Copyright © 2016 Mohit Kwatra, Ashok Jangra, Priyansha Choubey, Mangala Lahkar; NIPER Guwahati, Assam, India; E-mail: [email protected] 1 Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, Assam, India Lipopolysaccharide aggravates the restraint stress-induced behavioral deficits and hippocampal damage: Effect of Fisetin treatment Mohit Kwatra 1 , Ashok Jangra 1 , Priyansha Choubey 1 , Mangala Lahkar 1 P.1.g.003 NIPER - Guwahati Parameters Experimental groups MDA level (μM/g of tissue wt) Nitrite level (mM/mg of protein) Reduced glutathione (mg/g of tissue) Acetylcholinestrase activity (micromoles/min/ mg of protein) Control 32.48 ± 3.49 45.93 ± 5.32 62.55 ± 1.93 0.12 ± 0.01 LPS (0.83 mg/kg) 55.39 ± 4.54 a** 94.39 ± 3.33 a** 29.49 ± 4.44 a*** 0.23 ± 0.01 a** RS 65.32 ± 4.39 a*** 99.35 ± 6.39 a*** 32.44 ± 5.01 a*** 0.29 ± 0.03 a*** RS + LPS (0.83 mg/kg) 64.32 ± 2.34 a*** 96.34 ± 5.34 a*** 24.39 ± 3.54 a*** 0.33 ± 0.03 a*** Fisetin (15 mg/kg) + LPS 37.15 ± 1.39 b* 64.15 ± 4.39 b** 52.95 ± 2.99 b*** 0.15 ± 0.01 Fisetin (15 mg/kg) + RS 44.39 ± 1.30 c** 62.39 ± 5.32 c*** 49.95 ± 3.90 c* 0.17 ± 0.01 c** Fisetin (15 mg/kg)+ RS +LPS 43.49 ± 6.32 d** 68.11 ± 2.40 d** 45.95 ± 2.34 d** 0.22 ± 0.02 d** Results BDNF β-actin NF-κB Introduction Neuropsychiatric disorders like depression, anxiety, and dementia in humans often coexist together in certain medical conditions such as cancer, cardiovascular diseases, neurodegenerative disease which decreased the quality of life in patients and increase rate of mortality. Numerous reports provided insights for implications of chronic restraint stress and lipopolysaccharide (LPS) results in hippocampal neuronal damage and various neurobehavioral anomalies in rodents [1, 2]. In this present study, we have tried to establish a model where 28 days chronic restraint stress mediated neurobehavioral and neurochemical changes got aggravated by a single dose of LPS triggering various neuroinflammatory cascade resulting hippocampus neuronal damage. We further investigated the effect of Fisetin (3, 7, 3′,4′ -tetrahydroxyflavone) flavonol in preventing the restraint stress (RS) exposure and lipopolysaccharide (LPS) treatment induce neurobehavioral deficits and hippocampal damage. Methods β-actin HO-1 1. Effect of Fisetin on Restraint stress and LPS induced learning and memory function impairment 2. Effect on Fisetin on Restraint stress and LPS induced anxiety-like behavior in mice (1) Elevated Plus Maze (2) Open Field Test 3. Effect on Fisetin on Restraint stress and LPS induce depressive-like behavior in mice II. Effect on Fisetin treatment on Restraint stress and LPS induced Oxido-nitrosative stress, altered AchE activity in the hippocampus of mice 4. Effect on Fisetin treatment on Restraint stress and LPS modulated hippocampal gene expression levels β-actin IL-1β I. (1) (2) (3) (4) (5) β-actin Nrf-2 Acknowledgement We would like to thank the NIPER-Guwahati, Department of Pharmaceuticals, Ministry of Chemical and Fertilizers, Government of India for financial support to carry out this work. The authors are immensely thankful to Department of Biotechnology (DBT), Government of India for awarding international travel support. β-actin All the values are expressed as mean ± SEM (n = 6); a*** P < 0.001, a** P < 0.01, a* P < 0.05 compared with the normal control group; b*** P < 0.001, b** P < 0.01, b* P < 0.05 compared with the LPS group;,, c*** P < 0.001, c** P < 0.01, c* P < 0.05 compared with the RS group; d*** P < 0.001, d** P < 0.01, d* P < 0.05 compared with the LPS + RS group [1] Jangra, A., Dwivedi, S., Sriram, C.S., Gurjar, S.S., Kwatra, M., Sulakhiya, K., Baruah, C.C., Lahkar, M., 2016. Honokiol abrogates chronic restraint stress-induced cognitive impairment and depressive-like behaviour by blocking endoplasmic reticulum stress in the hippocampus of mice. Eur J Pharmacol 770, 25–32. [2] Sriram, C.S., Jangra, A., Gurjar, S.S., Mohan, P., Bezbaruah, B.K., 2016. Edaravone abrogates LPS-induced behavioral anomalies, neuroinflammation and PARP-1. Physiol Behav 154, 135–144. Discolosure: No potential conflict of interest Conclusion All experiments were performed in accordance with the CPCSEA, Government of India guidelines. Adult male Swiss albino mice (25–30 g) were divided into following experimental groups: Group 1: No stress/no LPS normal control Group 2: Restraint stress exposure for 6 h per day for 28 days (RS). Group 3: LPS injected (0.83 mg/kg, i.p.) on the 28 th day (LPS control). Group 4: Restraint stress (6 h per day for 28 days) and LPS injected (0.83 mg/kg, i.p.) on the 28 th day (RS + LPS). Group 5: Restraint stress (6 h per day for 28 days) and Fisetin (15 mg/kg, p.o.) treatment from 15 th to 28 th day. Group 6: Fisetin (15 mg/kg, p.o.) treatment from 15 th to 28 th day and LPS injected (0.83 mg/kg, i.p.) on the 28 th day Group 7: Restrained stress (6 h per day for 28 days) and LPS injected (0.83 mg/kg, i.p.) on the 28 th day; and Fisetin (15 mg/kg, p.o.) treatment for last 14 days. References The Combined paradigm of restraint stress and lipopolysaccharide represented behavioral deficits and hippocampus damage. Fisetin treatment significantly ameliorates the altered neurobehavioral and neurochemical alterations via inhibition of oxido-nitrosative stress and neuroinflammation through restoring of dysregulated inflammatory mediators gene expression level. 1 7 14 21 28 Restraint Stress (6 h per day) 3h Post LPS 29 LPS (0.83 mg/kg), i.p. Test for Depressive-like behavior -Forced Swim Test - Tail Suspension Test Animal Sacrificed Biochemical estimations - MDA level -Nitrite Level -Reduced Glutathione level -Acetylcholinesterase activiy Reverse transcriptase PCR -NFκB -Nrf2 -IL-1β -HO-1 -BDNF Fisetin (15 mg/kg, p.o.) Morris Water Maze Test (from 24 th to 28 th day) Hippocampus isolated ROS/RNS Production IL-1β and TNF-alpha production Toll like receptor (TLR) – 4 activation Oxido-nitrosative stress and neuroinflammation; microglia cells activation DNA damage DNA damage -Activation of NFκB proinflammatory signaling -Dysrupted BDNF signaling Neuroinflammation and Neuronal Cell death Neurobehaviorlal and Neurochemical deficits Chronic Restraint Stress LPS activation of innate immune response NF-κB Oxido-nitrosative stress and neuroinflammation Nrf-2 IL-1β, iNOS, COX-2 activation Antioxidants (GSH, GST, GPx, HO-1) Hypothesis Illustration of the study plan Test for Anxiety-like behavior -Elevated Plus Maze - Open Field Test Fisetin ? ? ?