@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2018;24(3):172-181

Background
Multiple sclerosis (MS) is a self-inflammatory and progressive disease of the central nervous system [1]. It has profound social and economic effects because of its severely debilitating nature and also because it involves people in their best-performing times for society (30 years).
Previous Studies
… [3-8]. It has been shown that in people with depression, the level of inflammatory cytokines such as IL-6 in nerve tissue has increased [9]. It has also been shown that Hypericum perforatum in In vivo conditions can modulate and reduce inflammatory cytokines such as IL-6, IL-17, and interferon gamma [10]. The anti-inflammatory effects of Hypericum perforatum are due to its inhibitory role in the expression of inflammatory precursor genes such as cyclooxygenase 2 (Cox2) and reusable nitric oxide synthesis [11] that, currently, evaluating nitric oxide levels is one of the most important indicators of oxidative stress due to the activity of nitric oxide synthesis. … [12]. Hypericin and pseudohypericin also have antibacterial and antiviral effects, as well as lipid lowering properties. Hypiran is prepared from hydroalcoholic extract of Hypericum perforatum, each ml containing 0.25 mg of Hypericin [10].
Aim(s)
The aim of this study was to investigate the potential beneficial effects of Hypiran on the relief of symptoms of Experimental Autoimmune Encephalomyelitis (EAE).
Research type
This research is an experimental study.
Research society, place and time
This research was carried out on adult male Wistar rats in the animal housing of Faculty of Sciences of the University of Urmia.
Sampling method and number
In this study, 30 male Wistar rats with the age of 6-8 weeks and weighing 20±100 g were used to induce the disease model. In this study, the rats were randomly divided into 3 groups (Each group contained 10 rats). Meanwhile, all stages of this research were approved by the Ethics Committee of Faculty of Sciences in observance of the ethics of working with laboratory animals under the code 2-370.
Used devices&materials
In order to induce EAE in the studied groups, spinal cord homogenate of Guinea pig with 1 to 1 was used with CFA: Compound Freund's Adjuvant, containing 10 mg/kg depleted form of mycobacterium to emulsion. The morbidity (severity of motor disability) was evaluated daily in each rat in the groups. The following scale was used to evaluate the severity of the disease: Zero: no disease, 1: disorder in the tail, 2: tail paralysis, 3: 2 legs paralysis, 4: 2 hands and 2 legs paralysis, and 5: death [13]. The rats were divided into 2 groups (Each group contained 10 rats) with the same age and weight. 10 rats were treated for 24 days after symptoms of EAE (day 12 after induction). This group received 110 mg/kg Hypiran extract as edible (gavage). 10 other rats were also considered as control group. These rats were treated with placebo (saline) in the same volume as in the previous group after symptoms in all of the rats (12 days after the induction of disease). Also, 10 male Wistar rats were similar in age, gender, and weight to the two previous groups, which were considered healthy group. On the 36th day after the beginning of the study, the rats were deeply anesthetized to examine the biochemical tests, and, then, each rat was fixed on the laboratory tray. After an autopsy and showing the animal's heart, blood samples were taken, using 5cc syringes; the blood was poured into laboratory tubes and placed at room temperature for 30 minutes; then, blood samples were taken to measure biochemical factors by centrifugation for 5 minutes at 3500 rpm and kept at 20 °C until freezing. Then, the rat head was removed with the help of a special scissor and the brain was removed from the skull. The right hemisphere of the brain was placed in 10% formalin solution for tissue studies, and the left brain hemisphere was transferred to a freezer with 80 °C temperature for biochemical studies. On the day of experiment, brain tissues were homogenized in phosphate buffer at 0 °C with pH=7.4 and with a sample concentration of 10% (w/v) with manual homogenization. The solutions were centrifuged with a 1000 g round (Sigma Laboratory Centrifuge, D-37520, Germany) and the supernatant was used to measure the production of lipid peroxidation products and enzymatic activity. Ferric Reducing Ability of Plasma (FRAP) was used to measure antioxidant capacity. … [14-16]. Nitric oxide production was determined by Griess test and using sodium nitrite standard curve [17]. … [18]. To evaluate the power of the interconnection, the most active form of Hypericum perforatum, Hypiran, was utilized by the molecular docking method of this compound with monoamine oxidase A [19]. Nonparametric Mann-Whitney U test was used to compare two data points and to compare several data, kurskall wallis was used with Bonferroni post hoc test.
Findings by Text
Finding by Text The mean severity of disability during the treatment period was lower in patient rats treated with Hypiran (2.1±0.6%) than in the patient rats without treatment (3.13±0.23; p<0.001). Mean total antioxidant capacity in the treated and induced groups did not significantly change. The measurement of malondialdehyde in treatment and control groups showed that there was a significant difference in EAE induction groups in all 3 groups (Diagram 1). The measurement of nitric oxide levels in the control group and comparison with the EAE induction group indicated a significant increase in nitric oxide (Diagram 2). Both the induction and the treatment group with Hypiran the values significantly changed, so that it was 0.5±0.2 in the EAE induction group and 1.62±0.3 in the control group. The structure and method of binding of Hypericin to monoamine oxidase A was generally demonstrated by Pymol software (Diagram 1). Investigating the interaction of Hypiran with monoamine oxidase A through Docking servers showed that this connection was completely specific and with ΔG equal to -7.5 kcal/mol and an estimated inhibitor constant, Ki of 3.3 μM showed that there was a high binding energy of Hypericin and monoamine oxidase A (Tables 1 and 2).
Main comparison to the similar studies
… [20-29]. Our results showed that the activity level of MPO and NO in the blood serum of animals treated with Hypiran was significantly decreased compared with the EAE group, which was consistent with the results of the previous study to reduce inflammatory cytokines [30]. Naziroglu et al. have shown that Hypericum Perforatum has changed the level of oxidative enzymes in patients with MS [24]. … [31-40]. The studies conducted in the Japanese patients showed that there was a direct relationship between changes in the level of MPO in the blood and recurrence and the decline of the disease, so that before the recurrence of the disease, the activity level of the enzyme was increased, and before the disappearing, the activity level of the enzyme was increased in patients [41]. In this regard, our study also showed that Hypiran was able to inhibit the activity of this enzyme in the affected and treated group. A study carried out by Mozafari et al. also showed that Hypericum perforatum extract was able to decrease the activity level of MPO in rats with ulcerative colitis [30]. … [42, 43]. Musqrave et al. have shown that taking Phenelzine as a classical inhibitor of the MAO has led to significant inhibitions of EAE disease symptoms in C57BL/6 mice [44]. Our analytical results also indicated a significant inhibitory effect of MAO enzyme on Hypiran extract.
Suggestions
In the pathogenesis of multiple sclerosis, cytokines play an important role. It is suggested to consider these issues in future investigations.
Limitations
Each research model compare to real human disease has its own limitations.
Conclusions
Hypiran may be used as a useful strategy for treating people with multiple sclerosis.
Acknowledgments
Hereby, the contributions of the Vice-Chancellor of the University of Urmia are appreciated.
Conflict of interest
There is no conflict of interest.
Ethical Permissions
The research protocol, based on international law on the protection of animal, was approved by the Ethics Committee of the University.
Funding Sources
This study is a part of MA thesis conducted by Mina Abdullahpouarsal, approved in Urmia University, and supervised by Dr. Shiva Khezri and Dr. Seyed Meysam Abtahiforooshani.

TABLES and CHARTS

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