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The second DDR binding partner identified
There exists a glucose residue in the structure of 5. Acid hydrolysis (Supplementary data) followed by TLC comparison with the reference compound as well as GC–MS analysis (l-cysteine methyl ester hydrochloride derivative: tR=22.22min for glucose residue of 5 and 22.46min for the reference compound) confirmed the presence of an α-d-glucose, in accordance the observed J value of H-1′ (3.7Hz). Taken together, the structure of 5 was identified and named as plancyol B.
It is notable that, apart from compounds 4 and 5, all the other compounds are N-containing substances. Compound 2 is a pyrazine, similar compounds have been characterized from the defensive spray of Phyllium westwoodii[13]. Compounds 3a and 3b are N-acetyldopamine analogues which were reported to take participation in insect cuticle sclerotization and have been also isolated by us from several insects such as Aspongopus chinensis and Blaps japanensis[14], [15], [16]. As far as plancyol B (5) is concerned, it is a phtalide derivative. Such type of compounds was normally found in plants or microorganisms [17], [18]. In this circumstance, whether compound 5 is inherent in the insects or from their dietary remains unclear.
Three known compounds were respectively identified as 3-acetamido-5-acetylfuran (6) [19], ginsenine (7) [20], and anoectochine (8) [20] by comparing their NMR data with those in the literatures.
Compounds 1–6 were evaluated for their inhibitory activities against JAK3 and DDR1 kinases. It was found that compound 4 exhibits potent inhibitory activities towards these kinases with IC50 values of 5.0μM for JAK3 and 4.87μM for DDR1. In contrast, compound 2 also exhibits inhibitory activity against JAK3 kinase (IC50 value: 12.6μM), less than that of 4. In conclusion, these results suggest the potential of compounds 2 and 4 in JAK3 or DDR1 associated disorders.


Acknowledgments
We are indebted to National Science Fund for Distinguished Young Scholars (81525026) for financial support.


Introduction
Discoidin domain receptor 1 (DDR1), which belongs to disk-shaped receptor tyrosine kinases (DDRs), mainly regulates collagen synthesis and degradation, and monitors extracellular matrix (ECM) component formation (Vogel et al., 1997). Previous studies showed that DDR1 is overexpressed in some rapidly growing tumors, such as in lung, liver and breast cancers (Valencia et al., 2012). DDR1 degrades the ECM by upregulating matrix metalloproteinase (MMP) expression in glioma and pituitary adenomas, thereby enhancing tumor invasiveness (Ram et al., 2006).
MMPs are a group of Zn2+-dependent endopeptidases that can degrade and remodel the extracellular matrix. Among the MMPs, MMP-9 is closely related to the degree of ischemic PR-619 injury (Wu et al., 2012). Various exogenous injury factors or brain ischemia–reperfusion (I/R) can cause an increase in oxygen free radicals and the inflammatory response, which could degrade the basement membrane and ECM components of the blood–brain barrier (BBB) by activating matrix metalloproteinases, such as MMP-9 and MMP-3, and thereby damaging the BBB (Lakhan et al., 2013).
Studies in a variety of tumor cells (including nervous system tumors) have confirmed that DDR1 activation causes tumor cells to upregulate the MMP-9 expression (Roig et al., 2010). However, the variation in DDR1 expression in cerebral I/R injury is still unclear, and whether it can cause BBB damage by regulating MMP-9 expression is yet to be confirmed.


Materials and methods


Results


Discussion
This study showed that phospho-DDR1 expression increased after cerebral I/R injury, and that inhibiting DDR1 expression using an intracerebroventricular siRNA injection downregulates MMP9 expression and activity and reduces the BBB permeability. Our results indicate that I/R injury does not induce an increase in DDR1 expression levels in the ischemic cerebral penumbra. However, it induces DDR1 tyrosine phosphorylation and the DDR1 activation extended for at least 48h. MMP-9 expression also increased to varying degrees, while inhibiting DDR1 expression using an intracerebroventricular siRNA injection downregulated DDR1, phospho-DDR1 and MMP-9 expression. Glial cells are a more important source of MMP-9 than neurons, under various CNS pathological conditions including ischemia and inflammation in the brain (Kaczmarek et al., 2002). Double immunofluorescence staining with DDR1 and NeuN showed that DDR1 was mainly expressed in neurons, and that its expression in astrocytes was low (Supplementary Fig. S1). This is not consistent with previous studies (Franco-Pons et al., 2006, Sánchez et al., 1994), which showed that in the adult brain, DDR1 is expressed in oligodendrocytes and astrocytes but not in neurons. We speculate that such differences may exist because our research focused on DDR1 expression in the ischemic penumbra in brain using a MCAO model, while previous studies have focused on DDR1 expression in normal brain tissue and its physiological function on myelination. In addition, previous studies showed that DDR1 activation up-regulates the production of chemokines in an NF-κB-dependent manner and is likely to contribute to the development of inflammatory responses in the tissue microenvironment (Matsuyama et al., 2004). Studies also showed that invading inflammatory cells can provide the major source of the MMP activity (Kaczmarek et al., 2002). Moreover, MMP-9 is expressed in adult brain neurons and is responsive to changes in neuronal activity (Dzwonek et al., 2004). Combined with the results of previous research (Castro-Sanchez et al., 2011), we hypothesized that MMP-9 overexpression and activation after cerebral ischemic injury may be regulated by its upstream molecule (DDR1) via inflammatory responses, which provided a reliable basis for our next experiments.
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