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Assessing neuroprotective efficacy of phytochemical saponin ruscogenin in both in vitro and in vivo model
⁎Corresponding author. maodewang@163.com (Maode Wang)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Parkinson disease (PD) is a multifactorial disease without aging, which is considered to be the main cause of PD apart from genetic, life-style, and environmental patterns. Phytochemicals play an immense role in treating various neurological diseases, and they possess immense antioxidant and anti-inflammatory properties. In the current work, the neuroprotective effect of ruscogenin in an in vitro and in vivo mouse model was examined. The mouse microglial BV-2 cell line was stimulated with lipopolysaccharides (LPS) and used to assess the cytotoxicity, antioxidant, and anti-inflammatory activities of ruscogenin. In the in vivo model, the PD was induced with MPTP and treated with 10 and 20 mg/kg of ruscogenin. The mice were then subjected to motor function analysis with the foot print test, grip strength test, and rota rod test. Antioxidant, anti-inflammatory, and neuroprotective activities of ruscogenin on PD-induced mice were assessed with biochemical analysis. To confirm the neuroprotective activity of ruscogenin, histopathological analysis of substantia nigra tissue was done. Our in vitro results confirm that ruscogenin did not disturb the viability of BV-2 cells. Ruscogenin decreased the oxidative stress and inflammatory marker levels in BV-2 cells. Ruscogenin treatment persuasively improved motor activities in PD-induced mice. The levels of inflammatory cytokines were considerably reduced by the ruscogenin treatment. Histopathological analysis revealed that ruscogenin has neuroprotective properties. In summary, ruscogenin is a potent neuroprotective agent that can be utilized to treat PD in the future. Furthermore, our study lacks the in-depth molecular analysis needed to understand the mode of action of ruscogenin on the PD, and hence we strongly recommend further molecular studies in the future.
Keywords
Parkinson disease
Oxidative stress
Neuroinflammation
Phytochemical
Ruscogenin
1 Introduction
The most prevalent disease that threatens the current elderly population is Parkinson’s disease, a neurodegenerative disease that has been diagnosed in more than 6 million populations (Singh et al., 2022; GBD2015, 2017). In recent decades, the prevalence of Parkinson's disease has risen dramatically, reaching approximately 74 % (Rai et al., 2022; GBD 2016, 2018). The prevalence of Parkinson's disease was higher in people over the age of 80. Men were more prone to PD compared to the female population (Rai et al., 2021; de Lau et al., 2004). Even though aging was considered the etiology of Parkinson disease, the exact cause was not yet clearly illustrated. The susceptibility of genes to environmental influences was also considered to be a cause of Parkison’s (Kieburtz & Wunderle, 2014; Ritz et al., 2014; Kieburtz & Wunderle, 2015; Rizek et al., 2016). Initially, motor dysfunctions like rigidity, posture instability, tremors, bradykinesia, and akinesia were considered to be symptoms of Parkinson disease (Ma et al., 2017).
Currently, there is no treatment to completely cure PD; most of the treatments provided are used to only slow the disease's progression (Rai and Singh, 2020; Church, 2021). Dopamine supplementation, such as levodopa, was provided to reduce the symptoms and improve the quality of life of the patients. However, long-term use of this medication causes motor fluctuations as well as non-motor symptoms (Dietrichs & Odin, 2017). Phytochemicals play a massive role as alternative medicine for various diseases (Farahani et al., 2015). Compounds such as phenol, flavonoids, stilbenes, terpenes, polyphenols, and flavonoids are proven to exhibit protective effects against the Parkinson's disease model. (Shahpiri et al., 2016).
Saponins, which are phytochemicals found in nature as glycosides, have a number of pharmacological properties, including antioxidant, anti-inflammatory, anti-cancer, and neuroprotective properties. They exist as steroidal or triterpenoid aglycones containing one or more sugar molecules (Sun et al., 2015). Ruscogenin is one such steroidal saponin that is present in the evergreen perennial plant Ophiopogon japonica, a native of China, India, and Japan. It is prescribed for ailments such as inflammatory diseases, thrombosis, cardiovascular disease, etc. (Facino et al., 1995; Kou et al., 2005; 2006; Huang et al., 2008). It is proven to inhibit LPS-induced lung injury and cerebral ischemia in in vivo models (Guan et al., 2013; Sun et al., 2012).
The role of ruscogenin on PD was not yet illustrated; hence, we assessed the neuroprotective effects of ruscogenin in both LPS-stimulated mouse microglial BV-2 cells and the MPTP-induced PD mice model. The effect of ruscogenin on neuroinflammation and oxidative stress was studied in both in vitro and in vivo Parkinson's disease models.
2 Materials & methods
2.1 Chemicals & reagents
Ruscogenin, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), dimethyl sulfoxide (DMSO), Lipopolysaccharides (LPS), 3-(3,4-dimethylthiazole-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT), 1X trypsin-EDTA were procured from Sigma Aldrich (USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin abtibiotic and culture wares were procured from Gibco (USA). All the chemicals used in the present study were of only analytical graded.
2.2 Culturing of BV-2 cells
Mouse microglial BV-2 cell line were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % FBS and 1 % antibiotic solution. The cells were incubated in humidified incubator maintained with 5 %CO2 and 95 % O2, temperature was maintained at 37 °C throughout the experiment period. The media was changed once every 48 h or whenever the pH of the media in cultured plates was changed. The cells were trypsinized upon obtaining 80 % confluency for further subculturing and other experimental procedures.
2.3 Cell cytotoxicity assay
4x103 BV-2 cells were cultured and challenged with different concentration of ruscogenin. 1 M ruscogenin stock solution was prepared with DMSO and it was diluted to different concentrations with DMEM. The final concentration of DMSO was maintained to be less 0.05 % to avoid any hindrance on cell growth. Control cells were untreated whereas the other cells were treated with 5 µM/ml − 100 µM/ml concentrations for 1 h followed by 200 ng/ml of LPS. After incubation period of 24 h, 20 µl of MTT solution (2 mg/ml) were added to the cells and incubated in dark for 4 h. The supernatant from the well were removed and with DMSO the formazan crystals formed were dissolved. The absorbance of the solution were read at 540 nm using a microplate reader. The experiments were performed in triplicates to avoid false results.
2.4 Quantification of nitric oxide (NO) production
Nitric oxide production in LPS stimulated BV-2 cells were measured with commercially available colorimetric kit procured from BioVision, USA. BV-2 cells were stimulated with 200 ng/ml of LPS. Control cells are untreated with cells either with LPS or ruscogenin. LPS control cells were treated only with 200 ng/ml LPS. Experimental cells were treated with 200 ng/ml LPS and 10, 25 and 50 µM/ml concentrations for 24 h. After 24 h, 100 µl of cell supernatant were removed and subjected to NO measurement was performed as per the protocol prescribed by the manufacturer. The absorbance was measured at 540 nm using microplate reader.
2.5 Measurement of ROS production
BV-2 cells were stimulated with 200 ng/ml of LPS. Control cells are untreated with cells either with LPS or ruscogenin. LPS control cells were treated only with 200 ng/ml LPS. Experimental cells were treated with 200 ng/ml LPS and 10, 25 and 50 µM/ml concentrations for 24 h. After 24 h, the cells were subjected to total ROS measurement with commercially available fluorometric kit procured from Sigma Aldrich, USA. The absorbance was measured at excitation 540 nm and emission 570 nm using microplate reader.
2.6 Measurement of proinflammatory mediators
The proinflammatory mediators Prostaglandin E2 (PGE2), Interleukin 6 and 1β were measured with ELISA kit purchased from PGE2 - MyBisource, USA and IL-6 and 1β - Abcam, USA. Control BV-2 cells are untreated cells either with LPS or ruscogenin. LPS control cells were treated only with 200 ng/ml LPS. Experimental cells were treated with 200 ng/ml LPS and 10, 25 and 50 µM/ml concentrations for 24 h. The assay was performed according to the instructions provided in the kit. The absorbance was measured at 450 nm using microplate reader.
2.7 Experimental animals
Young healthy male C57BL/6 mice weighing about 25 ± 10 g were used for the current study. The procedure performed in the current study was explained before the ethical committee members and the same was carried out. The mice were acclimatized for a week in laboratory condition with 24 ± 3 °C, 55 ± 5 % relative humidity and 12 h light dark cycle. The mice were housed in autoclavable polypropylene cages with husk bedding. The bedding was changed daily and the cages were changed thrice a week. Laboratory pellet diet was provided to the mice ad libidum. Animals were handled with utmost care and concern.
2.8 Induction of PD in mice
The PD was induced with MPTP in mice model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride. After acclimatization the mice were treated intraperitoneally with 30 mg/kg body weight (b.wt) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride for five consecutive days which disrupts substantia nigra thereby causes Parkinson like symptoms in mice.
2.9 Experimental design
The mice were divided into four groups each group with 10 mice, group I were control mice were treated with corn oil for 16 days. Group II were Parkinson induced untreated mice which were treated with 30 mg/kg b.wt MPTP for five consecutive days. Group III & IV are Parkinson induced ruscogenin treated mice which were treated with 30 mg/kg b.wt MPTP for five consecutive days and treated with 10 mg/kg b.wt & 20 mg/kg b.wt ruscogenin daily for a period of 16 days. After treatment period the blood was collected and the mice were euthanized. Brain tissue was collected for further analysis.
3 Analysis of motor Co-ordination
3.1 Footprint test
Footprint test was performed to analyze the gait behavior in Parkinson’s induced and simultaneously ruscogenin treated mice. A 50X10cm long with 10 cm height walls runway was prepared and white sheet was placed on the runway. The mice fore and hind feet were painted with non toxic red and blue paints respectively and were placed on the starting point of the runway. The mice were then allowed to walk through the runway. The stride length (distance between each stride), fore base width (distance between left and right footprints) were measured to assess the walking pattern of mice.
3.2 Grid performance test
Parkinson’s induced and ruscogenin treated mice were subjected to grid performance test to analyze the muscle strength. The experiment was performed with the grip strength test meter procured from Bioseb, France. The apparatus was horizontally placed and the mice were lowered to the apparatus with the tail and allowed to grasp the metal grid. Then the mice were pulled horizontally backwards. The force applied by the mice on to the grip before it lose its grip was recorded as the peak tension. Same researcher was performed the test for all the mice and the test was repeated five times to avoid false results.
3.3 ROTA rod test
The motor coordination in mice were evaluated with rota rod test using an automated rota rod instrument purchased from Panlab, Spain. The mice were placed on to the rotating rod for 5 min and the speed of rotation was adjusted as 5, 10 and 15 rpm. Initially it was set at 5 rpm and then the speed was increased. The time of to fall – the latency fall time for each mice at different rpm were measured. The mice were allowed for three trials before the initiation of experiment. The experiment was repeated thrice between the experiments with two different mice the apparatus were sanitized.
3.4 Quantification of dopamine & proinflammatory cytokines
Substantia nigra tissue of the mice was dissected and homogenated with homogenizing buffer. Neurotransmitter dopamine and proinflammatory cytokines TNFα, IL-1β and IL-6 were quantified using commercially available ELISA kits procured from Abcam, USA. The assay was performed according to the instructionns provided in the kit. The absorbance was measured at 450 nm using microplate reader.
3.5 Quantification of iNOS and COX2
The levels of neuroinflammatory proteins iNOS and COX 2 were quantified in mice serum using the kit purchased from MyBiosource, USA. The experiment was performed as per the instructions provided in the kit. The experiments were performed in triplicates and the absorbance was measured at 450 nm. The levels of iNOS and COX2 were determined with the standard curve plotted with absorbance of known concentrations.
3.6 Histopathological analysis
Substantia nigra tissue of the mice were subjected to histopathological analysis and haematoxilin eosin staining to assess the disruption induced by MPTP and the neuroprotection of ruscogenin against MPTP induced PD. The tissue was paraffin fixed in formalin immediately after dissection. The formalin fixed tissue was subjected to series of hydration and dehydration with series of alcohol and xylene. The processes tissue was the fixed with paraffin wax and subjected sectioning with microtome. 5micron tissue sections were deparaffinized and fixed on to the albumin coated slides. The tissues were then stained with haematoxilin and counter stained with eosin. The stained tissues were then observed and photographed under light microscope (Olympus).
3.7 Statistical analysis
The experiments were performed at least thrice and data were analyzed with one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison tests using the version 6 GraphPad Prism software. All the data were expressed as mean ± standard error mean and the p-values < 0.05 was considered as statistically significant.
4 Results
4.1 Cytotoxic effect of ruscogenin on mouse microglial BV-2 cell line
Fig. 1 depicts the results of MTT assay performed on mouse microglial BV-2 cell line. The cells were stimulated with lipopolysaccharides and then treated with different concentrations of ruscogenin. Ruscogenin does not exhibited increased cytotoxicity on mouse microglial BV-2 cell line. Even when cells were treated with a higher concentration (100 µM/ml) of ruscogenin, only a mild reduction in cell viability was observed.
Cytotoxic effect of ruscogenin on mouse microglial BV-2 cell line. 4x103 BV-2 cells were cultured in 96 well plates and treated with 5, 10, 25, 50, 75 & 100 µM/ml ruscogenin for 24 h. 1 h after ruscogenin treated the cells were stimulated with 200 ng/ml of LPS. The cells were then subjected to MTT assay and the absorbance were read at 540 nm. Data were assessed using statistical software Graphpad prism version 6 with ANOVA and Tukey’s multiple comparison post hoc test. Mean ± standard error mean, p-values < 0.05 statistically significant.
4.2 Antioxidant effect of ruscogenin on mouse microglial BV-2 cell line
Fig. 2A illustrates the production of nitric oxide on LPS-stimulated mouse microglial BV-2 cell line. LPS stimulation increased nitric oxide production in the BV-2 cell line significantly, whereas ruscogenin inhibited LPS-induced nitric oxide generation. Nitric oxide production decreased in a dose-dependent manner.
Antioxidant effect of ruscogenin on mouse microglial BV-2 cell line. (A) Nitric Oxide levels (B) Total ROS levels in control, LPS stimulated, LPS stimulated + ruscogenin treated BV2 cell line. BV-2 cell line were stimulated with LPS 200 ng/ml and then treated with 10, 25 and 50 µM/ml of ruscogenin for 24 h. Nitric oxide and total ROS levels were estimated with commercially available colorimetric and flurometric kit respectively. Data were assessed using statistical software Graphpad prism version 6 with ANOVA and Tukey’s multiple comparison post hoc test. Mean ± standard error mean, p-values < 0.05 statistically significant.
Fig. 2B depicts the total ROS production on LPS stimulated mouse microglial BV-2 cell line. LPS induced ruscogenin untreated cells produced increased levels of ROS than the control cells. Interestingly, a significant (p < 0.05) reduction in ROS generation was observed in ruscogenin-treated cells.
4.3 Anti-inflammatory effect of ruscogenin on mouse microglial BV-2 cell line
Fig. 3A illustrates the prostaglandin synthesis in the LPS-stimulated mouse microglial BV-2 cell line. LPS induction drastically increased the production of prostaglandin in BV-2 cells compared to the control cells. Ruscogenin treatment significantly (p < 0.05) decreased the levels of prostaglandin in a dose dependent manner. High dose (50 µg/ml) of ruscogenin-treated BV-2 cells showed decreased level of prostaglandin, which is comparatively similar to the control cells.
anti-inflammatory effect of ruscogenin on mouse microglial BV-2 cell line. (A) Prostaglandin E2 (PGE2), (B) Interleukin 6 and (C) Interleukin 1β in control, LPS stimulated, LPS stimulated + ruscogenin treated BV2 cell line. BV-2 cells were stimulated with 200 ng/ml of LPS and then treated with 10, 25 and 50 µM/ml of ruscogenin for 24 h. Prostaglandin E2 (PGE2), Interleukin 6 and Interleukin 1β were quantified with commercially available ELISA kit. Data were assessed using statistical software Graphpad prism version 6 with ANOVA and Tukey’s multiple comparison post hoc test. Mean ± standard error mean, p-values < 0.05 statistically significant.
Fig. 3B & C depicts the levels of interleukin 1β & 6 in control and treated BV-2 cells. Ruscogenin treatment significantly decreased the LPS-induced interleukins production in mouse microglial BV-2 cell line. Both interleukin 1β & 6 levels were significantly (p < 0.05) decreased in the ruscogenin-treated cells compared to the control.
4.4 Effect of ruscogenin on motor function in MPTP induced Parkinson mice model
Fig. 4A&B represents the motor functions in control and experimental mice. MPTP induction drastically decreased the length of strides and distance between fore paw in mice, which confirms the induction of PD in mice. Ruscogenin treatement significantly (p < 0.05) decreased both the length of strides and distance between fore paw in MPTP-induced mice. Fig. 4C depicts the results of grip strength in MPTP-induced and ruscogenin-treated mice. Ruscogenin treatment significantly (p < 0.05) increased grip strength in the MPTP-induced mice in a dose dependent manner.
Effect of ruscogenin on motor function in MPTP induced Parkinson mice model. (A) Stride length (B) Fore paw base width (C) Grip strength in control, MPTP treated, MPTP + 10 mg/kg b.wt ruscogenin treated and MPTP + 20 mg/kg b.wt ruscogenin treated mice. Stride length and fore paw base width were analyzed with footprint test and the grip strength was measured with grip strength meter. Data were assessed using statistical software Graphpad prism version 6 with ANOVA and Tukey’s multiple comparison post hoc test. Mean ± standard error mean, p-values < 0.05 statistically significant.
4.5 Effect of ruscogenin on motor co-ordination of MPTP induced mice
Fig. 5 depicts the results of latency fall time of control and treated mice mice in the rotating speed of 5 rpm (A), 10 rpm (B) and 15 rpm (C). MPTP-induced mice withstand only for 250 ± 20 sec at 5 rpm and it further decreased to 175 ± 10 sec and 150 ± 20 sec at 10 and 15 rpm respectively. Ruscogenin treatment significantly increased the motor co ordination in a dose dependent manner. 10 mg/kg b.wt ruscogenin treated mice had a latency fall time of 340 ± 20 sec, 270 ± 25 sec, 250 ± 10 sec and whereas the increased latency fall time of 380 ± 15 sec, 340 ± 15 sec, 310 ± 20 sec was observed in 20 mg/kg b.wt ruscogenin treated mice.
Effect of ruscogenin on motor co-ordination of Parkinson induced mice. Motor co-ordination was assessed with ROTA rod test. Control, MPTP treated, MPTP + 10 mg/kg b.wt ruscogenin treated and MPTP + 20 mg/kg b.wt ruscogenin treated mice were subjected to rota rod test with varying rotating speed of 5 rpm (A), 10 rpm (B) and 15 rpm (C). Data were assessed using statistical software Graphpad prism version 6 with ANOVA and Tukey’s multiple comparison post hoc test. Mean ± standard error mean, p-values < 0.05 statistically significant.
4.6 Effect of ruscogenin on neuroinflammatory markers in MPTP induced Parkinson’s mice model
Dopamine plays a key role in regulating inflammatory cytokines and preventing neuroinflammation. Dopamine levels were drastically decreased in MPTP-induced mice compared to the control mice. Whereas ruscogenin treatment significantly increased the levels of dopamine in MPTP treated mice (6A). Fig. 6B illustrates the levels of proinflammatory cytokines TNFα, IL-1β and IL-6 in control and experimental mice. Significant increase in the levels of TNFα, IL-1β and IL-6 were observed in MPTP treated mice, whereas ruscogenin treatment had significantly decreased the levels of TNFα, IL-1β and IL-6.
Effect of ruscogenin on neuroinflammatory markers in Parkinson’s induced mice. (A) Dopamine (B) Inflammatory cytokines. Control, MPTP treated, MPTP + 10 mg/kg b.wt ruscogenin treated and MPTP + 20 mg/kg b.wt ruscogenin treated mice were subjected to estimation of dopamine and inflammatory cytokines using commercially available ELISA kit. Data were assessed using statistical software Graphpad prism version 6 with ANOVA and Tukey’s multiple comparison post hoc test. Mean ± standard error mean, p-values < 0.05 statistically significant.
4.7 Anti-inflammatory effect of ruscogenin in Parkinson’s induced mice
Fig. 7 depicts the levels of iNOS and COX2 in control and experimental mice. Ruscogenin treatment significantly decreased the levels of iNOS and COX2 in MPTP treated mice compared to the MPTP alone treated mice. The decreased levels of iNOS and COX2 were in dose dependent manner 20 mg/kg b.wt ruscogenin treated mice shown significant decrease compared to 10 mg/kg b.wt ruscogenin-treated mice.
anti-inflammatory property of ruscogenin in Parkinson’s induced mice. (A) iNOS, (B) COX2. Control, MPTP treated, MPTP + 10 mg/kg b.wt ruscogenin treated and MPTP + 20 mg/kg b.wt ruscogenin treated mice were subjected to estimation of iNOS and COX2 using commercially available ELISA kit. Data were assessed using statistical software Graphpad prism version 6 with ANOVA and Tukey’s multiple comparison post hoc test. Mean ± standard error mean, p-values < 0.05 statistically significant.
4.8 Neuroprotective effect of ruscogenin on histoarchitecture of substantia nigra in MPTP induced Parkinson’s mice model
Fig. 8 illustrates the light microscopic images of haematoxylin & eosin stained substantia nigra tissue of control and experimental mice. Control (8A) and MPTP + 20 mg/kg b.wt ruscogenin treated (8D) shown normal histoarchitecture of substantia nigra region with normal dopamine neurons. Increased number of pyknotic cells and vacuolated cells were viewed in MPTP treated mice (Fig. 8B). Few shrunken, chromatolysed and necroti cells were observed in MPTP + 10 mg/kg b.wt ruscogenin treated. Compared to MPTP treated mice the MPTP + 10 mg/kg b.wt ruscogenin treated mice shown decreased number of necrotic, vacuolated and pyknotic cells. The substantia nigra tissue damage is decreased in 20 mg/kg b.wt ruscogenin treated mice (Fig. 8D) compared to the 10 mg/kg b.wt ruscogenin treated mice (Fig. 8C).
Neuroprotective effect of ruscogenin on substantia nigra’s histoarchitecture of Parkinson’s induced mice. (Group I) Control (Group II) MPTP treated Parkinson induced, (Group III) MPTP treated Parkinson induced + 10 mg/kg b.wt ruscogenin treated and (Group IV) MPTP treated Parkinson induced + 20 mg/kg b.wt ruscogenin treated mice. Substantia nigra tissue was dissected, 5micorn tissue section was subjected to hameatoxylin and eosin staining, and stained sections were observed for histopathological changes under light microscope and representative images were depicted.
5 Discussion
Neurological diseases tend to be the foremost cause of disability in the global elderly population, and the progression rate is drastic (GBD2015 2017). Since life expectancy has increased, the need for geriatric care has also increased. The population of individuals with Parkinson’s disease was doubled within a period of 15 years from 1990 to 2016, and it is expected to be doubled again in the coming years (Wanneveich et al., 2018; Rossi et al., 2018). Previously, PD was characterized as a motor system disorder, which is characterized by rigidity of limbs and trunks, bradykinesia, tremor, and instability in posture (Berganzo et al., 2016; Ferrazzoli et al., 2020), whereas non-motor disorders such as depression, sleep pattern disturbances, and psychosis are also diagnosed in most PD patients (Connolly and Lang, 2014; Church, 2021). Daily administration of Levedopa comprising the dopamine insufficiency was the traditional treatment given to the PD patients globally (Hall and Church, 2020). This treatment significantly reduces the motor symptoms in PD patients, but it fails to provide a complete cure and doesn’t cure the non-motor symptoms. Hence, the researchers started to explore a new strategy to discover a drug that completely cured PD patients.
We investigated the ability of the phytochemical ruscogenin to protect neuronal cells from inflammation. We have chosen two different models: a cell culture model and an animal model. Normal mouse microglial BV-2 cells were chosen for the study. The cells were first challenged with different concentrations of ruscogenin to assess the cytotoxicity of the drug. Our MTT results have proven that ruscogenin, even at a higher dose concentration of 100 µM/ml, doesn’t produce much cytotoxicity on mouse microglial cells.
Oxidative stress plays a key role in neurodegenerative diseases. The imbalance in oxidants and antioxidants was reported in most of the animal models and PD patients (Puspita et al., 2017; Kumar et al., 2012). An increase in the accumulation of free radicals in a cell can’t be scavenged by the natural antioxidant defense system, which induces damage to DNA, proteins, and lipids, eventually leading to cell death. Therefore, substituting an antioxidant can scavenge the free radicals and protect the cells from cell death. We stimulated the BV-2 cells with LPS, which increases free radical generation, and challenged the LPS-stimulated cells with 10, 25, and 50 µM/ml concentrations of ruscogenin. Ruscogenin significantly reduced the total ROS production in LPS-stimulated cells, thereby confirming its antioxidant activity. Nitric oxide is another factor that contributes to oxidative stress, and increased levels of nitric oxide were reported in the brains of PD patients in in vivo models (Aquilano et al., 2008). In our study, ruscogenin significantly reduced nitric oxide production in LPS-stimulated mouse microglial cells.
Activation of microglial cells by stimuli such as cytokines, endotoxins, and chemokines results in dopaminergic neuronal degeneration (Wilms et al., 2007). Activated microglia induced dopaminergic neuronal damage was observed in various brain regions, such as the cingulated cortex, hippocampus, and temporal cotex (Knott et al., 2000; Mancuso et al., 2007). NF-κB is translocated from the cell surface to the nucleus, where it forms DNA adducts, activating a variety of proinflammatory cytokines (Raza et al., 2003; Johnston et al., 2008). Flavonoids such as kaempferol, genistein, emodin, and naringenin reduced microglial activation and decreased the proinflammatory cytokines NF-κB, TNF-α, IL-6, and IL-1β. Our results correlate with the previous results. Ruscogenin effectively reduced the synthesis of prostaglandin-2, IL-6, and IL-1β during microglial activation.
Further, we assessed the neuroprotective efficacy of ruscogenin in an in vivo mouse model. The Parkinson model was created in mice with the administration of MPTP. Various behavioral analyses were used to investigate the induction of Parkinson's disease in mice. Gait was considered the characteristic indicator symptom in Parkinson patients, which occurs due to the neurodegeneration in the basal ganglia (Fathalla et al., 2016). Gait can be analyzed with the footprint test, which is widely accepted and measures the stride length of the animals in which white sheets are used and rodents are allowed to walk with paint coated feet (Carter et al., 2009; Mendes et al., 2015). Shorter stride length was observed in the rodents treated with neurotoxin (Ahmed et al., 2018). In our MPTP study, neurotoxin-treated mice had shorter strides than control, whereas ruscogenin treatment significantly increased stride length, confirming ruscogenin's neuroprotective potency against gait induction in MPTP-treated mice. Our analysis with a grip strength meter also confirms that ruscogenin significantly increased the grip strength in mice treated with MPTP.
The rotarod test was used to assess the motor coordination capability of neurotoxins MPTP and simultaneous ruscogenin treatment. Ruscogenin-treated mice can withstand longer periods on the rotarod even at higher rotational speeds than MPTP-treated mice. This may be due to a reduction in dopamine levels, which alters cortical and cerebellar functions and causes instability and movement control in mice (Georgiev et al., 2016; Salama et al., 2013; Seidel et al., 2017). The ruscogenin treatment significantly increased the dopamine levels in the MPTP-treated mice, which confirms its neuroprotective effect.
An increased number of microglial cells was found in the substantia nigra, and it increased along with age; they are more susceptible to oxidative stress and neuroinflammation (Beach et al., 2007). The substantia nigra of PD shows an increased number of activated microglial cells, which initiates the degeneration of neurons, particularly dopamine neurons (Jenner & Olanow, 1996). When microglial cells are activated, they release proinflammatory cytokines such as TNF-α, IFN-γ, IL-6, IL-1β, and iNOS, which cause inflammation in the substantia nigra. Increased expression of iNOS was reported in the substantia nigra of post-mortem PD patients and animal models of PD (Iravani et al., 2002; Broom et al., 2011). Inhibiting iNOS expression with a selective inhibitor was found to be effective in PD animal models (Broom et al., 2011; Barthwal et al., 2001). Overexpression of COX-2 was the other prominent factor observed in various PD animal models. COX-2 synthesis was initiated by proinflammatory cytokines and NF-κB (Teismann et al., 2003; 2012). Inhibition of COX-2 showed a reduction in neurodegeneration in animal PD models (Minghetti, 2004), and knocking out COX-2 reduced neurodegeneration in MPTP-treated mice (Feng et al., 2002). The ruscogenin treatment also effectively decreased the synthesis of proinflammatory cytokines, thereby reducing the synthesis of iNOS and COX-2 and thereby preventing neurodegeneration. Our histological findings confirm that ruscogenin significantly inhibited MPTP-induced neuroinflammation, preventing neurodegeneration in the mouse's substantia nigra.
6 Conclusion
In this study, the neuroprotective effect of the phytochemical saponin ruscogenin was investigated in an in vivo and in vitro model. Mouse microglial BV-2 cells were chosen as an in vitro model, and an MPTP-induced Parkinson mouse model was used for the in vivo study. Ruscogenin does not induce cytotoxicity in mouse microglial BV-2 cells, and it reduces the LPS-induced oxidative stress and proinflammatory cytokines. Ruscogenin improved neurodegeneration by increasing dopamine synthesis and decreasing proinflammatory cytokines, iNOS, and COX-2 levels in MPTP-treated mice. The neuroprotective properties of ruscogenin were authentically confirmed with motor coordination analysis and a histopathological study. Ruscogenin can be an effective supplement that not only reduces the symptoms but also inhibits the root cause of neurodegeneration.
Conflict of interest
The authors declare no conflict of interest.
References
- Neuroprotective Potential Of Polydatin Against Motor Abnormalities And Dopaminergic Neuronal Loss In Rotenone Induced Parkinson Model. Int. J. Morphol.. 2018;36(2):584-591.
- [Google Scholar]
- Role of nitric oxide synthases in Parkinson's disease: a review on the antioxidant and anti-inflammatory activity of polyphenols. Neurochem Res.. 2008;33(12):2416-2426.
- [Google Scholar]
- Role of nitric oxide in a progressive neurodegeneration model of Parkinson's disease in the rat. Redox Rep.. 2001;6(5):297-302.
- [Google Scholar]
- Marked microglial reaction in normal aging human substantia nigra: correlation with extraneuronal neuromelanin pigment deposits. Acta Neuropathol.. 2007;114(4):419-424.
- [Google Scholar]
- Motor and non-motor symptoms of Parkinson's disease and their impact on quality of life and on different clinical subgroups. Neurologia.. 2016;31(9):585-591.
- [Google Scholar]
- Neuroprotection by the selective iNOS inhibitor GW274150 in a model of Parkinson disease. Free Radic Biol Med.. 2011;50(5):633-640.
- [Google Scholar]
- Treatment Options for Motor and Non-Motor Symptoms of Parkinson's Disease. Biomolecules.. 2021;11(4):612.
- [Google Scholar]
- Pharmacological treatment of Parkinson disease: a review. JAMA.. 2014;311(16):1670-1683.
- [Google Scholar]
- Incidence of parkinsonism and Parkinson disease in a general population: the Rotterdam Study. Neurology.. 2004;63(7):1240-1244.
- [Google Scholar]
- Anti-elastase and anti-hyaluronidase activities of saponins and sapogenins from Hedera helix, Aesculus hippocastanum, and Ruscus aculeatus: factors contributing to their efficacy in the treatment of venous insufficiency. Arch Pharm (Weinheim).. 1995;328(10):720-724.
- [Google Scholar]
- Plant-derived natural medicines for the management of depression: an overview of mechanisms of action. Rev Neurosci.. 2015;26(3):305-321.
- [Google Scholar]
- Adenosine A2A Receptor Blockade Prevents Rotenone-Induced Motor Impairment in a Rat Model of Parkinsonism. Front Behav Neurosci.. 2016 Feb;29(10):35.
- [Google Scholar]
- Cyclooxygenase-2-deficient mice are resistant to 1-methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine-induced damage of dopaminergic neurons in the substantia nigra. Neurosci Lett.. 2002;329(3):354-358.
- [Google Scholar]
- Motor-cognitive approach and aerobic training: a synergism for rehabilitative intervention in Parkinson's disease. Neurodegener Dis Manag.. 2020;10(1):41-55.
- [Google Scholar]
- GBD, 2015. Neurological Disorders Collaborator Group. Global, regional, and national burden of neurological disorders during 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Neurol 2017; 16: 877–97.
- GBD, 2016. Parkinson's Disease Collaborators. Global, regional, and national burden of Parkinson's disease, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 2018 Nov;17(11):939-953. doi: 10.1016/S1474-4422(18)30295-3.
- Movement-related potentials in Parkinson's disease. Clin Neurophysiol.. 2016;127(6):2509-2519.
- [Google Scholar]
- Ruscogenin reduces cerebral ischemic injury via NF-κB-mediated inflammatory pathway in the mouse model of experimental stroke. Eur J Pharmacol.. 2013;714(1–3):303-311.
- [Google Scholar]
- Integrative medicine and health therapy for Parkinson disease. Top. Geriatr. Rehabil.. 2020;36:176-186.
- [Google Scholar]
- Possible mechanism of the anti-inflammatory activity of ruscogenin: role of intercellular adhesion molecule-1 and nuclear factor-kappaB. J Pharmacol Sci.. 2008;108(2):198-205.
- [Google Scholar]
- Involvement of inducible nitric oxide synthase in inflammation-induced dopaminergic neurodegeneration. Neuroscience.. 2002;110(1):49-58.
- [Google Scholar]
- Oxidative stress and the pathogenesis of Parkinson's disease. Neurology.. 1996;47(6 Suppl 3):S161-S170.
- [Google Scholar]
- Human interleukin-10 gene transfer is protective in a rat model of Parkinson's disease. Mol Ther.. 2008;16(8):1392-1399.
- [Google Scholar]
- Inflammatory regulators in Parkinson's disease: iNOS, lipocortin-1, and cyclooxygenases-1 and -2. Mol Cell Neurosci.. 2000;16(6):724-739.
- [Google Scholar]
- Anti-inflammatory activities of aqueous extract from Radix Ophiopogon japonicus and its two constituents. Biol Pharm Bull.. 2005;28(7):1234-1238.
- [Google Scholar]
- Antithrombotic activities of aqueous extract from Radix Ophiopogon japonicus and its two constituents. Biol Pharm Bull.. 2006;29(6):1267-1270.
- [Google Scholar]
- The role of free radicals in the aging brain and Parkinson's Disease: convergence and parallelism. Int J Mol Sci.. 2012;13(8):10478-10504.
- [Google Scholar]
- Mitochondrial dysfunction, free radical generation and cellular stress response in neurodegenerative disorders. Front Biosci.. 2007;1(12):1107-1123.
- [Google Scholar]
- Quantification of gait parameters in freely walking rodents. BMC Biol.. 2015;22(13):50.
- [Google Scholar]
- Cyclooxygenase-2 (COX-2) in inflammatory and degenerative brain diseases. J Neuropathol Exp Neurol.. 2004;63(9):901-910.
- [Google Scholar]
- Oxidative stress and cellular pathologies in Parkinson's disease. Mol Brain.. 2017;10(1):53.
- [Google Scholar]
- Advancement in the modelling and therapeutics of Parkinson's disease. J Chem Neuroanat.. 2020;26(104):101752
- [Google Scholar]
- Rai , S.N. , Tiwari , N. , Singh , P. , Mishra , D. , Singh , A.K. , Hooshmandi , E. , Vamanu , E. , Singh , M.P. , 2021 . Therapeutic Potential of Vital Transcription Factors in Alzheimer’s and Parkinson’s Disease With Particular Emphasis on Transcription Factor EB Mediated Autophagy . Front Neurosci. 14 ( 15 ), 777347 .
- Exploring the Paradox of COVID-19 in Neurological Complications with Emphasis on Parkinson's and Alzheimer's Disease. Oxid Med Cell Longev.. 2022;31(2022):3012778.
- [Google Scholar]
- Parkinson disease and smoking revisited: ease of quitting is an early sign of the disease. Neurology.. 2014;83(16):1396-1402.
- [Google Scholar]
- An update on the diagnosis and treatment of Parkinson disease. CMAJ.. 2016;188(16):1157-1165.
- [Google Scholar]
- Projection of the prevalence of Parkinson's disease in the coming decades: Revisited. Mov Disord.. 2018;33(1):156-159.
- [Google Scholar]
- Involvement of the cerebellum in Parkinson disease and dementia with Lewy bodies. Ann Neurol.. 2017;81(6):898-903.
- [Google Scholar]
- Phytochemicals as future drugs for Parkinson's disease: a comprehensive review. Rev Neurosci.. 2016;27(6):651-668.
- [Google Scholar]
- Deciphering the gut microbiome in neurodegenerative diseases and metagenomic approaches for characterization of gut microbes. Biomed Pharmacother.. 2022;156:113958
- [Google Scholar]
- Ruscogenin inhibits lipopolysaccharide-induced acute lung injury in mice: involvement of tissue factor, inducible NO synthase and nuclear factor (NF)-κB. Int Immunopharmacol.. 2012;12(1):88-93.
- [Google Scholar]
- Cyclooxygenase-2 is instrumental in Parkinson's disease neurodegeneration. Proc Natl Acad Sci U S A.. 2003;100(9):5473-5478.
- [Google Scholar]
- Teismann, P. 2012. COX-2 in the neurodegenerative process of Parkinson's disease. Biofactors. 2012 Nov-Dec;38(6):395-7. doi: 10.1002/biof.1035. Epub 2012 Jul 23. PMID: 22826171; PMCID: PMC3563218.
- Inflammation in Parkinson's diseases and other neurodegenerative diseases: cause and therapeutic implications. Curr Pharm Des.. 2007;13(18):1925-1928.
- [Google Scholar]