Potential of rosmarinic acid to ameliorate toxic effects of diethyl methoxy thio‑phosphoryl thio‑succinate on albino wistar rats’ lung, mast cell infiltration inhibitory pathway

Abstract Malathion (MA) is a widely used pesticide in agriculture. It can cause toxicity in different organs of the body. Rosmarinic acid (RO) is found in rosemary extract that can be absorbed through gastrointestinal tract mucosa with potent antioxidant, and anti‐inflammatory potential. The current study is designed to investigate the potential of RO to protect the lung after MA administration. Forty albino rats were allocated equally to four groups. C‐group received corn oil. RO‐group received RO orally. MA‐group received MA. MA‐RO‐group received RO in addition to MA. After three weeks the lungs were dissected for histopathological and biochemical investigations. MA‐group showed manifestations of severe inflammation with inflammatory cells infiltration in the lung. MA‐RO‐group showed limited inflammatory cell infiltration. C‐group and RO‐group appeared with weak anti‐survivin immunoreactivity. MA‐group showed strong positive immunoreactivity. The reactivity was weakly positive in MA‐RO‐group. MA‐group showed a significant decrease in SP‐D gene expression in comparison to the C‐group, in addition, MA‐RO‐group showed a significant increase in SP‐D expression. In conclusion, the current study approves that oral administration of MA causes lung injury as it has inflammatory effects, caused by oxidative stress and reports the potential of RO to protect lung tissue against toxic effects of MA through its anti‐inflammatory, antioxidant, and anti‐apoptotic potential.

in the pathogenesis of lung inflammatory diseases, such as acute lung injury and acute respiratory distress syndrome (Crimi et al., 2006).
Survivin, a member of the inhibitor of apoptosis family, inhibits caspasemediated cell death by increasing inhibition of caspase through binding the X-linked inhibitor of apoptosis. (Terasaki et al., 2013). They found clear evidence of survivin-positive epithelial cells of bronchioles and alveoli in bleomycin-injured lungs and suggested that it may be involved in lung regeneration and proliferation after acute lung injury. Because of the excellent bioactivity of Ro, authors hypothesized that supplementation with RO may protect against MA-induced lung injury in rats.
Therefore, the current study is designed to investigate the potential of RO to protect the lung after MA administration.

| Kits and chemicals
All chemicals and kits were purchased from Sino-pharm (China).

| Animals and experimental design
Forty albino Wistar rats were used with an average of weight 200 gm. Animals were allowed to acclimatize for one week under the following conditions (in accordance with national and institutional guidelines): free access to chow and water, 12 light/dark cycles, temperature 25°C, humidity 55%. Chow and water consumption in addition to mortality and health status were recorded daily. At the end of the first week, rats were allocated into four groups (n = 10). Control group (C-group) received 0.5 ml of corn oil/day by oral gavage. RO treated group (RO-group), received RO (50 mg kg −1 b.w. day −1 ) (Domitrović et al., 2013) in corn oil vehicle orally by oral gavage. MA treated group (MA-group) received MA (100 mg kg −1 b.w. day −1 ) in corn oil vehicle (Kalender et al., 2010). MA +RO treated group (MA-RO-group), received RO (50 mg kg −1 b.w. day −1 ) in addition to MA (100 mg kg −1 b.w. day −1 ) in corn oil vehicle. At the end of the treatment protocol (three weeks) all rats were euthanized by the help of sodium pentobarbital (intraperitoneal injection, 60 mg/kg b.w.). Lungs were dissected. The right lung was fixed in 10% formalin for histopathological examinations, while the left one was rapidly frozen (−80°C) for further biochemical studies.

| Histopathological examination
Hematoxylin and eosin staining was done in accordance with Li et al., (2018). Briefly, the fresh lung was cut into 0.5 cm 3 cubes immediately after extraction from the rats. It was placed in fixative 10% formalin and left for 48 hr then placed in tissue processing cassettes. By the help of ascending grades of alcohol, tissue is dehydrated to remove water and formalin traces from tissue then immersed in xylene to remove alcohol and facilitate paraffin wax infiltration into the tissue. Cassettes were placed on warm plates then tissue was removed and immersed in paraffin blocks. After paraffin solidification, the blocks were cut into 5 μm thick sections by using a manually operated rotary microtome. Tissue sections were placed on glass microscope slides, rehydrated, stained with hematoxylin and eosin. The stained tissue sections were dehydrated again by ascending grades of alcohol for 10 min then covered by a coverslip. Scoring was done as per lesion severity as shown in Table 1.

| Immunohistochemistry examinations
Immunohistochemistry was done in accordance with Magaki  After staining, slides were immersed in distilled water then counterstained with hematoxylin to stain nuclei in blue for better visualization. Ten fields per section were analyzed by Image J 1.24 version software.

| Real-time quantitative polymerase chain reaction (PCR) analysis
PCR (Thermo Fisher, USA) was used for the quantification of pulmo-  (Table 2).

| Statistical analysis
Statistical Package for Social Sciences (SPSS) software version 20 (SPSS Inc., USA) was used for data analysis. The statistical significance of differences between groups was validated using one-way analysis of variance (ANOVA) (Lee & Lee, 2018). Post hoc Tukey-Kramer test was used for group comparison (Kim, 2015). Data were expressed in mean ±standard deviation and probability value was considered significant if <0.05.

| Effect of RO on lung histological architecture after MA administration
Lungs of both C-group and RO-group had normal architecture with intact patent alveoli with spongy appearance in addition to normal

| D ISCUSS I ON
Currently, MA is an extensively used member of organophosphorus  kinase receptor c-kit antibody is a diagnostic marker of mast cell (Leong et al., 2003). This indicated the inflammatory effect of MA, as mast cells are important for immune responses as in allergy, asthma, and arthritis (Galli et al., 2008;Kaur et al., 2006). In addition, mast cell activation can enhance oxidative stress pathways (Zhao F I G U R E 2 (a-d) Photomicrographs of lung stained with anti-tyrosine-kinase receptor c-kit antibody (X 400), (n = 10). C-group (a) and RO-group (b) appear with weak immunoreactivity. MA-group (c) shows strong positive immunoreactivity. The reactivity is weak positive in MA- ROgroup (d). (e) Scoring shows a significant (p <.05) increase in MA-group if compared to C-group while MA-RO-group shows a significant (p <.05) decrease if compared to MA-group. * significant (p <.05) difference in comparison to C-group. # significant (p <.05) difference in comparison to MA-group. Data are presented as mean ± SD, (n = 10) with Clark et al., (2002) who linked between SP-D deficiency and apoptosis of pneumocytes, so significant increase in gene expression of MA-RO-group proved that RO has anti-apoptotic effect on F I G U R E 3 (a-d) Photomicrographs of lung stained with anti-survivin antibody (X 400), (n = 10). C-group (a) and RO-group (b) appear with weak immunoreactivity. MA-group (c) shows strong positive immunoreactivity. The reactivity is weak positive in MA- RO-group (d). (e) Scoring shows a significant (p <.05) increase in MA-group if compared to C-group while MA-RO-group shows a significant (p <.05) decrease if compared to MAgroup. * significant (p <.05) difference in comparison to C-group. # significant (p <.05) difference in comparison to MAgroup. Data are presented as mean ± SD, (n = 10) MA-induced lung injury. To the best of our knowledge, this is the first study to report the anti-apoptotic potential of RO against MA on lung tissue. Other studies had shown the anti-apoptotic effect on the cardiac muscles (Kim et al., 2005), and myoblast C2C12 cell line (Chen et al., 2014). Another study showed that RO arrested apoptosis induced by a high-fat diet (Cai et al., 2019). On the other hand, many studies demonstrated the apoptotic effect of RO on cancer cells such as lung, and prostate (Yesil-Celiktas et al., 2010).
In conclusion, the current study approves that oral administration of MA causes lung injury as it has inflammatory effects, caused by oxidative stress and reports the potential of RO to protect lung tissue against toxic effects of MA through its anti-inflammatory, antioxidant, and anti-apoptotic potential.

CO N FLI C T O F I NTE R E S T
Authors have declared that no competing interests exist.

E TH I C S S TATEM ENT
The study protocol was approved by Research and Ethics Committee, Quality Assurance Unit, Faculty of Medicine, Tanta University, Egypt.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

R E FE R E N C E S F I G U R E 4
Effect of RO on SP-D gene expression after MA administration. MA-group shows a significant (p <.05) decrease of SP-D gene expression if compared to C-group. There is a significant increase (p <.05) in gene expression of MA-RO-group if compared to MA-group. * significant (p <.05) difference in comparison to Cgroup. # significant (p <.05) difference in comparison to MA-group. Data are presented as mean ± SD, (n = 10)