2.3. MASM Prevents TBI-Induced Differential Expression of Many Genes
The liver was considered to be a radiosensitive organ [21
]. Vacuoles and mitochondrial swelling were significantly decreased by MASM in the liver (Figure 2
C3), indicating that MASM ameliorated TBI-induced liver tissues injury. To further investigate the potential molecular basis of the protective effects of MASM on irradiation damage, gene expression analysis was conducted on rats liver tissues using microarrays.
A heatmap was generated (Figure 3
) representing 3912 transcripts (probesets) comparing significantly differentially regulated genes (p
< 0.01) for the treatment groups (TBI, MASM + TBI) versus
untreated controls by using one-way analysis of variance (one-way ANOVA) (Supplementary File 1
). Cluster analysis of the microarray results revealed that three or four rats from the same group were the closest, indicating that all the experimental data in this study were reliable.
A combined algorithm with simple t
-test and fold change was used to find differentially expressed genes. By a threshold of p
< 0.05 and absolute fold change (FC) > 1.5, 2920 significant expressed probes were selected by comparison between radiation and wild-type (control) samples, involving 1445 unique genes (Supplementary File 2
). Among the genes originally displaying FC > 1.5, 414 were subsequently down-regulated and 1031 up-regulated. In the pretreatment with 30 mg/kg MASM groups, only 680 out of 1445 genes were still differentially expressed in response to TBI. These results indicated that the treatment with MASM prevented TBI-induced differential expression of 53% (765 genes) of genes (Supplementary File 3
). We focused on these genes for which TBI-induced alteration of expression were abolished or attenuated by MASM pretreatment, as these genes might be regulated by MASM and involved in the protective effects on radiation.
Gene ontology (GO) analysis was then applied to these genes in terms of molecular function, cellular component and biological process. Eleven enriched terms associated with these genes list were identified (Table 1
). The identified terms in molecular function were transporter activity, and chemorepellent activity. Only synapse part was in cell component. The terms included in biology process were death, growth, multicellular organismal process, locomotion, rhythmic process, response to stimulus, and localization.
Pathway enrichment analysis indicated that these genes were mainly involved in a total of 21 pathways (Table 2
). Most of these pathways were reported to be induced by radiation in the previous studies, such as olfactory transduction [22
], cytokine-cytokine receptor interaction [22
], neuroactive ligand-receptor interaction [23
], pathways in cancer [24
], MAPK signaling pathway [27
], PPAR signaling pathway [28
], GnRH signaling pathway [29
], calcium signaling pathway [25
], acute myeloid leukemia [30
], vascular smooth muscle contraction [32
], gap junction [33
], bladder cancer [34
] and circadian rhythm [36
]. Interestingly, recently a review article reported that targeting cellular metabolism can improve the efficacy of cancer therapy [38
For example, targeting metabolic enzymes, such as glucose transporters, fatty acid synthase and glutaminase can enhance the efficacy of common therapeutic agents or overcome resistance to chemotherapy or radiotherapy [38
]. In addition, it has been reported that nicotinamide sensitizes tumors, at least in part, by modulating vascular smooth muscle contraction [39
]. Matrine and its compounds have been widely used as adjuvant therapy in China to improve the 5-year survival rate and life quality of patients with cancer [14
]. However, little is known about the mechanisms underlying the therapeutic effects of matrine. Table 2
showed that 38, 10, 7, 5 and 12 genes were involved in metabolic pathways, purine metabolism, arachidonic acid metabolism, fatty acid metabolism and vascular smooth muscle contraction, respectively. It is very likely that MASM/matrine improve the efficacy of cancer therapy, at least in part, by targeting these cellular metabolism and modulating vascular smooth muscle contraction, and this area is of great interest for our further studies.
Since ionizing radiation induces simultaneous compensatory activation of multiple MAPK pathways [27
], we focused on these pathways. These pathways play critical roles in controlling cell survival or death and repopulation effects following irradiation, in a cell-type-dependent manner [27
]. The 13 genes presented in Table 3
were involved in multiple MAPK pathways including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK pathway.
This was consistent with previous reports that ionizing radiation activated all 3 MAPKs with different intensities and in a cell type-dependent context [27
]. 11 of among gene expressions were evidently up-regulated by TBI and these up-regulations were abolished or attenuated by pretreatment with MASM. These TBI-induced genes include fibroblast growth factor 3 (Fgf3), myelocytomatosis oncogene (Myc), phospholipase A2, group IIA (Pla2g2a or sPLA2), growth arrest and DNA-damage-inducible, beta (Gadd45b), mitogen activated protein kinase kinase kinase 10 (Map3k10), protein kinase, X-linked (Prkx), calcium channel, voltage-dependent, R type, alpha 1E subunit (Cacna1e), calcium channel, voltage-dependent, L type, alpha 1S subunit (Cacna1s), TAO kinase 2 (Taok2), activating transcription factor 4 (Atf4), nuclear receptor subfamily 4, group A, member 1 (Nr4a1). In contrast, the expressions of neurotrophic tyrosine kinase, receptor, type 1 (Ntrk1) and apoptosis signal-regulating kinase 1 (Ask1) were down-regulated by TBI and these down-regulations were abolished or attenuated by pretreatment with MASM. Interestingly, Nr4a1 was a growth factor-inducible member of the Nr4a1/Nur77 subfamily of the nuclear receptor superfamily of transcription factors. Nr4a1 was involved in determine whether cells undergo double-strand break (DSB) repair or apoptosis in response to irradiation [42
]. A previous study reported that radiation up-regulated Nr4a1/Nur77 phosphorylation and expression [42
]. In this study, our data indicated that Nr4a1 expression was evidently up-regulated by TBI and the TBI-induced increase was attenuated by pretreatment with MASM. In addition, activating transcription factor 4 (Atf4) was a member of the ATF/CREB (activating transcription factor/cyclic AMP response element binding protein) family of basic region-leucine zipper (bZip) transcription factors. Atf4 was induced by stress signals including endoplasmic reticulum stress, amino acid deprivation, anoxia/hypoxia and oxidative stress. Atf4 expression was regulated transcriptionally, translationally via the PERK pathway. Atf4 regulated the expression of genes involved in amino acid synthesis, oxidative stress, differentiation, metastasis and angiogenesis [43
]. As described by the earlier reports [44
], our data indicated that TBI increased the expression of Atf4/CREB. Myc was involved in a wide range of cellular processes including signal transduction, cell-cycle control, self-renewal, metabolism, maintenance of pluripotency, and control of cell fate decisions [46
]. Here, our data indicated that the TBI-induced increase in Myc was attenuated by pretreatment with MASM. This was consistent with previous reports that irradiation significantly increased Myc [47
]. Gadd45 proteins were implicated in stress signaling in response to environmental or physiological stressors, which resulted in either cell cycle arrest, DNA repair, cell survival and senescence, or apoptosis [49
]. Consistent with the earlier reports [50
], the expression of Gadd45b was up-regulated by radiation. Prkx was a member of an ancient family of cAMP-dependent serine/threonine kinases distinct from the classical PKA, PKB/Akt, PKC, SGK, and PKG families [52
]. As described by the earlier reports [53
], Prkx protein kinase was up-regulated by radiation. Ntrk1/Trk A was a member of the neurotrophic tyrosine kinase receptor (NTKR) family. Ntrk1 was a membrane-bound receptor that, upon neurotrophin binding, phosphorylates itself and members of the MAPK pathway. The presence of Ntrk1 leaded to cell differentiation and might play a role in specifying sensory neuron subtypes [55
]. In UV-irradiated normal skin, there was a significant reduction in Trk A/Ntrk1 tyrosine kinase receptor immunostaining after UV-irradiation [57
]. Albeit our data strongly support radiation-induced down-regulation of Ntrk1 [57
], there are also reports on UV-induced up-regulation of both nerve growth factor NGF and its high-affinity receptor Ntrk1 [58
]. cPLA2 was a member of the PLA2 enzyme superfamily, which included secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and other members. cPLA2, which activated AA hydrolysis, existed in three isoforms: α, β, and γ. cPLA2-α was known to be a major component of the arachidonate-releasing signal transduction pathway [59
]. Low level laser irradiation significantly inhibited phospholipase cPLA2-α mRNA expression, which was increased in response to mechanical stress [59
]. Here, our data showed that TBI-induced increase in sPLA2 was attenuated by pretreatment with MASM. These data showed that MASM pretreatment could attenuate TBI-activated all 3 MAPKs (Table 3
). In our previous study, MASM could inhibit the Akt pathway by suppressing the phosphorylation of Akt, glycogen synthase kinase 3β (GSK3β), and P70S6 kinase in cultured activated or TGFβ1-activated hepatic stellate cells [20
]. In addition, our data showed that radiation-induced phosphorylation of P38 and JNK (c-Jun NH2
-terminal kinase) was dose-dependently inhibited by pretreatment with MASM in murine macrophage RAW264.7 cells (unpublished data). Moreover, some agents were reported to provide the protective effects via the modulation of MAPKs pathway [61
]. Taken together, these data indicated that the pretreatment of rats with MASM modulated lethal TBI-induced multiple MAPK pathways, suggesting that MASM might provide the protective effects mainly or partially through the modulation of MAPKs pathway. Therefore, MASM has the potential to be used as an effective therapeutic or radioprotective agent to minimize TBI-induced damages.
A number of studies demonstrated that the combined modality therapy involving radiation and MAPK pathway inhibitors was a promising strategy for improving the treatment of patients with cancer [40
]. Preclinical and clinical evidence suggested that agents targeting aberrant tumor signals could effectively improve the therapeutic effect of ionizing radiation [24
]. Here, this study suggested that MASM could offer radioprotection mainly or partially through the modulation of TBI-induced MAPKs pathway. Therefore, it is very likely that MASM is used in combination with radiotherapy to improve the efficacy of cancer therapy, and this area is of great interest for our further studies.
To validate the consistency of microarray analysis in the present study, we compared gene expression levels of selected genes between microarray and real-time PCR. We determined the mean value of expression of the selected genes in five independent rats from each exposure group. This was compared with those in pooled RNA from 5 non-irradiated rats. The qualitative changes in gene expression levels were consistent between these analyses (Figure 4