Anti-inflammatory and Anti-oxidative Effects of Some Medicinal Herbs on Chronic Murine Toxoplasmosis: A Histopathological and Immunohistochemical Study

• Toxoplasma gondii (T. gondii) is a globally distributed parasite that was prioritized as one of the tops “Five Neglected Parasitic Infections”.
• There are few effective treatments for toxoplasmosis at the moment, because of its distinct pathophysiology as the parasite penetrates the blood-brain barrier and this makes treatment of persistent infection challenging.
• Herbal medicine or phytomedicine is the use of plants for curing diseases and improving human health.
• Anti-toxoplasma medicinal plants (OLE, LSSE, Eucalyptus and RSV) which were highly available an efficient alternative to the traditional treatment of Toxoplasma gondii.
• Neuron Specific Enolase (NSE) marker expression was a good assessment tool to measure the antioxidant effect of the used medicinal plants against experimental toxoplasmosis.

Nitric  oxide (NO) is a nitrogenous free radical secreted by a variety of  mammalian cells. It has very important functions both in helminths and mammalian hosts as it promotes the cytotoxic and microbicidal activities of macrophages. The killer molecule NO is synthesized by inducible nitric oxide synthase (iNOS) [5].  Interestingly, it was found that in experimental Toxoplasmic Encephalitis (TE), iNOS is severely expressed in focal gliosis, microglia/macrophages, and in particular glial cells in the surrounding vasculature [6]. Moreover, it was mentioned that Neuron Specific Enolase (NSE) may be used as a marker to identify the severity of neuronal damage after cerebral ischemia [7]. Dincel and Atmaca mentioned that NSE expression contributes more to the interpretation of TE-related neuropathology [4].

Currently, toxoplasmosis is treated with sulfadiazine and pyrimethamine (PRY) [8]. Unfortunately, the drug resistance in Toxoplasma is ongoing and the emergence of strains of T. gondii that are resistant to the current drugs represents a concern both for treatment failure and for increased clinical severity especially in those of immunocompromised patients [9]. Medicinal plants have been recognized as potential drug candidates [10]. Herbal medicine or phytomedicine is the use of plants for medicinal and therapeutic purposes for curing diseases and improving human health. Plants have secondary metabolites called phytochemicals (‘Phyto from Greek - meaning ‘plant’). These compounds protect plants against microbial infections or infestations by pests. Phytochemicals are active ingredients which possess therapeutic properties [11].

Lepidium sativum is a popular herb that is commonly known in Arabic as (Hab el Rashaad or Thufa), grown in many regions of Saudi Arabia like Hijaz, the Eastern province, and Al-Qaseem [12]. It has an antiparasitic efficacy against Eimeria tenella [13], Echinococcus granulosus [14], and T. spiralis [15]. Many beneficial biological effects of Eucalyptus are known such as anti-oxidant activities, anti-microbial, anti-hyperglycemic, and anti-Trichomonas activity [16]. Its extracts contain cineol, terpenoids, and polyphenols that can display remarkable antioxidant activity [17]. It was also found that eucalyptol (the principal compound of the essential oils of Eucalyptus species) exhibits antioxidant activity because of the presence of phenolic compounds [18]. Maslinic acid (MA) (2R, 3-dihydroxyolean-12-en-28-oic acid) which is found in numerous plants is a triterpenoid compound related to oleanolic acid [19]. It is also found in considerable amounts, especially in fruit and leaves of Olea europaea (olives) [20]. Maslinic acid acts like other protease inhibitors by inhibiting intracellular replications and growth of T. gondii as well as blocking the parasite entry into the cell [21].

Resveratrol (trans-3,4′,5-trihydroxystilbene, RSV), is a polyphenol found in many plants, especially peanuts, mulberries, and grapes [22]. It has a wide range of pharmacological activities, including antioxidant [23], anti-inflammatory [24], and anti-T. gondii [25]. Also, it helps in reducing oxidative stress contributes to prolonging the lifespan of organisms of different and also reduces the inflammation caused by the parasite [26]. We aimed in this research to assess the anti-inflammatory effects of medicinal herbs (Lepidium sativum, eucalyptus leaves extract, and olive leaves extract) and resveratrol against experimental Toxoplasma gondii infection in mice by histopathological and immunohistochemical study [5, 6].

PATIENTS/MATERIALS AND METHODS

Parasites and mice

Non-virulent ME49 strain of T. gondii was used for induction of chronic infection in mice. The strain was obtained from the Medical Parasitology Department at the Faculty of Medicine, Zagazig University in Egypt, maintained in the Animal House Center of the Faculty of Medicine at Zagazig University. Infected mice were sacrificed, and brains were removed under sterile conditions and homogenized with 1ml of normal saline, the number of tissue cysts was determined by placing 2 drops of each 20µl brain homogenate on slides and counted under light microscopy with magnifying (lens ×40) and the count was multiplied by 20 to obtain the number of tissue cyst per brain [27]. 176 healthy laboratory-bred male Swiss albino mice weighing about 20-25gm each, aged 5 weeks was selected from the Animal House Center of the Faculty of Medicine, Zagazig University were conducted in this study.

Drugs and Plant material

Mice received pyrimethamine (12.5mg/kg) and sulfadiazine (200mg/kg) (Sigma Aldrich). Their active ingredients were calculated for each mouse for every dose, and after that, its ingredients were dissolved in 0.5ml of 0.5% Tween-80 solution and given as a combination according to [28]. Resveratrol (100mg/kg) was purchased as a powder from (Sigma Aldrich). Its active ingredients were also calculated, and after that, its ingredients were dissolved in 0.5ml of 0.9% saline [29].

The fresh leaves of olive (Olea europaea) and Eucalyptus (Eucalyptus Camaldulensis) were collected from the experimental farm of the Pharmacognosy department, Faculty of Pharmacy, Zagazig University, Egypt. Dried seeds of cress (Lepidium sativum) were purchased from the local Egyptian market. The extract was prepared in the Pharmacognosy department, Faculty of Pharmacy, Zagazig University, Egypt. 500 g of fresh olive and Eucalyptus leaves and 300 g of dried cress seeds were cut into small pieces then, separately macerated into 80% methanol till complete exhaustion. The extracts were filtrated over filter paper and then the methanol was removed under reduced pressure at 50C to obtain semi-solid residues of crude plant extracts of 50 and 70 grams, respectively [30].

The suspensions of the dried extracts were prepared for oral administration using 0.5% Tween-80 (ADWIC, Egypt) as a suspending agent in normal saline. The concentration of each preparation was adjusted so that each 0.1 ml of the prepared suspension contains 1 mg of the plant extract, to achieve a dose of 200mg/kg for eucalyptus and olives leaves and Lepidium sativum seeds extract [31].

Experimental design

Mice were divided into eleven experimental groups (16 mice each) as follows Group (1) Control non-infected Group, (2) Control-infected non-treated, Group (3) infected and treated with pyrimethamine-sulfadiazine, Group (4) infected and treated with RSV, Group (5) infected and treated with Eucalyptus leaves extract, Group (6) infected and treated with Lepidium sativum seeds extract, Group (7) infected and treated with olives leaves extract, Group (8) infected and treated with both pyrimethamine-sulfadiazine and RSV, Group (9) infected and treated with both pyrimethamine-sulfadiazine and Eucalyptus leaves extract, Group (10) infected and treated with both pyrimethamine-sulfadiazine and Lepidium sativum seeds, and Group (11) infected and treated with both pyrimethamine-sulfadiazine and olives leaves extract. All drugs and plant extracts were given once daily for 2 weeks, started 24 hrs. post-infection, orally as a liquid suspension by gavage.

Mice inoculation and scarification  

Mice were orally infected with 10 cysts/ mouse using a 19-gauge gavage needle. Six weeks post-infection, mice were sacrificed, and their brain tissues were put in 10% formalin for both histopathology and immunohistochemistry studies.

Histopathology and immunohistochemistry (iNOs and NSE( Brain and liver tissues from the included groups were fixed in a 10% neutral buffered formalin solution. Paraffin blocks were prepared, sectioned between 3 and 5µm thickness then stained with hematoxylin and eosin for histopathological evaluation [32].

Other sections were prepared for immunostaining using Biotin-Streptavidin (BSA) [33]. To ensure proper deparaffinization, paraffin sections were put in Xylene overnight, and then these sections were brought to distilled water through 100%, 95%, 75%, and 50% ethanol. For antigen retrieval, Slides were placed in an unsealed plastic container filled with sufficient antigen retrieval solution (Citrate buffer solution, pH 6). The plastic container was placed in an open plastic tray to catch boil-over. Slides were put in a microwave oven (Samsung 800 Watts with digital control) at power 10 for 5 minutes. The amount of fluid in the container was checked and water was added if necessary to prevent slides from drying. Microwave for an additional 5 minutes on Power 10. The container was removed from the oven and allowed to cool for 15 minutes. Slides were then washed in deionized water several times then placed in phosphate buffer saline (PBS) for 5 minutes. Tissue sections were incubated with an endogenous peroxidase-blocking reagent containing hydrogen peroxide and sodium azide (DAKO peroxidase blocking reagent, Cat. No. S 2001). One to two drops of the supersensitive primary monoclonal antibody [against, Inducible nitric oxide synthase (iNOS, Cat. No. ABN26, Sigma-Aldrich) and Neuron-specific enolase (NSE, Cat. No. AB9698, Sigma-Aldrich) were then put on the sections. Slides were incubated horizontally in a humid chamber at room temperature for 60 min. After blotting off excess buffer, 2 drops of DAKO EnVision + system were applied for 25 minutes at room temperature. Sections were then rinsed with PBS as before and blotted. Chromogen used was DAB (diaminobenzidine), 1-2 drops for 10-20 min. until a desirable brown color was obtained, the slides were then washed in the buffer. Sections were taken to distilled water then nuclear counterstaining was done using Mayer's hematoxylin (Hx). Immunoreactive intensity was expressed by average grayscale. Values

RESULTS

DISCUSSION

Drug therapy for T. gondii is difficult because of its distinct pathophysiology [31]. To cause a persistent infection, the parasite penetrates the blood-brain barrier [35]. Treatment of persistent infection is challenging because the blood-brain barrier prevents the transfer of appropriate medication concentration [36].

There are few effective treatments for toxoplasmosis at the moment, and many of them have negative side effects. Therefore, it is essential to look for substitute chemicals with new modes of action [37]. In comparison to the current anti-toxoplasma medicines, natural chemicals, and traditional herbal medicine are highly available and have fewer adverse effects [38]. From our previous work, we found that natural plants (Lepidium sativum, Eucalyptus, and Olive leaf extract) and resveratrol showed anti-toxoplasma effect via significant reduction of the brain cysts count when used either as monotherapy or combined with PYR and SDZ (unpublished data). In the current investigation, we sought to assess the anti-inflammatory effects of OLE, LSSE, Eucalyptus, and RSV against chronic experimental T. gondii infection in mice. Drugs were used separately as well as in conjunction with PYR and SDZ. Analysis using, histopathological and immunohistochemical methods was used to evaluate the situation.

Regarding the histopathological and immunohistochemical finding, our results showed that G2 (infected non-treated group) had strong positive iNOS expression in hepatic Von Kupffer, inflammatory cells, and degenerated neurons. Further, a strong positive NSE immunostaining appeared as brownish cytoplasmic stainability in some degenerated neuronal cells and reactive glial cells. On the same side, Mahmoudvand et al. demonstrated that mRNA levels of iNOS significantly increased in chronic T. gondii infection [39]. They added that chronic T. gondii infection communication among immune cells promotes neuroinflammation through cytokine networks and induces pathological progression of Alzheimer’s disease (AD) in the mice brain.  Furthermore, Dincel and Atmaca showed a statistically significant increase in the expression of NSE, confirming the severity of degeneration in the CNS in TE [4]. They added that oxidative stress and expression of NSE might give an idea of the disease progress and may also have a critical diagnostic significance for patients infected with T. gondii.

 We observed an improvement in the histopathological appearance of liver and brain tissue evidenced by a decrease in inflammatory cells and an improvement in the tissue structure towards the normal, also, there was a decrease in iNOS level in the liver and brain, and NSE level in the brain detected by both photo-micrographs and morphometric analysis. The greatest improvement was found in OLE-treated groups. We proposed that this effect was attributed to the anti-inflammatory effect of the MA and oleuropein. According to Omar the anti-inflammatory effect of oleuropein was related to the reduction of lypoxygenase activity and the formation of leukotriene B4 [40].

This agreed with Abugomaa and Elbadawy who observed that OLE improved the histological appearance of renal glomeruli and renal tubules as well as retained the normal histological appearance of hepatic lobules in glycerol-exposed rats [41].

In LSSE-treated groups, the improvement in the histopathological and immunohistochemical findings of liver and brain tissue can be due to its anti-inflammatory and antioxidant activities. The primary component of L. sativum seeds is -linolenic acid, which suppresses the expression of iNOS and prevents NO generation. According to Ren and Chung linolenic acid may exert this action by preventing NF-B activity and the phosphorylation of mitogen-activated protein kinase (MAPK) in macrophages [42].

The ethanolic extract of L. sativum significantly decreased iNOS-2 expression and nitrate concentration. Nuclear factor kappa-B (NF-B) nuclear expression, NF-B DNA binding activity, and cytokines (TNF- and IL-6) were all considerably downregulated by the reduction in nitrosative stress in a dose-dependent manner [43]. This agreed with Al-Otaibi et al. who observed that LSSE improved histopathological changes in the liver of Trypanosoma evansi-infected mice [44], also, with Balgoon who observed that LSSE restored the normal hepatic and renal structure in rats exposed to aluminum-induced hepatic and renal toxicity [45]. As regards Eucalyptus extract treated groups, the obtained results may be attributed to the antioxidant and the anti-inflammatory potential of Eucalyptus extract and inhibition of TNFα, IL6, NO, iNOS, and COX-2 expression. This agreed with Mousa et al. who detected that Eucalyptus attenuated diclofenac sodium-induced pathological alterations in hepatic tissues of rats [46].

In RSV-treated groups, we proposed that the anti-inflammatory effect of RSV is due to the antioxidant properties of this compound and its ability to decrease IL1β. RSV decreased the generation of NO in the macrophages of mice with Leishmania infection, according to Mousavi et al [47]. This agreed with Highab et al. who detected that RSV reduced the hepatocytes injury and preserved the liver parenchyma in rats exposed to lead intoxication [48]. It has been proven that T. gondii elicits robust innate and TH1 adaptive immune responses in the CNS, where the expression of inflammatory cytokines and mediators such as NO has both protective and pathological effects [49]. Although these factors restrict parasite replication and spread, inflammatory responses can also cause considerable injury to uninfected neurons and can additionally influence neurotransmitter functions and synaptic transmission [50].

CONCLUSION

In conclusion, there was a great improvement in the histological appearance of the liver and brain in all treated groups compared to the control group (G2). All treated groups showed a significant effect on the expression of iNOS and NSE in both liver and brain mainly OlE treated groups (7 and 11). In addition, the medicinal plants (OLE, LSSE, Eucalyptus) and RSV could be an efficient alternative to traditional treatment of Toxoplasma gondii by ameliorating the histopathological changes that were caused by toxoplasmosis.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of Interest: None.

Ethical considerations

Mice were reared and sacrificed according to the protocol of The Institutional Animal Care and Use Committee of Zagazig University (ZU-IACUC) for Animal Use in Research and Teaching. All procedures were done under anesthesia and all efforts were made to ensure minimal animal suffering. As T. gondii is a bio-safety level 2(Bl-2) pathogen, appropriate precautions were followed when handling the parasite. Care was taken to avoid infection of assisting personnel during the parasite-animal passage. The study protocol was approved by the Medical Parasitology Department Review Board (Approval No, ZU-IACUC/3/F/95/2020).