Urtica dioica leaves modulates hippocampal smoothened-glioma associated oncogene-1 pathway and cognitive dysfunction in chronically stressed mice

Biomedicine & Pharmacotherapy 83 (2016) 676–686

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Urtica dioica leaves modulates hippocampal smoothened-glioma associated oncogene-1 pathway and cognitive dysfunction in chronically stressed mice Sita Sharan Patela , Neeraj Mahindrooa,b , Malairaman Udayabanua,* a b

Department of Pharmacy, Jaypee University of Information Technology, Waknaghat, 173234, Himachal Pradesh, India School of Pharmaceutical Sciences, Shoolini University, Solan, 173229, Himachal Pradesh, India

A R T I C L E I N F O

Article history: Received 23 April 2016 Received in revised form 30 June 2016 Accepted 13 July 2016 Keywords: Chronic stress Cognition Hypericum perforatum Sonic hedgehog Urtica dioica

A B S T R A C T

The present study was aimed to evaluate the effect of Urtica dioica (UD) extract against chronic unpredictable stress (CUS)-induced associative memory dysfunction and attempted to explore the possible mechanism. Male Swiss albino mice (25–30 g) were divided into six groups, viz. group-I received 0.3% carboxymethyl cellulose and served as control (CTRL), group II was exposed to CUS (21 days) and received vehicle (CUS), group III was subjected to CUS and received Hypericum perforatum extract (350 mg/kg, p.o.) (CUS + HYP), group IV received Hypericum perforatum extract (350 mg/kg, p.o.) (CTRL + HYP); group V was subjected to CUS and received UD extract (50 mg/kg, p.o.) (CUS + UD), group VI received UD extract (50 mg/kg, p.o.) (CTRL + UD). CUS significantly induced body weight loss (p < 0.05) and associative memory impairment in step down task (p < 0.05) as compared to control mice. CUS significantly downregulated Smo (p < 0.05), Gli1 (p < 0.01), cyclin D1 (p < 0.05), BDNF (p < 0.01), TrKB (p < 0.01) and MAPK1 (p < 0.01) mRNA expression in hippocampus as compared to control mice. CUS significantly increased the levels of TBARS (p < 0.01) and nitric oxide (p < 0.001), and decreased catalase (p < 0.001) and total thiol (p < 0.01) in plasma resulting in oxidative stress and inflammation. Chronic UD administration significantly reverted CUS mediated body weight loss (p < 0.05) and cognitive impairment (p < 0.05). UD administration significantly decreased the levels of TBARS (p < 0.01) and nitric oxide (p < 0.05), and increased the levels of catalase (p < 0.01) and total thiol (p < 0.05) in plasma. Chronic UD administration significantly upregulated hippocampal Smo (p < 0.05), Gli1 (p < 0.001), cyclin D1 (p < 0.05), BDNF (p < 0.05), TrKB (p < 0.05) and MAPK1 (p < 0.05) in stressed mice. Further, UD extract did not reverse cyclopamine induced downregulation of Gli1 and Ptch1 mRNA in hippocampal slices. UD modulated Smo-Gli1 pathway in the hippocampus as well as exerted anti-inflammatory and antioxidant effects. UD extract might prove to be effective for stress mediated neurological disorders. ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction Chronic unpredictable stress (CUS) is known to induce depression and cognitive deficit [1]. Chronic stress causes alterations in hippocampal neurogenesis [2], which is known to affect mood [3], synaptic plasticity [4] and memory [5]. Sonic hedgehog (Shh) is essential for adult hippocampal neurogenesis [6]. Activation of canonical Shh signaling is initiated by binding of Shh to its receptor patched (Ptch), mediating activation of smoothened (Smo) resulting in transcription of target genes

* Corresponding author. E-mail address: [email protected] (M. Udayabanu). http://dx.doi.org/10.1016/j.biopha.2016.07.020 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.

including Ptch, Gli1 (glioma-associated oncogene-1) and cyclin D1 [7,8]. The Shh/Gli1 signaling pathway is important in the maintenance and proliferation of neural progenitor cells in the adult hippocampus resulting in improved cognitive performance [9]. Gli1 is itself a Shh-target gene, induces cyclin D expression and result in cell cycle progression in hippocampus [10]. Shh signaling induces development of cholinergic neurons in brain [11] and deficits of Ptch1-Gli1 signaling in hippocampus contribute to depression and cognitive decline [12,13]. Hypericum perforatum (HYP) is a flowering plant belonging to the family Hypericaceae. HYP is significantly superior to placebo and its effectiveness is similar to standard antidepressant drugs [14]. HYP extract improves memory [15], proliferation of progenitor cells, restore synaptic plasticity [2] and Alzheimer's

S.S. Patel et al. / Biomedicine & Pharmacotherapy 83 (2016) 676–686

pathology [16]. HYP is known to ameliorate hippocampal neurogenesis and reverse cognitive dysfunction associated with depressive like behavior [2,17,18]. Urtica dioica (UD) is commonly known as stinging nettle belonging to the family Urticaceae. UD leaves are known to contain cholineacetyltransferase, 5-hydroxytryptamine, acetylcholine, gentisic acid, esculetin, scopoletin and rutin [19,20]. UD extract compensate astrocytes loss in the dentate gyrus [21] and reverse N-methyl-D-aspartate induced cognitive dysfunction [22]. UD extract modulate hippocampal insulin signaling and reverse memory dysfunction [23]. The aim of present study is to investigate the involvement of Shh signaling in the hippocampus during CUS mediated cognitive deficit and the effect of UD extract. 2. Materials and methods 2.1. Collection, identification and standardization of plant materials Plant materials were collected from the North Western Himalayan region and authenticated from Department of Forest Product, Dr. Y.S. Parmar University of Horticulture & Forestry, India. Leaves of UD (specimen number-12399) and aerial parts of HYP (specimen number-13505) were shade dried, pulverized and passed through 40 mesh sieve followed by extraction. The extraction of UD leaves and aerial parts of HYP (4
50 g) was performed at room temperature, with constant shaking during 48 h, using methanol and water (1:1) as solvent. The extract thus obtained was filtered, centrifuged, evaporated under reduced pressure and lyophilized. The yield of HYP and UD extracts were 8.6% (w/w) and 17.9% (w/w), respectively. The total phenolic content in UD and HYP extracts were determined by spectrophotometric method [24] and expressed as mg gallic acid equivalents (GAE) per g of dried sample. The total flavonoid content was determined from the standard curve of quercetin [25] and expressed as mg quercetin equivalents (QE) per g of dried sample. Identification and quantification of scopoletin in UD leaves extract was performed as per method described earlier [26]. The scopoletin was determined by reversed-phase high-performance liquid chromatography (RP-HPLC), using octadecylsilane (ODS2) column (Waters Spherisorb ODS2 Column, 80 Å, 5 mm, 4.6 mm X 250 mm), 0.5% glacial acetic acid in methanol-water (26:55) as mobile phase and UV detection at 310 nm. Calibration curve of standard scopoletin was prepared. Crude UD extract was then subjected to high-performance liquid chromatography (HPLC) analysis and quantified using calibration curve. In earlier study, we identified the active constituents in UD leaves extract by liquid chromatography–mass spectrometry (LC–MS) analysis [27]. Identification and quantification of active constituents in HYP extract was performed as per method described earlier [28]. The mobile phase consisted of water (A, containing 20% methanol and 0.5% trifluoroacetic acid) and acetonitrile (B, containing 10% methanol and 0.5% trifluoroacetic acid). Our analysis followed a linear gradient program. Initial conditions were 90% A; 0–20 min, changed to 30% A; 20–25 min, to 10% A; 25–30 min, to 0% A kept to 60 min; 60–65 min, went back to 90% A. The flow-rate was kept at 1 ml/min, injection volume was 10 ml and photo diode array detection at 200–800 nm. Calibration curve of standard hypericin and hyperforin were constructed from HPLC chromatogram. The amount of hypericin and hyperforin in HYP extract was determined. We further identified the active constituents in HYP extract by LC–MS analysis [29]. 2.2. Animals Male Swiss albino mice weighing 25–30 g was housed under a 12 h light/dark cycle at 24  2  C. The animals had access to food

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and water ad libitum. All animal experiments were carried out in accordance with Committee for the Purpose of Control and Supervision of Experiments on Animals (1716/Po/a/13/CPCSEA) and Institutional Animal Ethical Committee (6/UB/2014/JUIT/IAEC) clearance. 2.3. CUS procedure and drug administration Animals were divided into the following groups: group-I received 0.3% carboxymethyl cellulose and served as control (CTRL); group II was exposed to CUS and received vehicle (CUS); group III was subjected to CUS and received HYP extract (350 mg/ kg) (CUS + HYP); group IV received HYP extract (350 mg/kg) (CTRL + HYP); group V was subjected to CUS and received UD extract (50 mg/kg) (CUS + UD); group VI received UD extract (50 mg/kg) (CTRL + UD). All drugs used in the study were administered by oral route. Dose of HYP and UD used in the present experiment were selected from previous experiments [15,20]. The animals were subjected to CUS paradigm as described previously [30]. Animals undergo stress paradigm once a day over a period of 21 days as follows: day 1, cold swim (8  C, 3 min); day 2, tail pinch (1 min); day 3, food and water deprivation (24 h); day 4, immobilization (3 h); day 5, overnight illumination; day 6, foot shock (20 trials, 0.5 mA, 5.0 sec maximum duration, 1 min intervals); day 7, tail pinch (2 min); day 8, cold swim (10  C, 5 min); day 9, overnight illumination; day 10, foot shock (20 trials, 0.5 mA, 5.0 s maximum duration, 30 s intervals); day 11, food and water deprivation (24 h); day 12, immobilization (4 h); day 13, tail pinch (3 min); day 14, overnight illumination with wet cage; day 15, cold swim (6  C, 3 min); day 16, foot shock (20 trials, 0.5 mA, 5.0 sec maximum duration, 1 min intervals); day 17, food and water deprivation (24 h); day 18, tail pinch (2 min); day 19, immobilization (5 h); day 20, overnight illumination with tilted cage; day 21, cold swim (8  C, 3 min). Body weight was constantly measured throughout the study. After 3 weeks of CUS and drug treatment, animals were subjected to step down task. After cervical dislocation, blood was collected via retro-orbital puncture and plasma was separated for biochemical estimation. 2.4. Step-down task (SDT) The apparatus comprised a chamber (35
35
10 cm) with an elevated wooden platform (15
10
5 cm) at the centre. The floor consists of stainless steel bars placed in parallel (0.5 cm apart). On the first training day, mice were exposed to a 5-min learning trial, in which they were permitted to move freely in chamber prior to being placed on the platform. If the animals stepped down from the platform, they were exposed to an electric foot shock. After 24 h, memory retention was tested in similar manner but the shock was not delivered. 2.5. Real-time quantitative reverse transcription PCR (qPCR) After cervical dislocation, hippocampus was dissected and total RNA was isolated using TRIzol reagent. The integrity of RNA was checked on 2% agarose gel and quantified using spectrophotometer (mDropTM Plate-Thermo Scientific). The reverse transcription of 3 mg of RNA was performed using First strand cDNA kit (Fermentas life sciences). qPCR amplifications were performed in an CFX96TM Real-Time PCR Detection System (Bio-Rad) using iQTM SYBR green supermix (Bio-Rad). Reactions were carried out in total volumes of 12.5 ml, included 2.5pM of each primer (Table 1) and 1 ml of cDNA template containing 100 ng of cDNA. The thermal cycler conditions for cDNA amplification were as follows: Step 1, 95  C for 3:00 min;

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Table 1 Sequence of oligonucleotides used for qRT-PCR. S No.

Gene

Forward primer 5’!3’

Reverse primer 5’!3’

1 2 3 4 5 6 7 8 9 10

Shh Ptch1 Smo Gli1 Hhip Cyclin D1 BDNF TrkB MAPK1 GAPDH

CAA GTA CGG CAT GCT GGC TC AGG CGC TAA TGT TCT GAC CA GAC TCC GTG AGT GGC ATC TG AAG CCT GAG CCT GAG TCT GT GGG TCA CAT CTT GGG ATT TG GCG TAC CCT GAC ACC AAT CTC ATG TCT ATG AGG GTT CGG CG ACT GTG AGA GGC AAC CCC AA CCC TTA GAC ACT GTG ACG GT TTC ACC ACC ATG GAG AAG GC

AAG GTG AGG AAG TCG CTG TA CCT CCT GCC AAT GCA TAT AC GTG GCA GCT GAA GGT GAT GA GGT CAC TGG CAT TGC TAA AG GAG GCA CTT GTT CGG TCT GA ACT TGA AGT AAG ATA CGG AGG GC GCG AGT TCC AGT GCC TTT TG ATC ACC AGC AGG CAG AAT CC CAC AGT CCC AAA GCC ACA AA GGC ATG GAC TGT GGT CAT GA

Step 2, 95  C for 10 s, 52–58  C for 30 s and 72  C for 2:20 s (35 cycles). GAPDH was used as an internal control.

quantification of hyperforin, hypericin and scopoletin in brain homogenate was performed as described above.

2.6. Ex vivo assay

2.8. Biochemical analysis

Hippocampus was isolated from normal control mice and sliced into 500 mm sections using a tissue slicer. Slices were distributed into the 6-well tissue culture plates containing artificial cerebrospinal fluid (aCSF: 125 mM NaCl, 2.7 mM KCl, 1.25 mM NaH2PO4, 25 mM NaHCO3, 10 mM glucose, 0.5 mM CaCl2, 7 mM MgSO4, 4 mM ketamine, pH 7.4). The plates were then incubated with the supply of 95% O2 and 5% CO2 (Fisher Scientific, Model 371). After preincubation period, tissues were separated into different experimental groups. To study the effect of UD on Smo-Gli pathway, the tissues were divided into 13 groups: 1) control group, which was incubated with aCSF for 30 min; 2) cyclopamine (Cyc) 2.5 mM group, which was incubated with aCSF for 15 min and then with Cyc (2.5 mM) for additional 15 min; 3) Cyc 5 mM group, which was incubated with aCSF for 15 min and then with Cyc (5 mM) for additional 15 min; 4) purmorphamine (Pur) 1 mM group, which was incubated with aCSF for 15 min and then with Pur (1 mM) for additional 15 min; 5) Cyc 5 mM + Pur 1 mM group, which was incubated with Cyc (5 mM) for 15 min and then with Pur (1 mM) for an additional 15 min; 6) HYP 50 mg group, which was incubated with aCSF for 15 min and then with HYP (50 mg) for additional 15 min; 7) HYP 100 mg group, which was incubated with aCSF for 15 min and then with HYP (100 mg) for additional 15 min; 8) HYP50 mg + Cyc 5 mM group, which was incubated with Cyc (5 mM) for 15 min and then with HYP (50 mg) for an additional 15 min; 9) HYP 100 mg + Cyc 5 mM group, which was incubated with Cyc (5 mM) for 15 min and then with HYP (100 mg) for an additional 15 min; 10) UD 125 mg group, which was incubated with aCSF for 15 min and then with UD (125 mg) for additional 15 min; 11) UD 250 mg group, which was incubated with aCSF for 15 min and then with UD (250 mg) for additional 15 min; 12) UD 125 mg + Cyc 5 mM group, which was incubated with Cyc (5 mM) for 15 min and then with UD (125 mg) for an additional 15 min; 13) UD 250 mg + Cyc 5 mM group, which was incubated with Cyc (5 mM) for 15 min and then with UD (250 mg) for an additional 15 min. After incubation, slices were homogenized in TRIzol reagent and the expression level of Ptch1 and Gli1 mRNA were studied using qPCR.

The level of plasma thiobarbituric acid reactive substances (TBARS) was determined as per method described previously [32]. Nitric oxide (NO) level was determined as nitrite plus nitrate using Greiss reagent spectrophotometrically [33]. Catalase level was measured by the method described previously [33]. The level of total thiol in plasma was determined as per method described previously [34].

2.7. Identification of phytoconstituents in brain homogenate of stressed mice Quantification of phytoconstituents in brain homogenate was performed as described previously [31] with modifications. Whole brain tissue was homogenized in 2 ml of PBS and centrifuged at 10,000g for 10 min. Thereafter, 500 ml of the supernatant was extracted with 5 ml of dichloromethane. The dichloromethane extract was evaporated to dryness and dissolved in 100 ml of mobile phase. Thereafter, 20 ml was injected into HPLC system. The

2.9. Statistical analysis All data were expressed as mean  SEM values. Two-way ANOVA with Bonferroni post-hoc test was used to analyze body weight data. The statistical significance of other data were assessed by one-way ANOVA followed by Tukey’s post-hoc test with a confidence level of p < 0.05. 3. Results 3.1. Standardization of plant materials Phytochemical analysis revealed the presence of total phenolics (0.388  0.002 mg GAE/g, n = 6) and flavonoids (0.350  0.67 mg QE/g, n = 6) in UD leaves extract. Total phenolics and flavonoids content in HYP extract was 0.517  0.007 and 0.442  0.61, respectively. Mass calculated for C35H52O4 hyperforin, exact mass: 536.38, found 535.38 (M-1) (Suppl. 1A) and C30H16O8 hypericin, exact mass: 504.08, found 503.08 (M-1) (Suppl. 1B) in LC–MS analysis. HPLC chromatograms of standard hypericin (tR = 9.14) and hyperforin (tR = 31.00) were reported in Fig. 1A and Fig. 1B, respectively. The hydroalcoholic extract of HYP showed peak at tR = 9.19 (hypericin) and tR = 31.07 (hyperforin) (Fig. 1C). Quantitative HPLC analysis revealed the presence of hyperforin (6.12%) and hypericin (0.37%) in HYP extract. In HPLC analysis, the standard scopoletin showed peak at tR = 14.29 (Fig. 1D) corresponding to scopoletin in UD extract (tR = 14.25) (Fig. 1E). Herein, hydroalcoholic extract of UD contain 6.5% of scopoletin. 3.2. Body weight loss and cognitive deficit Chronic stress significantly decreased the body weight (p < 0.05 vs. CTRL) on week three. Chronic UD administration significantly reduced body weight loss in chronically stressed mice comparable to HYP extract (p < 0.05 vs. CUS) (Fig. 2A). In SDT task, stressed mice showed no alteration in step-down latency (SDL) on day 2 (memory retention trial) when compared with their respective day 1 (acquisition trail) SDL. During memory retention trial on day 2, the stressed animals showed significantly

S.S. Patel et al. / Biomedicine & Pharmacotherapy 83 (2016) 676–686 0.070

OH

(A)

0.060

O

679

OH

0.050

H3C H3C

Hypericin - 9.144

AU

0.040 0.030 0.020 0.010

OH OH

OH

O

OH

0.000 0.00

0.035

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H3C

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CH3

OH

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O

CH3

0.025

CH3

0.020

O

0.015

CH3

Hyperforin - 31.005

AU

30.00

CH3

0.010

H3C

0.005

CH3

CH3

0.000 0.00

5.00

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0.10 0.05 0.00 0.00

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(D) HO

0.020

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0.015 AU

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1 Scopoletin - 14.296

0.15

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Hyperforin - 31.078

Hypericin - 9.193

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(E)

0.0030 0.0025

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Scopoletin - 14.254

0.0035

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3.097

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0.0005 0.0000 2.00

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Fig. 1. HPLC chromatograms of the standard hypericin (A), hyperforin (B), 50% methanolic extract of Hypericum extract (C), standard scopoletin (D) and hydro-alcoholic extract of UD leaves (E).

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36

CTRL CUS

*

34

CUS+HYP CTRL+HYP

32

CUS+UD CTRL+UD

30 28 #

26 24 0

1

2

3

Weeks

(B) 200

Transfer latency (s)

Mean value of BW from 1-3 weeks in gms

(A)

150 100

CTRL CUS CUS+HYP CTRL+HYP CUS+UD CTRL+UD

50

**

*

#

0 Day 1

Day 2

Fig. 2. Effect of UD on CUS-induced alterations in body weight (A) and step down task (B). Data were mean  SEM values (n = 6). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + HYP and CUS + UD. *p < 0.05, **p < 0.01. CTRL = control; CUS = chronic unpredictable stress; HYP = Hypericum perforatum extract; UD = Urtica dioica extract.

decreased SDL as compared to normal animals (p < 0.05 vs. CTRL). Chronic treatment with UD significantly increased (p < 0.05 vs. CUS) the SDL in stressed mice on day 2. During memory retention trial, HYP treated stressed mice showed significantly increased SDL (p<0.01 vs. CUS) (Fig. 2B). 3.3. Smo-Gli pathway and synaptic plasticity Three weeks of CUS exposure did not modulate the expression level of hippocampal Shh mRNA (p>0.05 vs. CTRL) in stressed mice. Further, chronic UD and HYP administration did not alter the expression of Shh mRNA in control and stressed mice (p > 0.05) (Fig. 3A). Chronically stressed mice showed no significant alteration in hippocampal Ptch1 mRNA expression (p > 0.05 vs. CTRL). Chronic UD and HYP administration did not modulate hippocampal Ptch1 mRNA expression in stressed mice (p > 0.05 vs. CUS). UD and HYP administration significantly upregulated the mRNA expression of Ptch1 in control mice (p < 0.001 vs. CTRL and p < 0.01 vs. CTRL, respectivey) (Fig. 3B). Stressed mice showed significant downregulation in hippocampal Smo mRNA expression (p < 0.05 vs. CTRL). Chronic UD administration significantly upregulated the hippocampal Smo mRNA expression in stressed mice (p < 0.05 vs. CUS) comparable to HYP (p < 0.05 vs. CUS). Also, UD and HYP treatment significantly upregulated the mRNA expression of Smo in control mice (p < 0.05 vs. CTRL) (Fig. 3C). Chronically stressed mice showed significant downregulation in hippocampal Gli1 mRNA expression (p < 0.01 vs. CTRL). Chronic UD administration significantly upregulated the hippocampal Gli1 mRNA expression in stressed mice (p < 0.001 vs. CUS) comparable to HYP (p < 0.01 vs. CUS). Chronic UD and HYP treatment significantly upregulated the mRNA expression of Gli1 in control mice (p<0.01 vs. CTRL) (Fig. 3D). Three weeks of CUS exposure did not modulate the expression level of hippocampal hedgehog interacting protein (Hhip) mRNA (p>0.05 vs. CTRL). Further, chronic UD administration did not alter the expression of Hhip mRNA in control and stressed mice (p > 0.05). Chronic HYP administration significantly downregulated the mRNA expression of Hhip in stressed mice (p < 0.01 vs. CTRL, CUS + UD and CUS) (Fig. 3E). Chronically stressed mice showed significant downregulation in hippocampal cyclin D1 mRNA expression (p < 0.05 vs. CTRL). Chronic UD administration significantly upregulated the hippocampal cyclin D1 mRNA expression in stressed mice (p < 0.05 vs. CUS) comparable to HYP administration (p < 0.05 vs. CUS) (Fig. 3F). Hippocampal brain derived neurotrophic factor (BDNF) mRNA expression was significantly downregulated (p < 0.01 vs. CTRL) in chronically stressed animals. Chronic UD and HYP administration significantly upregulated the mRNA expression of BDNF in stressed

mice (p < 0.05 vs. CUS and p < 0.001 vs. CUS, respectively) (Fig. 3G). Chronically stressed mice showed significant downregulation in hippocampal tyrosine kinase B (TrkB) mRNA expression (p < 0.01 vs. CTRL). Chronic UD and HYP administration significantly upregulated the hippocampal TrkB mRNA expression in stressed mice (p < 0.05 vs. CUS) (Fig. 3H). CUS showed significant downregulation in hippocampal mitogen activated protein kinase-1 (MAPK1) mRNA expression (p < 0.01 vs. CTRL). Chronic UD administration significantly upregulated the hippocampal MAPK1 mRNA expression in stressed (p < 0.05 vs. CUS) and control mice (p < 0.01 vs. CTRL). Chronic HYP administration significantly upregulated the mRNA expression of hippocampal MAPK1 in control (p<0.01 vs. CTRL) and stressed (p<0.01 vs. CUS) mice (Fig. 3I). Cyc 2.5 mM did not alter the mRNA expression of Gli1 (p>0.05 vs. CTRL), while Cyc 5 mM significantly downregulated (p < 0.001 vs. CTRL) the mRNA expression of Gli1 in hippocampal slices. Acute Pur (Smo agonist) 1 mM significantly upregulated the mRNA expression of Gli1 in hippocampal slices (p < 0.001 vs. CTRL), while pre-treatment with Cyc 5 mM the level of Gli1 mRNA was significantly downregulated (p < 0.001 vs. Pur 1 mM). Both dosage (50 and 100 mg) of HYP significantly upregulated the mRNA expression of Gli1 in hippocampal slices (p < 0.001 vs. CTRL), while this effect was significantly blocked in presence of Cyc 5 mM (p<0.001 vs. Pur 1 mM) and (p<0.001 vs. HYP 50 and 100 mg). UD 125 mg treatment did not modulate the Gli1 mRNA expression in hippocampal slices (p>0.05 vs. CTRL). UD 250 mg treatment significantly upregulated the mRNA expression of Gli1 in hippocampal slices (p < 0.001 vs. CTRL), while pre-treatment with Cyc 5 mM blocked the effect of UD 125 mg and UD 250 mg on Gli1 mRNA expression (p < 0.001 vs. Pur 1 mM) and (p < 0.001 vs. UD125 and 250 mg) (Fig. 3J). 5 mM of Cyc treatment significantly downregulated (p < 0.001 vs. CTRL) the mRNA expression of Ptch1 in hippocampal slices. Acute Pur 1 mM significantly upregulated the mRNA expression of Ptch1 in hippocampal slices (p < 0.001 vs. CTRL), while pre-treatment with Cyc 5 mM, the level of Ptch1 mRNA was significantly downregulated (p < 0.001 vs. Pur 1 mM). Both dosage (50 and 100 mg) of HYP significantly upregulated the mRNA expression of Ptch1 in hippocampal slices (p < 0.001 vs. CTRL), while this effect was significantly blocked in presence of Cyc 5 mM (p<0.001 vs. Pur 1 mM) and (p<0.001 vs. HYP 50 and 100 mg). UD 125 mg treatment did not modulate the Ptch1 mRNA expression in hippocampal slices (p>0.05 vs. CTRL). UD 250 mg treatment significantly upregulated the mRNA expression of Ptch1 in hippocampal slices (p < 0.001 vs. CTRL), while pre-treatment with Cyc 5 mM blocked the effect of UD 125 mg and UD 250 mg on Ptch1 mRNA expression (p < 0.001 vs. Pur 1 mM) and (p < 0.001 vs. UD125 and 250 mg) (Fig. 3 K).

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Fig. 3. Effect of UD on CUS-induced alterations in hippocampal Shh (A), Ptch1 (B), Smo (C), Gli1 (D), Hhip (E), cyclin D1 (F), BDNF (G), TrkB (H) and MAPK1 (I) mRNA expression. Effect of UD on Gli1 (J) and Ptch1 (K) mRNA expression in hippocampal slices pre-treated with Smo antagonist Cyc. Data were mean  SEM values (n = 4). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + HYP and CUS + UD; CCTRL vs. CTRL + HYP and CTRL + UD; +CTRL vs. CUS + HYP; aCUS + UD vs. CUS + HYP; #CTRL vs. Cyc 5 mM; *CTRL vs. Pur 1 mM, HYP 50 mg, HYP 100 mg, UD 250 mg; aHYP 50 mg and UD 125 mg vs. HYP 50 mg + Cyc 5 mM and UD 125 mg + Cyc 5 mM; bHYP 100 mg and UD 250 mg vs. HYP 100 mg + Cyc 5 mM and UD 250 mg + Cyc 5 mM; +Pur 1 mM vs. Cyc 5 mM+ Pur 1 mM, HYP 50 mg + Cyc 5 mM, HYP 100 mg + Cyc 5 mM, UD 125 mg + Cyc 5 mM and UD 250 mg + Cyc 5 mM. *p < 0.05, **p < 0.01, ***p < 0.001. CTRL = control; CUS = chronic unpredictable stress; HYP = Hypericum perforatum extract; UD = Urtica dioica extract; Cyc = cyclopamine; Pur = purmorphamine.

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3.4. Oxidative and nitrative stress CUS significantly increased the level of plasma TBARS in mice (p < 0.01 vs. CTRL). Chronic UD administration significantly attenuated CUS induced elevated level of TBARS (p < 0.01 vs. CUS). UD treated control mice showed significantly decreased (p < 0.001 vs. CUS) level of TBARS as compared to stressed mice. Chronic HYP administration significantly decreased the level of plasma TBARS in stressed mice (p < 0.001 vs. CUS). HYP treated control mice showed significantly decreased (p < 0.001 vs. CUS) level of TBARS as compared to stressed mice (Fig. 4A). Level of plasma NO was significantly increased in stressed mice (p < 0.001 vs. CTRL). Chronic UD administration significantly attenuated CUS induced elevated level of NO (p < 0.05 vs. CUS). UD treated control mice showed significantly decreased (p < 0.001 vs. CUS) level of NO as compared to stressed mice. Chronic HYP administration significantly decreased the level of plasma NO in stressed mice (p < 0.01 vs. CUS). HYP treated control mice showed significantly decreased (p < 0.001 vs. CUS) level of NO as compared to stressed mice (Fig. 4B). CUS significantly decreased the level of plasma catalase in mice (p < 0.001 vs. CTRL). Chronic UD administration significantly increased the level of plasma catalase (p < 0.01 vs. CUS) in stressed mice. UD treated control mice showed significantly increased (p < 0.01 vs. CUS) level of catalase as compared to stressed mice. Chronic HYP administration significantly increased the level of plasma catalase in stressed mice (p < 0.001 vs. CUS). HYP treated control mice showed significantly increased (p < 0.001 vs. CUS) level of catalase as compared to stressed mice (Fig. 4C). Level of plasma thiols was significantly decreased in stressed mice (p < 0.01 vs. CTRL). Chronic UD administration significantly

increased the level of total thiol (p < 0.05 vs. CUS) in stressed mice. UD treated control mice showed significantly increased (p < 0.01 vs. CUS) level of total thiol as compared to stressed mice. Chronic HYP administration significantly increased the level of plasma total thiol in stressed mice (p < 0.05 vs. CUS). HYP treated control mice showed significantly increased (p < 0.001 vs. CUS) level of total thiol as compared to stressed mice (Fig. 4D). 3.5. Accumulation of UD and HYP constituents on whole brain HPLC chromatograms of standard hyperforin (tR = 31.10) and hypericin (tR = 9.15) were reported in Fig. 5A and Fig. 5B, respectively. The brain homogenate of HYP treated stressed mice showed peak at tR = 9.15 (hypericin) and tR = 31.09 (hyperforin) (Fig. 5C). Quantitative HPLC analysis revealed the presence of hyperforin (0.18%) and hypericin (0.06%) in rodent brain after CUS paradigm. In HPLC analysis, the standard scopoletin showed peak at tR = 18.31 (Fig. 5D) corresponding to scopoletin in brain homogenate (tR = 18.78) (Fig. 5E). Herein, UD treated stressed mice showed 0.2% of scopoletin in brain homogenate after CUS paradigm. 4. Discussion In the present study, we observed that CUS resulted in associative memory dysfunction evident from decreased step down latency, which is in line with previous report [35]. Chronic UD and HYP administration significantly increased the step down latency in chronically stressed mice. UD is known to improve learning and memory in hypercorticosteronemic mice [33].

Fig. 4. Effect of UD on CUS-induced alterations in TBARS level (A), nitric oxide level (B), catalase level (C) and total thiol level (D) in plasma. Data were mean  SEM values (n = 6). Significant differences: #CTRL vs. CUS; *CUS vs. CUS + UD and CUS +HYP;  CUS vs. CTRL + UD and CTRL +HYP. *p < 0.05, **p < 0.01, ***p < 0.001. CTRL = control; CUS = chronic unpredictable stress; HYP = Hypericum perforatum extract; UD = Urtica dioica extract.

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31.105

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Fig. 5. HPLC chromatogram of standard hyperforin (A), hypericin (B), brain homogenate of chronically stressed mice treated with Hypericum extract (C), standard scopoletin (D) and brain homogenate of chronically stressed mice treated with UD extract (E).

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Further, CUS significantly increased the body weight loss, whereas chronic UD and HYP administration significantly reversed stress mediated body weight loss on week three. Concurrent antidepressant administration during 21 days period alone is sufficient to reverse the effects of CUS [36]. Our earlier report suggest that hydroalcoholic extract of UD leaves showed the presence of scopoletin, gentisic acid, esculetin, quercetin and rutin (Suppl. 2) [27]. Quercetin, rutin and scopoletin are known to reverse cognitive dysfunction [37–39]. HYP extract is known to reduce depressive like behavior and cognitive dysfunction [14,15]. Chronic stress is known to affect hippocampal neurogenesis precipitating cognitive impairment [40], which is reversed by treatment with antidepressant drugs [41]. Shh signaling is a key determinant of hippocampal neurogenesis and cognition. In the present study, the mRNA expression of Shh in hippocampus was not altered in any of the group. Further, CUS did not modulate the level of Ptch1 mRNA expression in hippocampus but the level of Ptch1 mRNA was increased by chronic UD and HYP administration in control animals. CUS downregulated the mRNA expression of Smo and Gli1 in the hippocampus. A recent study suggests that depletion of amine transmitters, a clinical condition of depression resulted in decreased in Smo and Ptch receptor mRNA within the hippocampus [13]. Further, deficit of hippocampal Ptch1-Gli1 pathway is associated with cognitive dysfunction [12]. Chronic UD and HYP administration leads to significant improvement in Smo and Gli1 mRNA expression in both stressed and non-stressed mice. Chronic HYP administration induced hippocampal neurogenesis in stressed mice [2]. UD is reported to ameliorate muscarinic cholinergic system in hippocampus and cognition [27]. The coumarin scopoletin reduces anticholinergic and age impaired cognitive deficit [42]. Only HYP administered stressed mice showed downregulation of hippocampal Hhip mRNA. Hhip binds to Shh with high affinity and negatively regulate Shh signaling

[43,44]. Herein, CUS, HYP or UD treatment did not modulate canonical Shh signaling. CUS dysregulate non-canonical Shh signaling in hippocampus as observed through downregulation of Smo, Gli1 and MAPK1 mRNA. Shh signaling is modulated by noncanonical Src kinase pathway [45]. Activation of Src-MAPK pathway induces survival, repair and regeneration of hippocampal neurons [46]. Chronic UD and HYP administration significantly increased the mRNA expression of MAPK1. Pretreatment of hippocampal slices with Cyc (Smo antagonist) downregulated the mRNA expression of Gli1 and Ptch1. Neither UD nor HYP could reverse Cyc induced downregulation of Gli1 and Ptch1 mRNA in hippocampal slices, suggesting that UD and HYP treatment modulates Smo-Gli pathway. BDNF is reported to strongly influence synaptic morphogenesis, neuronal survival, long term potentiation of brain synapses as well as adult hippocampal neurogenesis [47,48]. CUS significantly downregulated the mRNA expression of BDNF and its receptor TrkB in hippocampus. Chronic UD and HYP treatment upregulated the mRNA expression of BDNF and its receptor TrkB in stressed mice. Upon binding to BDNF, the receptor TrkB activates MAPK/PI3 K/ cAMP-response element binding protein (CREB) pathway, resulting in cell survival, growth and synaptic plasticity [49,50]. Activation of CREB protein mediates the expression of cyclin D1 [51]. Shh signaling is known to induce cyclin D1 expression favour continue cell cycling [8] and hippocampal neurogenesis [52]. We observed that, cyclin D1 mRNA expression was downregulated, while chronic HYP and UD treatment significantly upregulated cyclin D1 mRNA expression in stressed mice. It could be hypothesized that UD and HYP might have mediated alteration in cyclin D1 expression partly by TrkB/MAPK/PI3 K/CREB and noncanonical hedgehog pathway. Mechanism of action of UD extract on hippocampal neurons is graphically represented in Fig. 6. Quercetin and rutin are known to enhance neurogenesis and

Fig. 6. Mechanism of action of UD extract on hippocampus: normal Shh signaling in hippocampus (A), effect of CUS on hippocampal Shh signaling (B), interaction of CUS and HYP/UD extract on hippocampal Shh signaling (C) and effect of UD/HYP extract on hippocampal Shh signaling (D).

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synaptogenesis in hippocampus [53,54]. Scopoletin reverse the downregulation of BDNF mRNA in brain resulting in amelioration of associative and spatial memory [55]. CUS induces neurological disorders by modulating antioxidant defense mechanism [56]. CUS resulted in elevation in the level of oxidative and nitrative stress markers in plasma, as indicated by raise in the levels of TBARS and NO, and depletion of antioxidants such as catalase and total thiol. Chronic UD and HYP administration reversed CUS-induced alteration in the level of catalase along with attenuation of oxidative and nitrative stress markers. UD and HYP extracts are known to possess antioxidant and anti-inflammatory effect [57,58]. Quercetin, esculetin, gentisic acid and rutin are known to reduce oxidative stress and inflammation [38,59,60,61– 63]. Orally administered HYP extract accumulated as hyperforin and hypericin in the brain tissue of depressed mice after last dose followed by 8hr fasting. Hyperforin and hypericin are well known antidepressant [14]. Hyperforin is known to possess cognitionenhancing and anxiolytic-like effects [64]. Similarly, orally administered UD extract accumulated as scopoletin in the brain tissue of depressed mice. Scopoletin inhibits monoamine oxidase [42] and attenuate depressive like behaviour [65] and cognition decline [66]. Shh mediated Gli1 transcription in hippocampus ameliorates neurogenesis and spatial memory performance in control animals [9]. Our observations indicate that chronic UD administration did not induce toxicity in control animals and relatively safe. Moreover, its clinical use is safe in humans [67].

[3] [4]

[5]

[6]

[7]

[8]

[9]

[10] [11]

[12]

[13]

[14]

5. Conclusion UD administration ameliorated Smo-Gli1 pathway in hippocampus along with attenuation of oxidative stress and inflammation. Phytochemical analysis revealed the presence of scopoletin, gentisic acid, esculetin, quercetin and rutin in UD extract, which might possibly be involved in the reversal of stress mediated neuronal dysfunction and needs further investigation. Results suggest that UD supplementation might prove to be effective for cognitive dysfunction associated with chronic stress. Conflict of interest

[15]

[16]

[17] [18]

[19]

The authors have declared that there is no conflict of interest. Acknowledgement Authors would like to thank Council of Scientific & Industrial Research for financial assistance as senior research fellowship (09/ 957(0002)/2012-EMR-I). This research was supported by partial funding from Defence Research and Development Organisation (Ministry of Defence, Govt. of India) (DLS/81/48222/LSRB-175/FSB/ 2008). Appendix A. Supplementary data

[20]

[21]

[22]

[23]

[24] [25]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. biopha.2016.07.020.

[26] [27]

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