STUDY THE EFFECT OF SILIBININ ON EXPERIMENTALLY INDUCED MEMORY IMPAIRMENT IN MALE RATS.

Aim of the work : is to study the effect of different doses of silibinin on D-galactose induced memory impairment in male rats. Methods: 60 male rats were divided into four groups, the first three groups were (10 rats each) and the fourth group (30 rats) was subdivided in to three subgroups (10 rats each)I-Control group was injected subcutaneously by physiological saline once daily for six weeks II- D-galactose group was injected subcutaneously by 150 mg/kg of D-galactose once daily for six weeks III-Placebo group 10 ml/kg of 0.5 % carboxymethylcellulose solution was injected intraperitonealy once daily for six weeks IV-Silibinin group include IVa(2 mg/kg),IVb(20mg/kg),IVc(200 mg/kg), silibinin was suspended in 10ml/kg of 0.5 % carboxymethylcellulose solution and injected intraperitonealy once daily for six weeks. Results: D-galactose group produced significant decrease step-through latency , increase time in the dark and increase in tissue malondialdehyde, nitrite, acetylcholine esterase, tumor necrosis factor-alpha level and caspase activity, it produced significant decrease in tissue catalase activity and brain-derived neurotrophic factor level when compared to control group.There was insignificant change in placebo group and silibinin (2 mg/kg) when compared to D-galactose group.While, it produced significant change in silibinin (20 mg/kg) and mg/kg) placebo group, it still produced significant change in silibinin (20 mg/kg) and silibinin (2 mg/kg) when compared to control group,it produced insignificant change in silibinin (200 mg/kg) when compared control group. Conclusion : Silibinin has role in memory disorders which develops efficient therapeutic approaches.

1. Group I: Control group: injected subcutaneously by1.5 ml physiological saline once daily for 6 week. 2. Group II: D-galactose group: 150 mg/kg of D-gal was subcutaneously injected once daily for 6 weeks (Ji et al., 2015). 3. Group III:(Placebo group): Carboxymethylcellulose (CMC) solution group: 10 ml/kg of 0.5 % CMC solution was given by intraperitoneal injection once daily for 6 weeks starting from the first day of D-gal injection (Duan et al., 2015). 4. Group IV: Silibinin treated group: Silibinin was given by intraperitoneal injection once daily for 6 weeks starting from the first day of D-gal injection was subdivided in to three subgroups according to the dose of silibinin (10 rats each): 5. Group Iva: Silibinin was suspended in10ml/kg of 0.5% CMC solution and was given by intraperitoneal injection once daily at the dose of 2 mg/kg (Duan et al., 2015). 6. Group Ivb: Silibinin was suspended in 10ml/kg of 0.5 % CMC solution and was given by intraperitoneal injection once daily at the dose of 20 mg/kg (Duan et al., 2015). 7. Group Ivc: Silibinin was suspended in 10ml/kg of 0.5 % CMC solution and was given by intraperitoneal injection once daily at the dose of 200 mg/kg (Duan et al., 2015).

At the end of the experiment:
Behavioral test was done in form of passive avoidance test to test memory function in all groups (Ji et al., 2015), the step through latency and time in the dark were estimated.
Passive avoidance test was performed using a two compartment chamber, one compartment was illuminated, the other was dark .At first there was training period during which each rat was received 3 trial of training with 30 minutes pause between them, in which each rat was first placed in the illuminated compartment (Ji et al., 2015). As soon as each rat entered the dark compartment, an electric shock with (frequency 50 Hz, duration 2 second and intensity 0.8 mA) was immediately delivered to the grid floor.
After 24 hour of training, each rat in the illuminated compartment, the time to enter the dark compartment (stepthrough latency in seconds) was estimated. Also, the time the rats spent in the dark compartment were estimated (Time in the dark in seconds)

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Then the rats of all groups were sacrificed, the hippocampus was dissected and following parameters were measured in its tissue: The sacrificed animals were packed in a special package according to safety precautions and infection control measures and were sent with hospital biohazard.

Statistical analysis:-
Results were expressed as Mean ± SD and all statistical comparisons were made by means of one-way ANOVA test, followed by Tukey's post hoc analysis, and p values less than 0.05 were considered statistically significant. Analysis was performed by statistical package for the social science software (SPSS version 22.0).

Results:-
Biochemical results from the present work revealed that D-galactose group produced statistically significant decrease step-through latency, significant increase time in the dark ands tatistically significant increase in tissue MDA, nitrite, AChE, TNF-α level and caspase 3 activity. But it revealed statistically significant decrease in tissue catalase activity and BDNF level when compared to control group.
However, there was statistically insignificant change between these parameters in placebo group when compared to D-galactose group.
Also, there was statistically insignificant change in silibinin (2 mg/kg) group when compared to placebo group. While, silibinin (20 mg/kg) and (200 mg/kg) groups produced statistically significant increase step-through latency, significant decrease time in the darkand decrease in tissue MDA, nitrite, AChE, TNF-α level and caspase activity. But it produced statistically significant increase in tissue catalase activity and BDNF level when compared to placebo group.
In addition, silibinin (20 mg/kg) and (200 mg/kg) groups produced same statistically significant change of these parameters when compared to silibinin (2 mg/kg) group, also silibinin (200 mg/kg) group produced same statistically significant change when compared to silibinin (20 mg/kg) group.
However, silibinin (20 mg/kg) and silibinin (2 mg/kg) groups still produced statistically significant change of these parameters as compared to control group, but silibinin (200 mg/kg) group produced insignificant change as compared to control group. High dose of D-gal also produces significant decrease in tissue BDNF level as compared to control group .Pratchayasakul et al., (2017)reported that BDNF is a biomarker for neurocognitive disorders. Oxido nitrosative stress with significant increase in nitrite induced by high dose D-gal lead to increase NOS expression which causes diminished BDNF. Also, increased pro inflammatory cytokines productions activate NF-κb which inhibits BDNF production (Sulakhiya et al., 2014).

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Another mechanism, that cognitive impairment in the D-gal model has been shown to be caused by generation of ROS, This overproduction of ROS cause memory impairment by increase in oxidative damage to many molecules including lipid, protein and DNA, also it cause lipid peroxidation, protein carbonyl formation and genome instability (Vida et al., 2014).This is evidenced by significant increase in tissue MDA (Aydın et al.,2016). Over production of free radicals show increase peroxynitrite level with also significant increase in nitrite (Pratchayasakul et al., 2017). Lu et al.,( 2012)added that NO production catalyzes a high-output pathway of NO production that is capable of causing neuronal peroxynitrite-mediated damage and dysfunction which share in memory damage ,also the increase in production of ROS can be detoxified by endogenous antioxidants, causing their cellular stores to be depleted resulting insignificant decrease in catalase activity (Jangra et al.,2015).  Other possible mechanism is that the effect of INN could be attributed to its potent antioxidant effects , alterations in AChE activity may be induced by the increased free radical formation which could provoke lipid peroxidation of cerebellar membranes causing changes in the conformational state of the AChE molecule and consequently inhibit its activity (Chtourou et al., 2012). We can conclude that INN can be used as therapeutic agents in the treatment of memory disorders and cognitive dysfunction.