TRPC6 Attenuates Cortical Astrocytic Apoptosis and Inflammation in Cerebral Ischemic/Reperfusion Injury

Transient receptor potential canonical 6 (TRPC6) channel is an important non-selective cation channel with a variety of physiological roles in the central nervous system. Evidence has shown that TRPC6 is involved in the process of experimental stroke; however, the underlying mechanisms remain unclear. In the present study, the role of astrocytic TRPC6 was investigated in an oxygen–glucose deprivation cell model and middle cerebral artery occlusion (MCAO) mouse model of stroke. HYP9 (a selective TRPC6 agonist) and SKF96365 (SKF; a TRPC antagonist) were used to clarify the exact functions of TRPC6 in astrocytes after ischemic stroke. TRPC6 was significantly downregulated during ischemia/reperfusion (IR) injury in cultured astrocytes and in cortices of MCAO mice. Application of HYP9 in vivo alleviated the brain infarct lesion, astrocytes population, apoptosis, and interleukin-6 (IL-6) and IL-1β release in mouse cortices after ischemia. HYP9 dose-dependently inhibited the downregulation of TRPC6 and reduced astrocytic apoptosis, cytotoxicity and inflammatory responses in IR insult, whereas SKF aggravated the damage in vitro. In addition, modulation of TRPC6 channel diminished IR-induced Ca2+ entry in astrocytes. Furthermore, decreased Ca2+ entry due to TRPC6 contributed to reducing nuclear factor kappa light chain enhancer of activated B cells (NF-κB) nuclear translocation and phosphorylation. Overexpression of astrocytic TRPC6 also attenuated apoptosis, cytotoxicity, inflammatory responses, and NF-κB phosphorylation in modeled ischemia in astrocytes. The results of the present study indicate that the TRPC6 channel can act as a potential target to reduce both inflammatory responses and apoptosis in astrocytes during IR injury, subsequently attenuating ischemic brain damage. In addition, we provide a novel view of stroke therapy by targeting the astrocytic TRPC6 channel.

The TRPC6 channel is emerging as an important target for the control of Ca 2+ currents in a wide range of disorders, including immune-mediated diseases, pulmonary arterial hypertension, atherosclerosis, and central nervous system (CNS)-related diseases, such as autism spectrum disorders, glioma, depression, traumatic brain injuries, seizure, Alzheimer's disease, and ischemic stroke (Hamid and Newman, 2009;Ding et al., 2010;Du et al., 2010;Griesi-Oliveira et al., 2015;Kim and Kang, 2015;Zhang et al., 2015Zhang et al., , 2016Pochwat et al., 2018;Ramirez et al., 2018;Chen et al., 2019). TRPC6 is abundantly expressed in various anatomical regions of the CNS, such as the cerebellum, hippocampus, middle frontal gyrus, and cortex (Riccio et al., 2002;Du et al., 2010). As a regulator of Ca 2+ influx, TRPC6 is involved in neuronal survival, synapse formation, neuronal nerve-growth-cone guidance, and sensory transduction (Li et al., 2005;Jia et al., 2007;Zhou et al., 2008;Quick et al., 2012). Dysfunction of the TRPC6 channel may trigger a series of downstream events and neurobiological disorders.
Ischemic stroke is a life-threatening condition caused by a vascular embolism due to cardiac events, artery-to-artery embolism, or in-situ small artery disease (Hankey, 2017). Several underlying mechanisms, including excitotoxicity, ionic imbalance, oxidative and nitrative stress, inflammation, and apoptosis, are involved in the pathophysiological process of cerebral ischemia (Khoshnam et al., 2017). Ca 2+ overload has a critical role and initiates the ischemic cascade during brain ischemia/reperfusion (IR) injury. Increasing evidence indicates an important role of TRPC6 in cerebral IR injury . TRPC6 is identified on cortical neurons and astrocytes, and is downregulated in neurons after brain ischemic injury (Du et al., 2010;Guo et al., 2017;Qu et al., 2017;Shirakawa et al., 2017). Notably, maintaining the TRPC6 protein level in neurons improves neuronal survival and behavioral performance, thus alleviating ischemic brain damage (Du et al., 2010;Guo et al., 2017). However, the roles of the TRPC6 channel in mouse cortical astrocytes following IR injury have not been evaluated.
In the present study, the specific effects of astrocytic TRPC6 on ischemic stroke were investigated. TRPC6 protein expression in primary mouse astrocytes was downregulated following IR injury. Inhibition of TRPC6 downregulation via HYP9 or TRPC6 overexpression protected astrocytes and the brain against IR insults. These results indicate that the TRPC6 channel contributes to neuroprotection in cerebral ischemia by promoting astrocyte survival. Furthermore, this study provides therapeutic evidence for the treatment of ischemic stroke by targeting the TRPC6 channel.

Animals
C57BL/6J male mice 8-10 weeks of age were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). All animal experiments were approved by the Animal Care Committee of the First Affiliated Hospital at Zhejiang University. Mice were housed in polypropylene cages and maintained at 25 ± 1 • C under 12 h light/12 h dark cycles with free access to rodent chow and water. Mice were randomly allocated to each group before any treatment. Proper anesthetic procedures were used to ensure that the mice did not suffer unnecessarily during or after the experimental procedure. A total of 82 mice were used in this study (including 10 mice that died).

In vivo Model of Focal Cerebral Ischemia
Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Animals were anesthetized with 1% sodium pentobarbital (75-100 mg/kg, intraperitoneal injection); body temperature was maintained at 37 ± 0.5 • C during the operation using a heating pad. Transient MCAO was generally performed as previously reported (Lin et al., 2013a). Briefly, the left common carotid artery was exposed to separate the internal carotid artery (ICA) and the external carotid artery (ECA). Next, a 6-0 monofilament nylon suture (RWD Life Science Co., LTD, Shenzhen, China) with a rounded tip was inserted through the exposed left ICA and advanced into the middle cerebral artery. After 1 h of occlusion, the filament was gently withdrawn to allow reperfusion. At 24 h after reperfusion, animals were sacrificed and the brain tissues were obtained for future assays.

Intracerebroventricular Injection
Mouse intracerebroventricular injection was performed using a stereotaxic instrument (RWD Life Science Co., Ltd., Shenzhen, China) with a micro-syringe pump under anesthesia. Drugs or vehicle (5 µL in total) were slowly injected (1 µL/min) into the left ventricle at a depth of 2.5 mm below the brain surface, 1.0 mm lateral and 0.5 mm posterior to the bregma. After injection, animals were allowed to recover from anesthesia under a heating pad.

Oxygen-Glucose Deprivation and
Re-oxygenation (OGD/R) OGD/R experiments were established to mimic the in vitro condition of IR injury. The cultures were incubated for 1-4 h in DMEM medium without glucose in a humidified incubator chamber at 37 • C with 95% N 2 and 5% CO 2 . Subsequently, the astrocytes were returned to the original culture conditions and then incubated under normoxic conditions for the next 24 h.

TTC Staining
Animals were sacrificed at 24 h after reperfusion. The infarct volume was tested based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. The brains were sectioned into 2-mm coronal slices using a brain matrix. The brain slices were incubated in 2% TTC solution staining for 20 min at 37 • C and then fixed in 4% paraformaldehyde (PFA). The ischemic volume was quantified using ImageJ software (version 2.0). The infarct percentage was shown as the ratio of the ischemic area to the entire slice area.

Analysis of Cell Viability and Lactate Dehydrogenase (LDH) Release
An LDH release quantification kit (#11644793001; Sigma-Aldrich) was used to evaluate cell viability. The cell-free supernatant was obtained by centrifugation at ∼250 × g for 10 min. Subsequently, the supernatant was incubated in working solutions in the dark for 1 h at room temperature (∼22 • C). The absorbance of the samples was measured at 490 nm using a microplate reader (SpectraMax i3x; Molecular Devices, Sunnyvale, CA, USA). To determine the cytotoxicity percentage, all groups were compared with the control groups.

Flow Cytometry
The flow cytometry measurement of astrocyte apoptosis and Ca 2+ concentration was performed using a BD flow cytometer (BD Biosciences, San Jose, CA, USA). Astrocytes were digested with trypsin. The harvested cells were washed with phosphatebuffered saline (PBS) twice before incubating in buffer containing 5 µL of fluorescein isothiocyanate-annexin V and 5 µL of propidium iodide or 0.5 µg/ml DAPI for 15 min at room temperature in the dark. Subsequently, the cells were detected and analyzed.
The Ca 2+ concentration was also measured using the BD flow cytometer. Cortical astrocytes were loaded with 2 µM Fluo-4 AM (#F14217; Invitrogen, Carlsbad, CA, USA) in culture medium for 1 h at 37 • C in an incubator with 5% CO 2 . Next, the medium was changed to Hank's balanced salt solution (#14025092; Gibco) and the astrocytes were incubated for another 30 min at 37 • C. Then, the astrocytes were collected and analyzed using flow cytometry.

Enzyme-Linked Immunosorbent Assay
The release of interleukin-6 (IL-6) and IL-1β from cortices and cultured astrocytes was quantified using enzyme-linked immunosorbent assay (IL-6, #70-EK206HS; IL-1β, #70-EK201BHS-96; MULTI SCIENCES, Hangzhou, China). The experiments were performed according to the manufacturer's instructions. The final absorbance was detected at 450 nm and the background correction was set at 570 nm on a microplate reader (SpectraMax i3x; Molecular Devices).

Statistical Analyses
Data are presented as the means ± standard error of the mean (SEM) of at least three independent analyses. SPSS ver. 25.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) were used for statistical analyses. One-way analysis of variance with Tukey's test for post-hoc analysis were performed. P-value <0.05 was considered statistically significant.

TRPC6 in Mouse Cortical Astrocytes Is Downregulated After Ischemia
In several studies, TRPC6 in neurons reportedly declines after cerebral ischemia, and maintaining the expression of neuronal TRPC6 protein contributes to neuronal survival and reduced cerebral ischemic insult (Du et al., 2010;Lin et al., 2013a;Guo et al., 2017). However, the role of the astrocytic TRPC6 channel after stroke remains unclear.
Astrocytes have provided important insights into ischemic stroke (Liu and Chopp, 2016). To confirm the vital functions of the astrocytic TRPC6 channel in cerebral ischemia, we established an in vitro model of stroke in primary mouse astrocytes using an OGD/R experiment. Astrocytes were exposed to 1-4 h of OGD followed by 24 h reperfusion. The apoptosis rate was elevated and cell viability was decreased in astrocytes following hypoxia injury (Figures 2A-C). The TRPC6 protein levels in astrocytes after OGD/R were significantly downregulated (Figures 2D,F). Conversely, the expression of the apoptosis-specific biomarker, cleaved caspase-3, was gradually increased after stroke compared with the control groups (Figures 2D,E). As shown in Figures 2A-D, 3 h OGD and 24 h reperfusion triggered appropriate hypoxic damage in astrocytes; thus, the subsequent in vitro OGD/R experiments were performed under this condition.
The in vivo stroke model is mimicked by MCAO in mice. Immunofluorescence signals showed that astrocytic TRPC6 was downregulated in the ischemic penumbra (Figures 2G,H). In parallel with the in vitro results, the TRPC6 protein level in peri-infarct areas was decreased compared with that in contralateral cortices (Figures 2I,K). In addition, after ischemic stroke, the cleaved caspase-3 expression in the ipsilateral hemisphere was upregulated compared with the contralateral hemisphere (Figures 2I,J). Furthermore, immunofluorescence FIGURE 2 | TRPC6 in the cortical astrocytes is downregulated in ischemia. (A) Cultured astrocytes were exposed or not to OGD/R, and their apoptosis and death was detected by FITC/PI flow cytometry analysis. The percentage of apoptosis in scatter plots was presented as the proportion of apoptotic cells vs. total cell (Continued) Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 February 2021 | Volume 8 | Article 594283 FIGURE 2 | population (H1R24: 1 h OGD followed by 24 h reperfusion; H2R24: 2 h OGD followed by 24 h reperfusion; H3R24: 3 h OGD followed by 24 h reperfusion; H4R24: 4 h OGD followed by 24 h reperfusion). (B) Bar chart showed the statistical results of apoptosis rate measured by flow cytometry in each group in three independent experiments. Data were shown as mean ± SEM (n = 3). (C) LDH assay was used to measure the cytotoxicity of astrocytes in control and OGD groups. Data were shown as mean ± SEM (n = 4). (D) The protein level of TRPC6 and cleaved caspase-3 in astrocytes were tested by western blot analysis. β-actin was used as a loading control. (E,F) Quantifications of cleaved caspase-3 and TRPC6 protein levels shown in (D). Data were shown as mean ± SEM (n = 3). (G) Brain slices with TTC staining. Green rectangle: contralateral cortex; Black rectangle: peri-infarct cortex. (H) Representative images of the cortex after sham or MCAO operation (peri-infarct cortex) double-stained with the TRPC6 (Red) and GFAP (Green) antibodies (Scar bar: 100 µm; 600× magnification). (I) Mice were exposed to sham operation or MCAO procedure (n = 3). The expressions of TRPC6 and cleaved caspase-3 in sham group (left hemisphere) and in contralateral (C) or peri-infarct (P) cortex were detected by western blotting. β-actin was used as a loading control. The number "1," "2," "3" represents cortices in each group were extracted form three different mice. (J,K) Quantifications of cleaved caspase-3 and TRPC6 protein levels shown in (I). Data were shown as mean ± SEM (n = 3). MCAO-C, contralateral cortices in MCAO mice; MCAO-P, peri-infarct cortices in MCAO mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (all comparisons to control group or sham group). # p < 0.05 (comparisons to MCAO-C group).
signals showed that astrocytic TRPC6 was downregulated in the ischemic penumbra ( Figure 2H).
These results indicate that IR injury-induced changes in TRPC6 channel protein levels may have important functions in brain ischemia.

Reducing IR-Mediated TRPC6 Downregulation With HYP9 Alleviates Ischemic Insults in the Mouse Cortex
Consequently, to determine the role of TRPC6 in an in vivo model of ischemic stroke, intraventricular administration of HYP9 or SKF was performed in mice. MCAO resulted in increased infarct size and pro-inflammatory cytokine release compared with the sham groups (Figure 3). Pretreatment with HYP9 alleviated cortical injury, reduced infarct volume (Figures 3A,B), astrocytic population (Figure 3C), cleaved caspase-3 expression (Figures 3D,E), and inhibited proinflammatory cytokine (IL-6 and IL-1β) generation after MCAO (Figures 3G,H). However, the effects of SKF on an animal model of stroke regarding infarction volume, astrocytic population, cleaved caspase-3 protein level, and cytokine generation were not significantly different. In conclusion, the results show that application of the TRPC6 agonist HYP9 can alleviate IR-induced cerebral injury in an animal model of stroke.

Dose-Dependent HYP9 Inhibition of TRPC6 Downregulation in Astrocytes Promotes Astrocyte Survival During IR Injury
HYP9 is a specific TRPC6 channel activator (Leuner et al., 2010). Treatment with HYP9 (0, 1, 5, 10, 15, 20, or 30 µM) was used to verify whether TRPC6 channel degradation is responsible for IR-induced astrocyte insults. Astrocytes were pre-incubated with different concentrations of HYP9 for 12 h in a CO 2 chamber and then exposed to normal or OGD/R conditions. Application of 15 µM HYP9 contributed to a significant decrease in OGD/Rinduced astrocyte apoptosis and cytotoxicity (Figures 4A-C).
Western blot results showed that HYP9 (15 µM) suppressed the downregulation of TRPC6 and decreased cleaved caspase-3 protein levels in primary mouse astrocytes in the OGD group (Figures 4D-F). These results indicate that maintaining the protein level of the TRPC6 channel in astrocytes with HYP9 diminishes OGD/R-induced astrocyte apoptosis and improves cell vitality.

TRPC Antagonist SKF Aggravates
Astrocyte Damage Dose-Dependently in the OGD/R Experiment Next, whether blockage of the TRPC6 channel by SKF results in increased astrocyte damage after OGD/R was investigated. Cultured astrocytes were pretreated with 0, 0.05, 0.5, 5, 10, 20, 30, or 40 µM SKF for 12 h before the OGD/R experiment. As shown in Figures 5A,B, the apoptosis of astrocytes was markedly increased in the OGD group supplemented with 30 or 40 µM SKF compared with the OGD group without SKF pretreatment. In addition, the effects of SKF on astrocyte cytotoxicity in the control and OGD groups were investigated using the LDH assay. Notably, IR injury-mediated astrocyte cytotoxicity was elevated after pre-incubation with 20, 30, or 40 µM SKF in OGD groups ( Figure 5C). In addition to the increased cleaved caspase-3 expression, the OGD/R-induced degradation of TRPC6 protein was not rescued after treatment with SKF (Figures 5D-F). Taken together, pretreatment of cultured astrocytes with 30 or 40 µM SKF aggravates apoptosis and cytotoxicity in OGD groups.

TRPC6 Protects Astrocytes Against IR Injury in Modeled Ischemia
To further clarify the protective role of astrocytic TRPC6 during IR injury, we then infected primary cortical astrocytes with lentivirus vectors to overexpress (WT-TRPC6) or knock down (sh-TRPC6) TRPC6. The efficiency of lentiviral infections was shown in Figure 6D. OGD/R notably decreased the protein levels of TRPC6 compared with that in control groups in astrocytes ( Figure 6D). Overexpression of TRPC6 strikingly reduced OGD/R-induced astrocyte apoptosis (Figures 6A,B), cytotoxicity (Figure 6C), and cleaved caspase-3 protein levels (Figures 6D,E). However, knocking down TRPC6 via sh-TRPC6 did not exacerbate IR injury-mediated apoptosis, cytotoxicity or cleaved caspase-3 protein levels in astrocytes (Figure 6). In general, TRPC6 attenuates astrocytes IR injury in modeled ischemia in vitro.
addition to cell apoptosis and vitality, the expression of the pro-inflammatory cytokines IL-6 and IL-1β were measured to confirm the effects of HYP9 and SKF on ischemic astrocytic inflammation. The IL-6 and IL-1β protein levels were significantly increased in the OGD groups compared with the control groups. Notably, HYP9 and WT-TRPC6 reduced the elevated level of IL-6 and IL-1β in astrocytes in the OGD groups (Figures 7A-F).
The data show that inhibition of TRPC6 downregulation with HYP9 or WT-TRPC6 reduces inflammatory responses in astrocytes after OGD/R.

Inhibiting TRPC6 Downregulation Suppresses Intracellular Ca 2+ Overload in Primary Astrocytes
The above reported results indicate the TRPC6 channel is closely associated with apoptosis, vitality, and inflammatory response in cultured cortical astrocytes exposed to IR damage. As a Ca 2+ channel, TRPC6 modulates the concentration of intracellular Ca 2+ ([Ca 2+ ] i ) (Wang et al., 2020a). To understand the potential mechanism of TRPC6 channel against cerebral ischemic insults in astrocytes, the effects of HYP9 and SKF on [Ca 2+ ] i in astrocytes was measured. Consistent with previous research (Li et al., 2015;Rakers and Petzold, 2017), OGD/R dramatically increased astrocytic [Ca 2+ ] i compared with control conditions (Figures 8A,B). Notably, application of HYP9 statistically reduced the elevated [Ca 2+ ] i in the OGD group (Figures 8A,B). In addition, SKF raised [Ca 2+ ] i in ischemic astrocytes (Figures 8A,B). These results indicate that astrocytic TRPC6 channel triggers a variety of protective cellular responses by inhibiting brain ischemia-induced Ca 2+ overload.

TRPC6 Diminishes NF-κB Nuclear Translocation and Phosphorylation
NF-κB phosphorylation and nuclear translocation are considered vital pathways that mediate the inflammatory cascade, including cytokine generation (Kopitar-Jerala, 2015;Shih et al., 2015). Therefore, the influence of HYP9 and SKF on NF-κB nuclear translocation and phosphorylation in primary mouse astrocytes exposed to ischemic damage was investigated. Immunofluorescent signals showed the nuclear distribution of NF-κB was increased after OGD/R (Figure 9). Notably, the TRPC6-specific activator HYP9 attenuated IR injury-induced NF-κB nuclear translocation (Figure 9). Furthermore, HYP9 and WT-TRPC6 inhibited the phosphorylation of NF-κB in astrocytes subjected to OGD/R (Figures 10A-G). Collectively, the findings indicate that NF-κB phosphorylation and nuclear translocation may be pivotal downstream pathways that contribute to the protective effects of TRPC6 in astrocytes after stroke.

DISCUSSION
In cerebral ischemia, several clinical trials have failed due to narrow therapeutic window, adverse effects, and individual differences (Petrovic-Djergovic et al., 2016). A breakthrough in stroke therapeutic strategies is considered urgent. In the present study, significant downregulation of TRPC6 protein was observed in both cultured astrocytes and cerebral cortices after IR injury. In vivo results confirmed that the TRPC6 channel-specific activator HYP9 attenuated the infarct volume, astrocytes population, apoptosis, and the generation of proinflammatory cytokines (IL-6 and IL-1β) caused by MCAO. However, significant effects of SKF on infarct lesion or cortical IL-6 and IL-1β release in MCAO mice were not observed. In addition, HYP9 was shown to attenuate apoptosis rate, improve cell vitality, and reduce the generation of IL-6 and IL-1β in cultured astrocytes subjected to OGD/R. Furthermore, the TRPC channel antagonist SKF aggravated astrocytic apoptosis and cytotoxicity in OGD groups. In addition, inhibition of the TRPC6 channel downregulation with HYP9 suppressed the increase in [Ca 2+ ] i in ischemic astrocytes. The IR-mediated increased NF-κB phosphorylation and nuclear translocation were inhibited by HYP9 pretreatment in primary mouse astrocytes. In parallel, overexpression of TRPC6 also decreased IR injury-induced astrocytic apoptosis, cytotoxicity, inflammatory responses and NF-κB phosphorylation in modeled ischemia in vitro.
Previously, neuron-specific-related mechanisms are primarily considered in the pathogenesis of brain ischemia. As knowledge increased, astrocytes and other CNS cells are shown to be closely involved in ischemic stroke (Pekny et al., 2016;Werner et al., 2020). Astrocytes interact with neurons and participate in structural support, neuronal metabolism, synaptogenesis, synaptic transmission, axonal remodeling, and neurogenesis (Halassa and Haydon, 2010;Liu and Chopp, 2016). IR injuryactivated astrocytes initiate the CNS inflammatory response, which in turn exacerbates brain insults during ischemic stroke (Cekanaviciute and Buckwalter, 2016). Thus, maintaining the normal function of astrocytes likely promotes post-ischemic neurological recovery. Therapeutic targeting of astrocytes is expected to be a future area of research regarding treatment and prognosis of brain ischemia (Neuhaus et al., 2017).
Hyperforin, a main compound of St. John's wort, can activate the TRPC6 channel and induce changes in [Ca 2+ ] i currents (Leuner et al., 2007); however, hyperforin is chemically unstable when subjected to oxygen and light. HYP9 is a stable synthetic phloroglucinol derivative of hyperforin that maintains the biological function of hyperforin in activating the TRPC6 channel (Leuner et al., 2010). Consequently, HYP9 was used in the present study to explore its function in the TRPC6 channel and the associated downstream cellular mechanisms in ischemic stroke. Inflammatory responses and apoptosis in ischemic astrocytes were inhibited by maintaining the protein level of the TRPC6 channel via HYP9. Overexpression of TRPC6 also reduced IR injury-induced astrocytic apoptosis, cytotoxicity, and inflammatory responses in experimental stroke in vitro. Meanwhile, SKF is a TRPC channel blocker (Song et al., 2014;Jing et al., 2016). SKF also affects other channels belonging to the TRPC channel subfamily. Evidence shows that TRPC1, TRPC3, FIGURE 8 | TRPC6-specific agonists HYP9 suppresses intracellular Ca 2+ overload in primary astrocytes. Astrocytes were pre-incubated with Vehicle (DMSO, the final concentration was 0.025%), 15 µM HYP9 or 30 µM SKF for 12 h, and then exposed to control condition (CON) or 3 h OGD and 24 h reperfusion (OGD). (A) Representative charts of average fluorescence intensity. The level of intracellular Ca 2+ in astrocytes was measured by Ca 2+ fluorescence probe Fluo-4 AM through FITC-488 flow cytometry. (B) Quantification of Ca 2+ concentration in astrocytes by flow cytometry. Data were shown as mean ± SEM (n = 4). ****p < 0.0001 vs. CON + Naive group; #### p < 0.0001 vs. OGD + Naive group. and TRPC7 are involved in cerebral ischemic stroke Xu et al., 2018). In the present study, SKF aggravated astrocyte injury after ischemia in vitro. However, significant changes in infarct volume, cleaved caspase-3 expression, and cytokine release were not observed in the MCAO model after SKF application. The inconsistent results between cell and animal models of stroke may be due to the complex effects of SKF on cortical non-astrocytic cells. Noticeably, knocking down TRPC6 via sh-TRPC6 shown no significant effects on apoptosis, cytotoxicity, and inflammatory responses in primary astrocytes after OGD/R. Since TRPC6 is strikingly downregulated in astrocytes exposed to IR insults, knocking down of TRPC6 did not significantly aggravate astrocytic damage in OGD group. The dissimilar effects of SKF and sh-TRPC6 on ischemic astrocytes might be related to the side-effect of SKF on other TRPCs (TRPC3/7). Overall, further in vivo studies using astrocytic TRPC6-, siRNA-, or shRNA-targeted transgenic mice are needed to fully clarify the role of the astrocytic TRPC6 channel in stroke.
A crucial relationship exists between Ca 2+ and NF-κB signaling pathways . As a vital second messenger, Ca 2+ governs a variety of functions causing cellular pathological and physiological changes. Modulation of the Ca 2+ current has attracted interest as a promising medical strategy for ischemic stroke (Kalogeris et al., 2016;Secondo et al., 2018). The TRPC6 channel, a Ca 2+ permeable cation channel, has been associated with Ca 2+ entry and brain ischemic pathogenesis . In the present study, TRPC6 was dramatically downregulated during IR injury. The TRPC6-specific agonist HYP9 rescued TRPC6 degradation in primary cultured astrocytes and reduced [Ca 2+ ] i . Consistently, the TRPC channel antagonist SKF contributed to increased [Ca 2+ ] i . How a Ca 2+ entry activator reduces and a Ca 2+ channel blocker increases the Ca 2+ influx in primary astrocytes is notable. TRPC6 has been shown to block the NMDA receptor (NMDAR)-triggered [Ca 2+ ] i increase and neurotoxicity in neurons after brain IR damage (Li et al., 2012). Overexpression of TRPC6 inhibits [Ca 2+ ] i overload, prevents neuronal death, lessen infarct size, and improves behavior performance after ischemia. Similarly, another research team suggests that the TRPC6 channel selectively suppresses the NMDAR-mediated current in hippocampal neurons, which is increased via TRPC6 knockdown or interference with SKF (Shen et al., 2013). In another study, SKF increases intracellular Ca 2+ by promoting the reverse mode of the Na + /Ca 2+ exchanger (Song et al., 2014). Thus, we hypothesized that the underlying mechanism of TRPC6-induced [Ca 2+ ] i decrease in astrocytes after ischemic insults may be associated with the complex interaction between the TRPC6 channel and other ion exchangers.
Although the present study showed a protective role of the TRPC6 channel in astrocytes after ischemia, various questions remain unanswered. The effects of the astrocytic TRPC6 FIGURE 9 | TRPC6 diminishes NF-κB nuclear translocation in astrocytes after ishcemia. Astrocytes were pre-incubated with Vehicle (DMSO, the final concentration is 0.025%), 15 µM HYP9 or 30 µM SKF for 12 h, and then expose to control condition (CON) or 3 h OGD and 24 h reperfusion (OGD). Representative images of cortical astrocytes after OGD/R double-stained with the NF-κB (red) and GFAP (green) antibodies (Scar bar: 100 µm; 630× or 1,890× magnification). channel on ischemic neurons will be addressed in our future work. Furthermore, the underlying mechanisms of TRPC6triggered astrocytic Ca 2+ changes in ischemic stroke warrant further investigations.
a mouse model of ischemic stroke. Inhibition of TRPC6 channel downregulation via HYP9 or TRPC6 overexpression alleviates apoptosis and inflammatory response in astrocytes exposed to ischemic damage. Furthermore, the protective role of TRPC6 in stroke is associated with Ca 2+ /NF-κB-dependent pathways. The results of this study indicate that the astrocytic TRPC6 channel and TRPC6 agonist HYP9 might be a novel therapeutic approach to prevent ischemic stroke induced by brain injury.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Care Committee of the First Affiliated Hospital at Zhejiang University.

AUTHOR CONTRIBUTIONS
LL wrote the manuscript. LL, MC, and KL finished the experiment. SZ and XXio designed the general idea. All authors edited the drafts of the manuscript.

FUNDING
This study was supported by the National Natural Science Foundation of China No. 81971008 to SZ and No. 81870939 to XXio.