Roles Of Kir6.1/K-ATP Channels In Ischemia/Reperfusion-induced Brain Injury In Mice

Stroke is a central nervous system disease with highest morbidity, highest mortality and disability rate. Stroke is mainly classified into ischemic stroke and hemorrhagic stroke. Nowadays, the progressive injury following stroke originates from the complex pathologic mechamisms, including energy exhaustion, excitotoxicity, calcium overloading, oxidative stress, inflammation and apoptosis. Though there is some progress in animal experiments, it’s not successful in the stroke translational researches. At present, there is no ideal drug for stroke. Currently, tissue plasminogen activator (tPA) is the main treatment drugs for stroke. However, this treatment is only appropriate for a very small number of patients, due to a narrow time window and adverse reactions. So, it is necessary to clarify the pathophysiological mechanisms of stroke, which is essential for drug development, all these are urgent for stroke treatment.ATP-sensitive potassium (K-ATP) channels are an unsual and non-voltage dependent potassium channel, which provide a unique link between cellular energetics and electric excitability. They are eight heteromultimers composed of four inwardly rectifying potassium channel (Kir6.x) and four sulfonylurea receptors (SURs). These channels widely distribute all round the organism. For a long time, as to the study of K-ATP channels in stroke, there are only some researches about the roles of Kir6.2/K-ATP channels, which mainly express in neurons. These results showed that Kir6.2/K-ATP channels, which affected brain ischemic injury via potentiating the depolarization of cellular membrane potential. In the past few years, scientists focused on the neuroprotection of neurons against stroke. Recently, more and more studies clarified the importance of glia in stroke. In particular, astrocytes, which are abundant in the brain, appear to be important regulators of post-ischemic inflammatory responses and glial scar formation, all of them are significant in the neurogenesis and repair after stroke. In our previous studies, Kir6.1/K-ATP channels widely distribute in the brain, among astrocytes, microglia and neural stem cells, all these play essential roles in the development of stroke. However, whether Kir6.1/K-ATP associates with stroke or not? Up to now, there is no research about them in stroke. Therefore, this topic is to reveal the roles of Kir6.1/K-ATP channels in brain ischemic injury, and discovery new targets for stroke treatment.So, in part I, we subjected both wild-type (WT) mice and Kir6.1heterozygote knockout (Kir6.1+/-) mice to cerebral ischemia/reperfusion injury with middle cerebral artery occlusion (MCAO) model, and to investigate the impacts of Kir6.1subunit knockdown on ischemic stroke. Our data reveals that the downregulation of Kir6.1/K-ATP channels is more susceptible to brain ischemia, which is related to enhancing ER stress and inflammatory responses during the brain ischemic injury. According to part Ⅰ, we found that Kir6.1/K-ATP channels played an essential part in post-ischemic inflammation. Then, in part Ⅱ, we focused on the roles of Kir6.1/K-ATP channels expressed in BV-2cell lines in OGD/R-induced inflammation. These results showed that opening Kir6.1/K-ATP channels could attenuate OGD/R-induced inflammation in BV-2cell lines, which implied that Kir6.1/K-ATP channels expressed in microglia were an important target for regulating post-ischemic inflammation. As to astrocytes play an important role in the brain. Accordingly, in part III, we concentrated on the roles of Kir6.1/K-ATP channels expressed in astrocytes in the brain ischemic injury. We found that opening Kir6.1/K-ATP channels expressed in astrocytes could inhibit OGD/R-induced ER stress and inflammation. Then, we subjected Kir6.1conditional knockout mice to MCAO model, and found astrocytes null for Kir6.1aggravated brain ischemic injury. These results suggested that Kir6.1/K-ATP channels expressed in astrocytes might be an essential target for stroke treatment.Part I Effects of Kir6.1gene heterozygote knockout on the focal cerebral ischemia/reperfusion injury in miceAIM:To investigate the effects of Kir6.1gene heterozygote knockout on focal cerebral ischemia/reperfusion injury in mice.METHODS:We subjected two to three-month-old WT and Kir6.1+/-male mice to cerebral ischemia by MCAO model with a modified intralumenal filament technique as described previously. They were all peformed by ischemia1h and reperfusion24h or72h. The mortality rate and neurological deficits were measured in each group after cerebral ischemia. The infarct size was determined by staining coronal brain slices with235-triphenyltetrazoliumchloride (TTC), the extravasation of Evan’s blue (EB) made known the BBB disruption. Moreover, we observed the changes of neurons, astrocytes, microglia and neurovascular substrates by immunohistochemistry staining, and applied Western blot assay to analyse expression of matrix metalloproteinases9(MMP-9), ER stress related proteins, including glucose-regulated protein78(GRP78), C/EBP-homologous protein (CHOP), caspase-12, and inflammatory related proteins, such as nuclear factor κB (NF-κB), NOD-like receptor pyrin domain containing three (NLRP3) and caspase-1. Furthermore, we detected the contents of interleukin-lbeta (IL-1β) and tumor necrosis factor-alpha (TNF-a) in the ischemic brain tissues by enzyme-linked immunoabsorbent assay (ELISA).RESULTS:1. Kir6.1gene knockdown exacerbated acute neural injury induced by cerebral ischemia/reperfusion injury in miceKir6.1gene knockdown aggravated the neurological disorder and infarct size after brain ischemic injury in mice. Kir6.1gene knockdown exacerbated BBB extravasation, increased expression of activated MMP-9and advanced Collagen-IV degradation. Kir6.1gene knockdown exacerbated neuronal death in the core infarct area, and promoted glial responses in the peri-infarct area after cerebral ischemia/reperfusion injury.2. Kir6.1gene knockdown exacerbated brain ischemic injury via enhancing ER stress and post-ischemic inflammationKir6.1gene knockdown enhanced ER stress induced by ischemic injury, by upregulating ER stress-related proteins, including molecular chaperone GRP78, transcription factor CHOP and apoptosis executor caspase-12. Kir6.1gene knockdown intensified post-ischemic inflammation, and promoted production of inflammatory factors TNF-a and IL-1β.CONCLUSIONS:1. Kir6.1/K-ATP channels knockdown aggravates the brain ischemic injury induced by focal cerebral ischemia/reperfusion in mice.2. Kir6.1/K-ATP channels knockdown exacerbates cerebral ischemia and reperfusion injury via advancing ER stress and post-ischemic inflammation. Part II Roles of Kir6.1/K-ATP channels in OGD/R-induced inflammation in BV-2cell linesAIM:To investigate the roles of K-ATP channels expressed in microglia in the OGD/R-induced inflammation.METHODS:BV-2cell lines were used in the following experiments. Firstly, we used immunofluorescence images to validate K-ATP channels subunits expressed in BV-2cell lines. Secondly, we analysed the changes of K-ATP channels subunits after OGD/R by Western blot, and then observed inflammatory cytokine IL-1β release and inflammasome activation, which cleaved proIL-1β into mature IL-1β, by ELISA and Western blot assays respectively. Finally, we pretreated BV-2cell lines with K-ATP channels opener (Cromakalim)1h before OGD, compared the production of IL-1β and the expression of NLRP3and caspase-1by ELISA and Western blot assays respectively.RESULTS:1. Oxygen-glucose deprivation induced inflammasome activation and promoted IL-1β production in BV-2cell linesOGD/R induced K-ATP channels Kir6.1subunit downregulation. OGD/R upregulated NLRP3and caspase-1, and induced inflammasome activation, which lead to an increase of IL-ip release from BV-2cell lines.2. Opening K-ATP channels inhibited OGD/R-induced inflammation in BV-2cell linesK-ATP channels opener (Cromakalim) pretreatment could ameliorate OGD/R-induced IL-1β release from BV-2cell lines. Pretreatment with Cromakalim could inhibit OGD/R-induced upregulation of NLRP3and caspase-1, and impeded activation of inflammasome, all of these resulted in the decrease of IL-1β. CONCLUSIONS:1. OGD/R results in activation of inflammasome and increases IL-1β production in BV-2cell lines.2. Opening Kir6.1/K-ATP channels inhibites inflammasome activation, which decreases production of IL-1β in BV-2cell lines.Part III Roles of Kir6.1/K-ATP channels expressed in astrocytes in the brain ischemic injuryAIM:To investigate the effects of Kir6.1/K-ATP channels specificly expressed in astrocytes on brain ischemic injury.METHODS:We selected two to three-month-old WT and Kir6.1homozygote knockout (Kir6.1-/-) male mice, and screened the expression of microRNAs between both genetype mice by Real-time PCR analyses. Next, we subjected WT mice to brain ischemia by MCAO model as described previously and analysed changes of related microRNAs. Thirdly, we isolated primary astrocytes from the cortex of WT mice, and observed the expression of miR-181a and miR-7after OGD by Real-time PCR assays. Moreover, we pretreated primary astrocyte with Nicorandil1h before OGD, and detected release of pro-inflammatory factors TNF-α, IL-1β and IL-10by ELISA. Expression of GRP78, CHOP, caspase-12, NLRP3and caspase-1, which were relevant to ER stress and inflammation, were determined by Western blot. Finally, in order to verify the influence of Kir6.1/K-ATP channels expressed in astrocytes deficiency on brain ischemic injury in vivo, we introduced Kir6.1conditional knockout mice and subjected them to MCAO model. We utilized the neurological score and TTC staining to assess the neurological disorder and infarct size. RESULTS:1. Either Kir6.1gene deficiency or brain ischemic injury caused downregulation of miR-7At the baseline, Kir6.1knockout caused downregulation of miR-7and miR-124. Brain ischemia induced downregulation of miR-7, upregulation of miR-181a and miR-290-5p. Moreover, OGD/R caused downregulation of miR-7and upregulation of miR-181a in primary astrocytes.2. Opening K-ATP channels decreased ER stress and inflammation induced by OGD/R in primary astrocytesPretreatment with K-ATP channels opener (Nicorandil) could inhibited OGD/R-induced downregulation of Kir6.1subunit and high-mobility group boxl (HMGB1). Nicorandil decreased expression of ER stress-related proteins, including molecular chaperone GRP78, transcription factor CHOP and apoptosis executor caspase-12. So it could attenuate ER stress induced by OGD/R model. Nicorandil decreased OGD/R-induced TNF-α and IL-1β release from astrocytes, it could also inhibit OGD/R-induced upregulation of NLRP3and caspase-1, impeded inflammasome activation and decreased production of IL-1β.Nicorandil downregulated OGD/R-induced the increase of miR-7, and targeted the NLRP3inflammasome, which inversely correlated to the expression of NLRP3.3. Kir6.1/K-ATP channels deficiency in astrocytes aggravated cerebral ischemia/reperfusion injuryCompared to contol group mice, there was larger infarct size in the mice, which were deficient by Kir6.1/K-ATP channels expressed in astrocytes. Moreover, it’s also accompanied by severer neurological disorder.CONCLUSIONS:1. Opening Kir6.1/K-ATP channels attenuates OGD/R-induced ER stress and inflammatory responses in primary astrocytes.2. Kir6.1/K-ATP channels deficiency in astrocytes aggravates focal cerebral ischemia/reperfusion injury in mice. SUMMARY:1. Kir6.1/K-ATP channels deficiency exacerbates acute neural injury induced by focal cerebral ischemia/reperfusionKir6.1gene knockdown aggravated cerebral ischemia/reperfusion injury, and enhanced ER stress and post-ischemic inflammation. These results indicate that Kir6.1/K-ATP channels are important targets for the acute brain ischemic injury, which expands the study of Kir6.1/K-ATP channels to stroke.2. Kir6.1/K-ATP channels inhibites OGD/R-induced inflammation in BV-2cell linesCromakalim opening Kir6.1/K-ATP channels decreased OGD/R-induced inflammatory responses in BV-2cell lines, including inhibiting IL-1β release and inflammasome activation. These results suggest Kir6.1/K-ATP channels expressed in microglia might be a new target for OGD/R-induced inflammation.3. Kir6.1/K-ATP channels expressed in astrocytes are an important target for post-ischemic inflammation in brain ischemiaOpening Kir6.1/K-ATP channels could inhibite inflammation induced by OGD/R in astrocytes. Kir6.1conditional knockout in astrocytes could exacerbate cerebral ischemia/reperfusion injury. Both Kir6.1/K-ATP channels deficiency and cerebral ischemia could induce downregulation of miR-7. While miR-7targeted NLRP3inflammasome and regulated astrocytic inflammation. These results indicate that Kir6.1/K-ATP channels expressed in astrocytes are an essential target for the post-ischemic inflammation of stroke, and it also implies that miR-7might be a regulator of Kir6.1/K-ATP channels.