A STUDY OF THE EFFECTS OF DIFFERENT DOSES OF SILYMARIN ON ROTENONE-INDUCED RAT MODEL OF PARKINSONISM.

Yasmeen Mohamed El-Harty, Rizk Mahmoud El-Khouly, Hala Fouad El-Baradey and Ahmed El-Sayed Abdelfattah. Department of medical physiology, Faculty of Medicine, Tanta University, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 16 October 2018 Final Accepted: 18 November 2018 Published: December 2018


ISSN: 2320-5407
Int. J. Adv. Res. 6 (12), 925-937 927 received daily IP injection of silymarin at doses of 0, 100, 200 and 300 mg/kg ,dissolved in 50% PEG, (Haddadi et al., 2014) respectively for 2 weeks (Plangár et al., 2013) Neurobehavioral Tests: At the end of the experiment, the following behavioral tests were carried out on all groups for neurobehavioral analysis of parkisnonian features. Rats were trained on these tests for three days, and then the tests were carried out: 1-Bar test for catalepsy: in which rats were placed with both forepaws on a bar which was 10 cm above the base in half rearing position and the time of removal of one or both paws was recorded and considered as the bar test elapsed time. Cut-off time for descent latency time was considered as 180 s (El-Horany et al., 2016).
2-Forepaw stride length to assess the shuffling gate observed in PD patients: In this test, rats were trained to walk down a narrow corridor which was lined by clean white paper. Their forepaws were dipped in ink and they were allowed to walk along the corridor again to analyze any deficits in stride length through measuring the distance between the footprints (Taylor et al., 2010).

Blood and tissue collection:
All rats were anaesthetized by i.p injection of phenobarbital (50 mg/kg) (Samson et al., 1957). Retro-orbital blood samples were collected from all animals for serum preparation. Then, all animals were sacrificed by cervical dislocation. Brains were dissected very quickly and carefully to avoid any mechanical trauma, washed with ice cold saline, and then divided into pieces, wrapped in aluminum foil and stored at -80°C till used for preparation of brain tissue homogenate The sacrificed animals were packed in special package according to safety precautions and infection control measures and 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 version 22.00 for windows (SPSS Inc., Chicago, IL, USA,2013) and GraphPad prism software version 7.0 (San Diego, USA, 2016).

Effect of silymarin on rotenone induced neurobehavioral deficits:
As shown in figure 2 , the results of this work clearly indicate that rotenone could induce the characteristic motor parkinsonian features including catalepsy & shuffling gate as shown by the significant change induced by rotenone in the bar test & the forepaw stride length tests respectively when compared to the control group. On the other hand, silymarin injection cause significant decrease in descent latency time in bar test and significant increase in forepaw stride length when compared to rotenone group. Silymarin effect was shown to be graded and dose-dependent as evidenced by the significant difference between the results of silymarin injection at a dose of 100 mg/kg and 300 mg/kg which eventually showed insignificant change when compared to the control.

Effect of silymarin on rotenone induced oxidative, nitrostative and inflammatory stress:
As shown in figure 4 (A & B) & figure 5 (A &B), rotenone injection induced a significant increase of MDA levels in the brain tissue indicating increased lipid peroxidation, decreased catalase activity in the brain tissue indicating 928 decreased antioxidant capacity, increased TNF-α indicating inflammatory stress and increased nitrite/nitrate in brain tissue indicating nitrosative stress. Silymarin injection showed significant change when compared to rotenonetreated groups in a dose dependant manner as aforementioned.

Effect of silymarin on rotenone-induced apoptosis:
As shown in figure 6, the results of this work showed that rotenone injection induced significant increase of brain tissue levels caspase-3 when compared to control respectively. Silymarin showed significant decrease of caspase-3 when compared to rotenone in a dose dependent manner. These levels showed insignificant change when compared to control at silymarin dose of 300 mg/kg.

Effect of silymarin on rotenone-induced DNA oxidative damage:
As shown in figure 7, our results showed that rotenone causes significant oxidative DNA damage indicated by the significant increase in serum 8-OHdG levels when compared to control. Silymarin treatment resulted in significant decrease of 8-OHdG levels when compared to rotenone reaching a result insignificant from control at the dose of 300 mg/kg in a dose dependent manner.

Effect of silymarin on BDNF:
As shown in figure 8, rotenone significantly lowered the BDNF level when compared to control. Silymarin, however could elevate the BDNF level significantly starting at a dose of 100 mg/kg, which showed significant increase compared to rotenone results, in a dose-dependent manner as mentioned before. BDNF levels increased to reach an insignificant change when compared to control at doses of 200 mg/kg and 300mg/kg.

Effect of silymarin on dopamine
As shown in figure 9, rotenone also induced significant decrease in dopamine levels in brain tissue which was alleviated by silymarin starting at a dose of 200 mg/kg which showed significant increase when compared to rotenone and showed insignificant change when compared to control. The effect of silymarin showed a dosedependent manner as mentioned before. The results of (Haddadi et al., 2015) were similar to our results regarding catalepsy. All 3 doses of silymarin significantly attenuated the severity of induced catalepsy, indicated by bar test results, but 300 mg/kg showed an earlier response, after a single dose, than 200 and 100 mg/kg. Moreover, (Haddadi et al., 2013) showed that pretreatment with silymarin in the same three doses was also capable of preventing catalepsy in 6-hydroxydopamine

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The results of this work revealed that rotenone injection induced a state of oxidative, inflammatory and nitrostative stress condition as evidenced by the significant increase of MDA, decreased catalase activity, increased TNF-α and increased nitrite/nitrate in brain tissue respectively. These effects were alleviated by silymarin injection. rotenone has been suggested as an inhibitor of mitochondrial complex I activity uniformly throughout the brain (Xiong et al., 2009). Complex I inhibition causes generation of OH and ROS initially leading to depletion of antioxidant molecules such as glutathione in substantia nigra pars compacta (SNpc) (Saravanan et al., 2005). the enzymes that remove both O2-and H2O2 protect the cells against intermediates of oxygen generated during normal aerobic metabolism (Zelko et al., 2002). Impairment of catalase activity by rotenone, as evident in this study, hinders this antioxidant process. under neuropathological conditions, microglia are activated in response to dopaminergic neuronal damages. Additionally, activated microglia produce various potentially neurotoxic molecules, including iNOS and proinflammatory cytokines, such as TNF-α and IL-1b ( showed that unlike the lower dosage levels, long-term administration (8 weeks) of silymarin at a dose of 100 mg/kg might cause pro-oxidant effects on organs such as hippocampus which showed augmentation of NO and MDA content and upregulation of proinflammatory mediators such as IL-1b at mRNA level. This controversy might be attributed to the prolonged duration of administration. Also, (Johnson, et al., 2003) showed that SM at a dose of 250 mg/kg results in increased expression of TNF-α, IL-1B, IL-6 and iNOS in mice spleenocytes. Also, in contradiction to our study, the results of (Baluchnejadmojarad et al., 2010) who showed that nitrate/nitrite level was non-significantly lowered by silymarin at a dose of 200 mg/kg when compared to the PD group which may be attributed to the shorter duration of treatment. Also (Lee et al., 2015) reported that silibinin, the major active constituent of silymarin, had no clear modulatory effect on neuroinflammation suggesting that suggest that silibinin has a direct neuroprotective role rather than indirect effects via modulating neuroinflammation.
The results of this work also showed that rotenone injection induced apoptotic changes as evidenced by the significantly increased brain tissue levels caspase-3 whereas Silymarin showed an anti-apoptotic effect. (Baluchnejadmojarad et al., 2010 andHuang et al., 2016) reported that oxidative stress also compromises mitochondrial oxidative phosphorylation which leads to decreased energy output and secondary cell death. The critical damage to mitochondria, enhanced ROS formation and provoked oxidative damage caused by rotenone exposure mediates rotenone-induced apoptosis. Inhibition of apoptosis by silymarin is dependent on inhibition of NF-kB activation (Manna, et al., 1999).
Our results also showed that rotenone causes significant oxidative DNA damage indicated by the significant increase in serum 8-OHdG levels while Silymarin treatment resulted in significant decrease of 8-OHdG levels. Rotenoneinduced mitochondrial dysfunction leads to increased oxidative stress as evident in the present study. Hence, the oxidative damage to lipids, proteins and DNA can be detected as in the reports by (Dias et al., 2013 andHwang, 2013) who reported oxidative damage in lipids, proteins and DNA in brain tissue from PD patients.
Overall, in all three studies (Alam et al., 1997;Zhang et al., 1999 andHegde, 2006), late stage PD cases were used, and therefore the increase in DNA damage was found in the remaining small fraction of surviving neurons (or perhaps glia), as it is likely that by this point, the vast majority of dopaminergic neurons have already degenerated. It is unclear whether DNA damage is responsible for neuronal loss or is an epiphenomenon of the disease in the surviving neurons or both. In addition, mtDNA damage was not investigated (Coppedè & Migliore, 2015).

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To our knowledge, this is the first study to demonstrate the effect of silymarin on DNA oxidative damage in rotenone-induced PD. Our results show no contradiction with the work of (Di Cesare Mannelli et al., 2013) who reported that silibinin decreased oxidative DNA damage in oxiplatin-induced oxidative stress in nervous systemderived cellular models.
The results of this work revealed that rotenone significantly lowered the BDNF level whereas silymarin, however could elevate the BDNF level significantly. To our knowledge, this study was the first to report the effect of silymarin on BDNF in rotenone-induced rat model of PD. (Song et al., 2016) reported the reduced expression of BDNF and High-affinity tyrosine kinase beta (TrkB) induced by lipopolysaccharide-injection was reversed by silibinin-treatment in memory impairment rat model which correlates with our results. Also, (Jangra et al., 2015) reported that silibinin ameliorated the aluminum-induced decrease in BDNF in hippocampus.
The results of this work revealed that rotenone induced significant decrease in dopamine levels in brain tissue which was alleviated by silymarin starting at a dose of 200 mg/kg which showed significant increase when compared to rotenone and showed insignificant change when compared to control. Regarding silymarin's efect on dopamine, (Perez et al., 2014) reported that silymarin protected dopaminergic neurons starting at a dose of 100 mg/kg proposing that this was possibly due to its antioxidant and anti-inflammatory properties. They also showed that silymarin alone had no effect on dopamine levels in control mice which is shown in this study as well. Controversially, Perez et al., 2014 highlighted that silymarin doses of 200 and 300 mg/kg showed less dopamine preserving effect when compared to doses of 50 and 100 mg/kg which was attributed to the pro-oxidant and pro-inflammatory effect of silymarin at high doses as previously reported by .

Conclusion:-
We can conlcude that silymarin is a potent neuroprotective flavonoid via its antioxidant, anti-inflammatory and antiapoptotic effect which eventually enhanced dopamine level and the characteristic motor deficits of PD. The investigated neuroprotective effect was shown to be dose-dependent.

Recommendations:
Our data suggests that silymarin can be used as a promising adjuvant therapy in treatment of PD. However, further clinical trials are required with special reference to the dose in order to avoid any undesirable side effects of high dose.

Disclaimer:
The authors report no conflict of interest. This study was self-funded. Values are mean ± SEM (n = 10). *, # and ** Denote a statistically significant difference at p ≤ 0.05, * denotes statistical significance when compared to control group, # denotes statistical significance when compared to Rotenone-treated group, ** denotes statistical significance when compared to 100 mg/Kg SM-RT group using one way ANOVA with Tukey post hoc test. Values are mean ± SEM (n = 10). *, # and ** Denote a statistically significant difference at p ≤ 0.05, * denotes statistical significance when compared to control group, # denotes statistical significance when compared to Rotenone-treated group, ** denotes statistical significance when compared to 100 mg/Kg SM-RT group using one way ANOVA with Tukey post hoc test :-Effect of different doses of SM on on brain tissue homogenate nitrite/ nitrate level (μmol/g) (A) and brain tissue homogenate tumor necrosis factor alpha level (TNF-α) (pg/g) (B) in Rtinduced PD: Values are mean ± SEM (n = 10). *, #, ** and *** Denote a statistically significant difference at p ≤ 0.05, * denotes statistical significance when compared to control group, # denotes statistical significance when compared to Rotenone-treated group, ** denotes statistical significance when compared to 100 mg/Kg SM-RT group, *** denotes statistical significance when compared to 200 mg/Kg SM-RT group using one way ANOVA with Tukey post hoc test 933 Figure 6:-Effect of different doses of SM on brain tissue homogenate caspase-3 level (OD/μgm protein) in Rt-induced PD: Values are mean ± SEM (n = 10). *, # and ** Denote a statistically significant difference at p ≤ 0.05, * denotes statistical significance when compared to control group, # denotes statistical significance when compared to Rotenone-treated group, ** denotes statistical significance when compared to 100 mg/Kg SM-RT group, using one way ANOVA with Tukey post hoc test Values are mean ± SEM (n = 10). *, # and ** Denote a statistically significant difference at p ≤ 0.05, * denotes statistical significance when compared to control group, # denotes statistical significance when compared to Rotenone-treated group, ** denotes statistical significance when compared to 100 mg/Kg SM-RT group, using one way ANOVA with Tukey post hoc test 934 Figure 8:-Effect of different doses of SM on brain tissue homogenate brain derived neurotrophic factor (BDNF)(pg/g) in Rt-induced PD: Values are mean ± SEM (n = 10). *, # and ** Denote a statistically significant difference at p ≤ 0.05, * denotes statistical significance when compared to control group, # denotes statistical significance when compared to Rotenone-treated group, ** denotes statistical significance when compared to 100 mg/Kg SM-RT group, using one way ANOVA with Tukey post hoc test Figure 9:-Effect of different doses of SM on brain tissue homogenate dopamine level (µg/g) in Rt-induced PD: Values are mean ± SEM (n = 10). *, # and ** Denote a statistically significant difference at p ≤ 0.05, * denotes statistical significance when compared to control group, # denotes statistical significance when compared to Rotenone-treated group, ** denotes statistical significance when compared to 100 mg/Kg SM-RT group, using one way ANOVA with Tukey post hoc test 935