MATERIALS AND METHODS

This study was performed in Cukurova University Medicine Faculty Department of Pharmacology, between the dates of 01.01.2018-01.05.2018.

Chemicals: Culture media were purchased from GIBCO BRL (Grand island, NY, U.S.A.) and calf serum was from HyClone Laboratories, Inc. (Logan, UT, U.S.A.). Rat tail collagen type 1was purchased from Becton Dickinson Laboratories (Bedford, MA, U.S.A.). RIPA buffer, fetal bovine serum, PBS, NaCl, TritonX-100, EGTA, dithiothreitol, NaF, Tris-Cl, Na3VO4 were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Inc. bax, bcl-2, wee 1, AIF, gadd153 and grp78 of ELISA kit were purchased from Shanghai Sunred Biological Technology Co., Ltd. p-erk and p-jnk were purchased from My BioSource, Inc.(San Diego, U.S.A.). SOD and GPX assays obtained from BioVision (USA). TAS and TOS assays were purchased from Rel Assay Diagnostics Inc.(Gaziantep, TURKEY).In addition, the Bradford dye reagent was purchased from Bio-Rad Laboratories, Inc.(California, U.S.A.).

Cell culture: The mouse osteocyte-like cell line MLO-Y4 was obtained from Kerafast, Inc. The cells were cultured according to the previously described protocol by Kato and Bonewald^21,22. MLO-Y4 cells were incubated with MTX (10^–5 M) and MTX (10^–5 M) plus HP extract (100 μmol L^–1) for 48 h and then cells were homogenized for ELISA experiments

Cell homogenization: Cells (5×10⁴ cells/cm²) were exposed to 100 μmol L^–1 MTX for 48 h. They were then washed in Phosphate Buffer Solution (PBS) and lysed in RIPA buffer (150 mmol L^–1 NaCl 0.5%, TritonX-100, 20 mmol L^–1 EGTA, 1 mmol L^–1 dithiothreitol, 25 mmol L^–1 NaF, 50 mmol L^–1 Tris-HCl [pH 7.4], 1 mmol L^–1 Na₃VO₄) for 15 min on ice followed by centrifugation at 15000 rpm for 20 min. and supernatants are taken and pellets are discarded²³.

Total protein determination: Bradford method is used to determining of the total protein in homogenized tissues. Protein determination (μg μL^–1) was done according to the standard curve drawn in Prism software²³.

ELISA (Enzyme linked immunosorbent assay) tests: ELISA test is used to examine the activity of caspase-3 and expression of bax, bcl-2, wee 1, gadd153 AIF protein which are apoptotic pathway’s mediators. Active ERK (p-ERK) and active JNK(p-JNK) also analyzed by ELISA.

The detection of TAS, TOS, SOD and GPx was performed as previously described²⁴.

Statistical analysis: Results were expressed as Mean±SEM. and n refers to the number of cell culture flask used for each groups. Differences in results between tissues were tested by Analysis of Variance (ANOVA) corrected for multiple comparisons (Bonferroni corrections). P values less than 0.05 were considered to be significant.

RESULTS

Apoptotic mediators: We evaluated mediators of apoptosis which is also known as programmed cell death. While caspase and bax have pro-apoptotic effect, bcl-2 has anti-apoptotic effect. Increased activity of caspase 3 and disruption in the bax/bcl-2 ratio in the direction of bax initiates apoptosis in the cell. MTX treatment increased activity of caspase-3 (Fig. 1a) and expression of bax (Fig. 1b), wee 1, gadd153 and AIF (Table 1) decreased bcl-2 (Fig. 1c) significantly and HP treatment reduced these effects significantly.

MAPK mediators: JNK and ERK are mediators of MAPK signaling pathway which transfer information to the nucleus. The activity of JNK (p-JNK) and ERK (p-ERK) found higher in the MTX group when compared to the control group. HP treatment reduced the MTX induced activity of JNK and ERK. (Fig. 2a-b).

Oxidative stress parameters: The Total Antioxidant Status (TAS) levels were found significantly lower in the MTX group than in the control group (p<0.05). And the TAS levels were found significantly higher in HP treated group when compared to MTX group (Fig. 3a).

The total oxidant status (TOS) levels were found higher in the MTX group than the control group (p<0.05) and the TOS levels were found significantly lower in HP treated group when compared to MTX group (Fig. 3b).

The SOD and GPx (endogen ROS scavenger) levels were found significantly lower in the MTX group when compared to the control group (p<0.05). The SOD and GPx levels were found higher in the HP treated group than in the MTX group (p<0.05) (Fig. 4a-b).

Fig. 1(a-c):	Effect of MTX and HP on (a) caspase-3 activity (b) bax expression and (c) bcl-2 expression (n = 8), Statistical analysis: ANOVA, Post hoc: Bonferroni
	*: For control p<0.05, #For I/R p<0.05

Table 1:	Effects of HP on expression of mitotic division inhibitors (Wee1 and GADD153), proapoptotic AIF and dual function GRP78 proteins
Results are presented as Mean±SE, Statistical analysis: one way anova (Bonferroni corrections), *p<0.05 for the comparison between the Control and MTX, #p<0.05 for the comparison between the MTX group and MTX+HP, MTX: Methotrexate, HP: Hypericum perforatum

Fig. 2(a-b):	Effect of MTX and HP on (a) p-JNK expression and (b) on p-ERK expression (n = 8), Statistical analysis: ANOVA. Post hoc: Bonferroni
	*: For control p<0.05, #For I/R p<0.05

Fig. 3(a-b):	Concentration of (a) TAS levels and (b) TOS levels in groups (n = 8), Statistical analysis: ANOVA, Post hoc: Bonferroni *: For control p<0.05, #For I/R p<0.05

Fig. 4(a-b):	Activity of (a) GPx level and (b) SOD level in groups (n = 8), Statistical analysis: ANOVA, Post hoc: Bonferroni
	*: For control p<0.05, #For I/R p<0.05

DISCUSSION

This study was designed in vitro acute chemotherapy with MTX by using osteocyte-like MLO-Y4 cells, which are regarded to be the most compatible in vitro model available to clarify the pathophysiological responses of in vivo osteocytes^21,25. MTX induced apoptosis by increasing caspase-3 activity and expression of pro-apoptotic proteins and decreasing anti-apoptotic proteins. MTX together HP treatment ameliorated the apoptotic effect of MTX via inhibiting MAPKs and oxidative stress.

There are lots of extracellular and intracellular stimuli have been shown to activate MAPK pathways²⁶. One of them are Reactive Oxygen Species (ROS) that activates MAPK pathways²⁷, but the mechanism(s) for this effect is unclear ROS may have important roles as a modulator of cell as signaling molecules. Indeed, lots of evidence supported that ROS has a physiological role as a “second messenger” in intracellular signaling pathways that control cell growth, proliferation, migration and apoptosis²⁸.

Many protective features of HP have been shown in studies^16,19,29,30. This plant has both antioxidant and anti-inflammatory effects. Chemotherapeutic drugs, including methotrexate used in cancer treatment, have side effects. Methotrexate-induced toxicity the duration of treatment with methotrexate and the interaction of many factors such as dose schedules, type of disease and patient risk factors as well as genetic and molecular apoptotic factors³¹. MTX therapy is contraindicated in rheumatology authorities²⁴. For these reasons, it is emphasized that if the use of medication is necessary for patients with rheumatoid arthritis and cancer, the dose should be reduced by 75% and be given by close monitorisation³². MTX treatment induces oxidative stress. Increasing superoxide radicals with oxidative stress cause decrease of endogen antioxidant such as glutathione (GSH). With the reduction of the level of GSH due to methotrexate therapy decrease of the effectiveness of the antioxidant defense system, which protects against reactive oxygen radicals such as superoxide anion, hydroxyl radicals, hydrogen peroxide and hydrochloric radicals³¹. A study has shown that oxidative stress, plays a role in methotrexate-induced small bowel injury due to neutrophil infiltration³³. The most serious side effect of methotrexate is renal toxicity³¹. Recent studies showed that MTX treatment causes bone loss and the depletion of bone-forming osteoblasts and osteocyte cell death³⁴. HP is frequently used by local people in wound healing. Studies have shown that this plant is a potent anti-inflammatory and antioxidant¹⁶. Studies have shown that the antioxidant properties of HP also contribute synergistically to its anti-inflammatory properties. It has been shown that hypericin is a free radical scavenger and inhibits arachidonic acid release from phospholipids via blocking the 5- and 12-lipoxygenase pathway and IL-la and IL-12 formation. Furthermore, it has been reported that NF- [kappa] B, the regulator of inflammatory mediators and MAPK, are inhibited by the hypericin^35,36. It has been shown that the formation of another active ingredient, hyperphorine inhibits formation of free oxygen radicals, elastase release, cyclooxygenase-1, 5-lipoxygenase and IL6 release from leukocytes. Hiperosid and isoquercitrin which are flavonoid of HP inhibit the neutrophil elastase which has a role in the pathogenesis of inflammation and nitric oxide synthase. Isoquercitrin has also been shown to inhibit prostaglandin biosynthesis and secretion³⁷. In addition, the flavonoid, amentoflanon, has been shown to elicit arachidonic acid release from cyclooxygenase-2, phospholipase A2, iNOS and neutrophils. Recent toxicological studies with anticancer drugs have focused on oxidative stress. Despite these wide indications for use, MTX is confronted with side effects, especially nephrotoxicity and hepatotoxicity and these side effects are often responsible for the oxidative damage caused by reactive oxygen species^32,33. For this reason, it is suggested that the osteo-protective effect of HP may have resulted from its antioxidant and anti-inflammatory properties. This study has shown the cell-protective effect of the HP, but the studies have also shown the toxic effects of the HP on cancer cells^17,23,38. In this respect, the importance of HP is increasing. Because chemotherapeutic agents also harm normal cells and there is no chemotherapeutic agent that does not harm normal cells and has no side effects.

CONCLUSION

In conclusion, MTX treatment induces apoptosis and increases MAPK activity in osteocytes via inducing oxidative stress. HP treatment attenuates MTX induced apoptosis by reducing oxidative stress and MAPK activity. HP has a protective effect on MTX induced osteo-toxicity. Our study has shown that it can be beneficial to use MTX with HP, which is frequently used in the clinic and whose toxic effects are known.

SIGNIFICANCE STATEMENT

The protective effect of the HP against the MTX has been discovered. Hypericum perforatum has strong anti-inflammatory and antioxidant agent. This study predicts that it is effective against other chemotherapeutics that increase oxidative stress. This study will help the researchers to uncover the importance of alternative plant-based remedy based on Hypericum perforatum in the treatment of MTX induced osteo-toxicity that many researchers were not able to explore. Thus, a new theory that the extract of Hypericum perforatum is effective and sufficient in the fight against MTX induced osteo-toxicity may be arrived at.