ASH-WEX and FIV were observed to inhibit production of ROS and nitrite, thus resulting in reduced mitochondrial stress. It may be suggested that both extracts may prevent the oxidative damage during neuroinflammation by regulating the mitochondrial homeostasis. Various previous studies also reported anti-oxidative potential of
Withania leaf extract in physiological abnormalities seen in the PD [
33]. These observations are also supported by the upregulated expression of the mitochondrial stress response proteins like HSP 70 and mortalin in the ASH-WEX- and FIV-pretreated LPS-activated microglia. The overexpression of these chaperone proteins are known to protect the mitochondria and reduce oxidative stress [
34,
35]. Upregulation of HSP 70 is also reported to suppress LPS-induced cytokine expressions by inhibiting the IkBα degradation and NFkB nuclear translocation [
36]. Shikonin is reported to attenuate the production of NO/iNOS, TNF-α, IL-1β, and COX-2 in LPS-stimulated microglia by inhibiting Akt phosphorylation [
37].
Enhanced production of ROS amplifies the inflammatory reaction and contributes to the subsequent neuronal damage in neurodegenerative diseases. This was evident from the sandwich ELISA-based immunoassay data showing dramatic increase in TNF-α, IL-1β, and IL-6 production in LPS-treated microglial cells. This increase in the release of these cytokines was significantly inhibited by ASH-WEX and FIV pretreatment to these LPS-treated cells. Experiments carried out in vitro and in vivo have demonstrated that TNF-α promotes neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in PD [
38]. Experiments using different mutant mice deficient in iNOS or TNFα receptors have shown reduced neurotoxicity [
39,
40]. A recent study reported that LPS activates the microglia with immediate superoxide release and later enhances the production of TNF-α, NO, prostaglandin E
2 (PGE
2), and IL-1β [
41]. The current data may suggest that ASH-WEX and FIV inhibited microglial-mediated neuroinflammation by preventing the release of the ROS, RNS, and pro-inflammatory cytokines which work synergistically to induce the neurotoxicity and increase the duration of the chronic inflammation [
42]. Wit A also has been reported to inhibit the iNOS expression and NO production in Raw 264.7 cells [
43].
LPS-induced inflammatory cascades in microglial cells include activation of pathways such as NFkB, MAPK, and PI3K-Akt pathways, which have high implication in neurodegenerative processes [
23]. Downregulated expression of the transcription factor NFkB in the ASH-WEX- and FIV-pretreated cells was also accompanied by inhibition of its translocation from cytoplasm to nuclear compartment. NFkB activation and its translocation are critical for the expression of the various cytokines, chemokines, receptors required for neutrophil adhesion and migration, MHCs, iNOS, and COX-2 in microglia in response to LPS [
44,
45]. Our results are in line with the recent reports suggesting that anti-inflammatory activity of 30 % methanolic extract of the
Withania leaves in stainless steel implant induced inflammation in zebrafish by inhibiting the NFkB transcriptional activity [
46]. Further, its active component, i.e., Wit A has also been reported to inhibit the NFkB activity in cellular model of cystic fibrosis inflammation [
47].
ASH-WEX and FIV pretreatment to these microglial cells also inhibited LPS-induced AP1 expression. The transcriptional activation of the AP1 via JNK/MAPK pathway has been reported to induce the production of the pro-inflammatory factors in LPS-activated microglia [
48,
49]. The concerted action of both AP1 and NFkB signaling pathways together results in the induction of pro-inflammatory cytokines [
50,
51]. Therefore, simultaneous inhibition of both NFkB and AP1 transcriptional activity by ASH-WEX and FIV may be responsible for their anti-neuroinflammatory potential of ASH-WEX and FIV. Many other plant products like brevicompanine, curcumin, and isooreintin have been reported to attenuate the LPS-induced production of the inflammatory mediators through inhibiting both NFkB and AP1 pathways [
52‐
54]. Further, ASH-WEX- and FIV-pretreated cells also showed significant downregulation in the expression of p-Akt
SER473 as compared to LPS alone treated group. Phosphorylation of Akt leads to changes in the catalytic activity of downstream targets, such as GSK-3, mTOR, COX-2, and mPGES-1, and results in secretion of various prostaglandins and leukotrienes having inflammatory activity [
55‐
57].
During any insult to the brain or injury, the activation of microglia leads to induction of proliferation and migration of these cells towards the site of the lesion with elevated production of the various inflammatory molecules [
58,
59]. ASH-WEX and FIV pretreatment was observed to downregulate the expression of the cell cycle regulatory proteins PCNA and Cyclin D1 along with cell cycle arrest of the LPS-activated BV-2 microglia at G
0/G
1 and G
2/M phase. The inhibition of proliferation was also accompanied by induction of apoptosis as depicted by AnnexinV-FITC flow cytometry data. ASH-WEX- and FIV-mediated induction of apoptosis may be the result of inhibition of anti-apoptotic protein Bcl-xl. Upregulated Bcl-xl expression has been detected in reactive microglia of the patient with neurodegenerative diseases [
60]. Both the extracts were also seen to elevate the levels of the PARP expression, whose cleavage into 27 and 87 kDa fragments leads to the apoptosis progression. Apoptosis of the inflamed microglial cells is one of the mechanisms of the anti-inflammatory compounds to inhibit the neuroinflammation [
61].
Further, ASH-WEX and FIV also inhibited the production of the MMP 2 and MMP 9 suggesting their role in maintaining the blood-brain barrier (BBB) integrity as illustrated by gelatin zymogram data. MMPs are microglial inflammatory factors that degrade components of the basal lamina, leading to the disruption of the BBB, and contribute to the neuroinflammatory response in many neurological diseases [
62]. Further released proteases from activated microglia result in proteolytic degradation of the BBB which further enhances the recruitment of the microglia to the lesion site [
63], as depicted by maximum migration of cells to the scratched area in LPS-treated cultures. ASH-WEX and FIV were observed to reduce rate of migration of cells in scratched area which may also be due to collective effect of the controlled proliferation, apoptosis, and cell cycle arrest by the ASH-WEX and FIV pretreatment. Based on these observations, it may be suggested that ASH-WEX and especially FIV may prove to be potential agent for preventing the uncontrolled migration of the microglia to the lesion site during CNS injury.
Abbreviations
AP1, activator protein 1; ASH-WEX, Ashwagandha leaf water extract; DIC, differential interference contrast; FITC, fluorescein isothiocyanate; FIV, fraction IV; LPS, lipopolysaccharide; IL-1β, interleukin-1 β; MMPs, matrix metalloproteinases; MHC II, major histocompatibility complex II; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide; NFkB, nuclear factor-kB; PCNA, proliferating cell nuclear antigen; PBS, phosphate-buffered saline; RNS, reactive nitrogen species; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; SEM, standard error of the mean; TNF-α, tumor necrosis factor-α
Acknowledgements
Muskan Gupta is thankful to the Council of Scientific and Industrial Research (CSIR), India, for the fellowship. Infrastructure provided by the University Grants Commission (UGC), India, under UPE and CPEPA schemes and Department of Biotechnology (DBT), India, under DISC facility is highly acknowledged.