Icaritin Promotes Brain Functional Rehabilitation in Ischemic Stroke Rats by Regulating Astrocyte Activation and Polarization Via GPER

doi:10.21203/rs.3.rs-3219595/v1

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Icaritin Promotes Brain Functional Rehabilitation in Ischemic Stroke Rats by Regulating Astrocyte Activation and Polarization Via GPER

https://doi.org/10.21203/rs.3.rs-3219595/v1

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Cerebral ischemic injury induces the polarization of astrocytes toward two different phenotypes, i.e., the proinflammatory A1 phenotype and the protective, anti-inflammatory A2 phenotype, affects the prognosis of cerebral ischemia. To explore the neuroprotective effect of phytoestrogens Icaritin (ICT) on cerebral ischemic rehabilitation and the preliminary mechanism of regulating astrocyte polarization. The Transient middle cerebral artery occlusion (tMCAO)/reperfusion was performed on male adultsand then treated with ICT (i.p.) once daily for 28 days. Intervention of G protein-coupled estrogen receptor (GPER) specific inhibitor G15 was repeated. The body weight, Garcia JH scale, right/left brain weight ratio, CatWalk gait test and Y maze test to assess overall neural function in rats. Cytokines in ischemic cortical were detected by ELISA. And the double immunofluorescence staining and Western blotting to evaluated the activation and A1 and A2 polarization of astrocytes. The results showed that ICT treatment markedly perfected functional outcomes on a long-term basis after ischemic stroke, it also improved learning and memory and gait. ICT inhibited the polarization of A1 type astrocytes and promoted the polarization of A2 type astrocytes, promote neuron regeneration in hippocampus dentate gyrus (DG) region. G15 removes some of the protective effects of ICT. The experimental results show that ICT exerts neuroprotective effects and regulates astrocyte polarization through GPER, suggesting that it may be a potential therapeutic agent for ischemic stroke during the recovery period.

cerebral ischemia

icaritin

astrocyte

GPER

neuroprotection

ICT improved neurological function, promoted recovery of learning, motor function and gait.

ICT promote neuron regeneration in hippocampus DG region.

ICT regulates astrocyte polarization through GPER.

Stroke is the second leading cause of death and disability worldwide, and it is a serious threat to human health and life expectancy (Saini et al., 2021). Stroke is an acute cerebrovascular disease, and ischemic stroke is the main cause (Barthels and Das, 2020). The tPA is only one Food and Drug Administration-approved drug for treating this disease, but it has a short treatment window of only 4.5 hours (Fukuta et al., 2017). The narrow effective therapeutic window of tPA is a major limitation to its use after stroke and leads to plentiful patients with stroke not receiving thrombolysis therapy in a timely manner (Boese et al., 2018; (Venkat et al., 2018). Thus, many patients experience functional impairments, including motor dysfunction (Cramer and Chopp, 2000; (Suri et al., 2018), impairment of learning and memory ability (Lugtmeijer et al., 2021), language and speech disorders (Baker et al., 2018), emotional deficits (Schöttke and Giabbiconi, 2015), etc. However, drugs that can effectively treat ischemic stroke and improve disease prognosis while exhibiting few side effects remain scarce. Therefore, novel and more effective candidates for treating ischemic stroke are urgently needed.

Astrocytes are the neuroglia with the highest content in the brain and play a crucial role in maintaining the physiological function of the CNS (Lezmy et al., 2021). Acute cerebral ischemia leads to rapid activation of astrocytes and induces their polarization, a process known as reactive astrogliosis (Burda et al., 2016). Active astrocytes are rapidly polarized toward two phenotypes: the proinflammatory A1 phenotype and the anti-inflammatory A2 phenotype (Liddelow et al., 2017). Studies indicate that it can promote the recovery of late stroke by regulating the transformation of astrocytes (Abeysinghe et al., 2016; (Guo et al., 2021). In addition, astrocytes can secrete many kinds of cytokines after cerebral ischemia, which participate in the regulation of neuroinflammation (Linnerbauer et al., 2020). For instance, A1 astrocytes secrete many inflammatory cytokines that promote neuroinflammation and seriously affect neuronal survival (Liu and Chopp, 2016). However, A2 astrocytes can secrete many neurotrophic factors that promote neuronal survival (Turnquist et al., 2016) and neurogenesis (Wang et al., 2022). Therefore, astrocyte polarization is a novel target for the recovery of ischemic stroke.

The GPER is a receptor with seven transmembrane structures, and natural endogenous estrogen estradiol is its main ligand. In the CNS, GPER is widely distributed in neurons, microglia and astrocytes (Wang et al., 2021). Studies have found that activation of GPER can alleviate the activation of astrocytes in acute cerebral ischemia, reduce the release of pro-inflammatory factors TNF-α, IL-1β and IL-6, and alleviate neuroinflammation (Yuan et al., 2021). It can be seen that GPER is an important target for regulating astrocyte activation and neuronal protection, but whether GPER is involved in regulating astrocyte activation during cerebral ischemic rehabilitation is still unclear.

As a phytoestrogen, ICT binds to the estrogen membrane receptor and plays a biological role. Studies have found that ICT has antitumor (Yu et al., 2020), antiosteoporosis (Jiang et al., 2014), anti-inflammatory (Bi et al., 2022) and immunomodulatory effects (Wu et al., 2015). ICT can protect against apoptosis and oxidative stress injury by activating Nrf2 signaling (Xu et al., 2021) and inhibiting STAT3 signaling (Yang et al., 2017) to exert a neuroprotective effect. Moreover, our previous studies demonstrated that ICT alleviates glutamate-induced neuronal damage in vitro and exerts a therapeutic effect against cerebral ischemia/reperfusion injury by inhibiting excitatory toxicity in vivo (Liu, S. et al., 2021). These studies show that ICT is a potential candidate compound for treatment of ischemic encephalopathy. Based on the above clues, we explore the effect of ICT on the recovery period after cerebral ischemia and the possible mechanism of regulating astrocyte polarization.

2.1 Experimental animals and drugs

Healthy Sprague-Dawley rats aged 6–8 weeks (weighing 240–260 g) were obtained from Slack Jingda Laboratory Animal Company, Ltd. (License No. SCXK 2019-0004) and fed in a 21–25°C, 50–60% humidity atmosphere under a 12 h light-dark cycle. All rats were adapted to the laboratory environment for five days and could freely acquire water and food. All animal procedures were performed following the China Council on Animal Care guidelines and were approved by the Gannan Medical Ethics Committee (Ganzhou China).

Icaritin (3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl) chromen-4-one is a yellow powder with a chemical formula of C₂₁H₂₀O₆, molecular weight of 368.38, and purity of 98% (Fig. 1A) and was procured from Co., Ltd. Shanghai Chenxiang Medical Technology. Before use, ICT at a final concentration of 0.5 mg/mL was dissolved in 90% DMSO (Cat No. d2650, Sigma, USA). G15 (Cat No. 14673, Cayman Chemica, USA) was dissolved in DMSO.

2.2 Experimental protocol

In order to observe the effects of ICT on rats during cerebral ischemia recovery, animals were randomly divided into four groups: the sham, ICT, tMCAO, and tMCAO + ICT groups. According to our previous study, we chose the dose of ICT (0.5 mg/ kg) for the spread experiment. Rats in the ICT and tMCAO + ICT groups were treated with ICT by intraperitoneal injection (i.p.) once daily for 28 consecutive days after surgery. Rats in the sham and tMCAO groups were treated with an equal volume of vehicle (90% DMSO). In the intervention experiment, rats were divided into two groups: the DMSO group and G15 group. Both groups were treated with ICT by intraperitoneal injection (i.p.) once daily for 28 consecutive days after surgery. G15 (0.25 mg/ kg) was given intraperitoneally every 3 days for G15 group, the other group was given a vehicle as a control.

2.3 tMCAO rat model

Based on previous studies, we used the suture occlusion method to create a rat tMCAO model (Li et al., 2017; (Liu, C. et al., 2021; (Liu et al., 2017; (Xie et al., 2021). Briefly, rats were made a 3 cm incision in the neck along the anterior midline after anesthetization by intraperitoneal injection of 1% pentobarbital sodium at a dose of 4.5 mL/ kg. The glands and muscles were separated until the carotid sheath was exposed. The common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were bluntly separated with a glass minute hand. A 4.5-cm nylon filament (ϕ 0.38 ± 0.02 mm, Beijing Cinontrch Co., Ltd. China) was inserted via the ECA stump into the middle cerebral artery (MCA) and obstructed blood flow in the MCA for 2 h. After that, the nylon filament was removed to allow restoration of blood flow to the MCA. Finally, a small amount of penicillin was dropped, and the incision was sutured. Sham rats underwent a similar surgery but without occlusion of blood flow for MCA. The operation room temperature was strictly controlled between 23–25°C. Twenty four hours after tMCAO, rats with a Longa score of 1–3 were included in the experiment (Xie et al., 2021).

2.4 The survival and body weight of the rats were recorded.

All rats were weighed on days 1, 3, 7, 14, 21 and 28 after tMCAO. The survival of rats was recorded for 28 consecutive days after tMCAO according to previous studies (Li-Sha et al., 2015).

2.5 Neurological test

All rats underwent neurological function assessment with the Garcia JH scale on days 1, 3, 7, 14, 21 and 28 after tMCAO. The evaluation included six tests, and scores ranged from 3 to 18 (Garcia et al., 1995). The following six parameters were evaluated: (1) spontaneous activity, (2) symmetry of the extremities, (3) forelimb movements, (4) climbing, (5) body proprioception and (6) response to stimulation.

2.6 Assessment of the right/left brain weight ratio

The right/left brain weight ratio was measured as previously described for the analysis of brain edema (Xie et al., 2021). The rats were anesthetized and sacrificed after neurological assessment on day 28. The rat brain was carefully obtained and photographed. The whole brain was cut in half along the sagittal plane, and the left and right hemispheres were weighed after the cerebellum was removed. The formula for calculating the right/left brain weight ratio was as follows: (right brain weight/left brain weight)× 100%.

2.7 Y-maze test

Spatial learning was assessed through the Y-maze test as previously described (Carroll et al., 2007). The Y-maze had three arms at 120° to each other, and the rats were placed in the maze and allowed to explore freely for 5 minutes. A video camera mounted above the maze recorded the movements of the rats for analysis. Noldus animal behavior analysis software (Noldus Information Technology, Netherlands) was used to record and analyze the trajectories of the rats. The total number of times the rats entered the three arms and the percentage of alternations (entry into an arm that differed from the previous two entries) were recorded. The formula for the spontaneous alternation performance rate was as follows: [alternations/(total times-2)] ×100%.

2.8 Gait test

We used the CatWalk XT gait analysis system (XT, Noldus Information Technology, Netherlands) to analyze the gait of each rat on days 1, 3, 7, 14, 21 and 28 after tMCAO according to previous studies (Cao et al., 2017). Before tMCAO, the rats were trained on the system (twice a day for 2 consecutive days). The rats were moved to a dark and quiet environment at least 1 h before the formal test. The original cage was placed in the target box on the other side of the corridor, and the smell of feces and its companions in the cage were used to induce the rat to walk. Walking continuously for 8 s without turning back or stopping was considered a successful trial. Data from at least 3 successful trials for each rat were used for statistical analysis.

2.9 Immunofluorescence staining

In study, immunofluorescence staining detection was performed according to previous studies (Liu, C. et al., 2021). Phosphate buffered saline (PBS) and 4% paraformaldehyde were sequentially perfused through the heart to fix the rats after anesthetization. After removing the brain, it was placed overnight in 4% paraformaldehyde and placed in 20% and 30% sucrose at 4°C until it sank. The brains were embedded and stored at -80°C. Then, the brains were cut into 30 µm thin slices using a cryo-ultramicrotome (HM525, Thermo Fisher, USA). After washing the brain slices with PBS, the brain slices were blocked in 3% BSA (containing 0.3% Triton X-100) for 60 min at 37°C. The brain slices were incubated overnight at 4°C with 1% BSA and primary antibodies against GFAP (1:300, MA5-12023, Invitrogen, Carlsbad, CA), GBP2 (1:300, 11854-1-AP, Proteintech, China), S100A10 (1:150, PA5-95505, Invitrogen, Carlsbad, CA), NeuN (1:1000, ab104224, Abcam, UK) and BrdU (1:300, ab6326, Abcam, UK). The brain sections were washed 3 times using PBS followed by incubation with fluorescently labeled anti-mouse IgG (1:500, A11029, Invitrogen, Carlsbad, CA) and anti-rabbit IgG (1:500, A21429, Invitrogen, Carlsbad, CA) secondary antibodies for 60 min in the low light environment at 37°C. 4′,6′-Diamidino-2-phenylindole (DAPI, 32670, Sigma, USA) was used to stain the nuclei. Laser scanning confocal microscopy (Carl Zeiss Lsm880, Germany) was used to acquire immunofluorescence graphics. Three sections from each rat and one nonoverlapping 40× field in the ischemic penumbra zone were chosen at random. The mean GFAP fluorescence intensity and GFAP-positive cells were measured by ImageJ (National Institutes of Health, Bethesda, MD, USA).

2.10 Western blotting

Our Western blotting method refers to previous studies in this study (Zeng et al., 2019). After 28 days of reperfusion, the cerebral ischemic penumbra tissues were collected and dissolved in protein lysis buffer. The samples were centrifuged after mixing fully. The total amount of protein in the samples was determined by the BCA protein assay (23228, Thermo Fisher, USA). Thirty micrograms of protein extract from each sample was distributed on a 10–12% SDS-PAGE gel (R33400, Invitrogen, Carlsbad, CA) and electrophoresed (Mini-protean, Bio-Rad, Hercules, CA), and the protein in the gel was transferred to a PVDF membrane (88520, Thermo Fisher, USA). The PVDF membrane was blocked in 5% nonfat milk liquid for 1 h and then bound to primary antibody at 4°C for 12 h. The primary antibodies included GFAP (1:1000, 12389s, Cell Signaling Technology, USA), C3 (1:1000, ab200999, Abcam, UK), GBP2 (1:1000, 11854-1-AP, Proteintech, China), Nrf2 (1:1000, WL02135, Wanleibio, China), BDNF (1:1000, ab108319, Abcam, UK), TNF-α (1:1000, WL05181, Wanleibio, China), IL-1β (1:500, WL00891, Wanleibio, China), IL-6 (1:500, WL02841, Wanleibio, China), IL-10 (1:500, WL03088, Wanleibio, China), TrkB (1:1000, 4603s, Cell Signaling Technology, USA), Doublecortin (1:1000, 4604s, Cell Signaling Technology, USA), Synapsin-1 (1:1000, 5297s, Cell Signaling Technology, USA), PSD-95 (1:1000, 9450s, Cell Signaling Technology, USA) and β-actin (1:1000, MA5-15739, Invitrogen, Carlsbad, CA). Afterward, the membrane was washed and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5000, SA245916, Thermo Fisher, USA) and an anti-mouse secondary antibody (1:5000, SG253594, Thermo Fisher, USA) at 37°C for 1 h. Finally, the membrane was washed three times with TBST, and photos were taken with the instrument after a drop of color-substrate solution. The gray value of the PVDF membrane was analyzed by ImageJ.

2.11 Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0.2 software. All of the data are presented as the mean ± SD. Data differences between the two groups were tested by t-test, differences between groups were compared by one-way ANOVA followed by the Newman–Keuls post hoc test or two-way ANOVA followed by Tukey’s post hoc test. The significance level was considered to be P < 0.05.

After the above experiments, the results show that ICT can promote the improvement of neurological function and reduce brain volume deficits after stroke, promote the improvement of learning function, motor coordination and gait in tMCAO rats. In addition, we found that ICT could inhibit the polarization of astrocytes to the A1 phenotype and promote the polarization of astrocytes to the A2 phenotype and ICT promotes neuron regeneration in the hippocampus. G15 intervention cancels the therapeutic effect of ICT, suggesting that ICT regulates astrocyte polarization through G15 and promotes the recovery of nerve function after cerebral ischemia.

3.1 The protective effect of ICT in tMCAO rats during recovery period

The experimental process was shown in the Fig. 1B. The body weight of the tMCAO group was significantly decreased on days 1, 3, 7, 14, 21 and 28 after tMCAO compared with that of the sham group. Compared with that of the tMCAO group, the body weight of the tMCAO + ICT group was consistently higher (Fig. 1C). The whole brain revealed that in the tMCAO group, distinct black infarct areas were present in the right hemisphere. Compared with that of the sham group, the right/left brain weight ratio of the tMCAO group was significantly decreased, while that of the tMCAO + ICT group was obviously increased (Fig. 1D and E). Y-maze test showed that the spontaneous alternation rate of rats in the tMCAO group was obviously decreased compared to that of rats in the sham group, while the spontaneous alternation rate of rats in the tMCAO + ICT group was markedly enhanced compared to that of rats in the tMCAO group (Fig. 1F).

The Garcia JH scale score of the tMCAO group was visibly decreased on days 1, 3, 7, 14, 21 and 28 after tMCAO compared with that of the sham group. Compared with that of the tMCAO group, the Garcia JH scale score of the tMCAO + ICT group was consistently higher and then significantly higher on days 3, 7 and 21 after tMCAO (Fig. 1G). The gait test results showed that run duration was significantly increased in the tMCAO group rats on days 1 and 21 and markedly decreased by ICT treatment on day 21 (Fig. 1H). The RH and LH stand time were significantly increased in the tMCAO group compared with the sham group on days 1, 14 and 21, and this change was suppressed by ICT treatment and markedly decreased on day 21 (Fig. 1I and J). These results demonstrate that ICT can successfully ameliorate the impairment of spatial learning, motor coordination balance, and gait in tMCAO rats. These results indicate that ICT can improve neurological and motor function in tMCAO rats during recovery period.

3.2 The effect of ICT on astrocyte activation and polarization in tMCAO rats during the recovery period

We investigated the activation and polarization of astrocytes in the ischemic cortex at 28 d after tMCAO. These results showed that compared with those in the sham group, the numbers of GFAP-positive and GFAP/GBP2 immunofluorescence colocalized cells in the tMCAO group were obviously increased, indicating that cerebral ischemia can promote the activation of astrocytes and their polarization toward the A1 phenotype. However, these changes were reversed by ICT treatment (Fig. 2A–D). Then, we further investigated the expression of A1 astrocyte-associated proteins. The results showed that the levels of GFAP, GBP2 and C3 were obviously increased in the tMCAO group compared with the sham group; however, ICT-treated rats had remarkably lower levels of GFAP, GBP2 and C3 than tMCAO rats (Fig. 2E–G).

The results showed that the number of GFAP/S100A10-colocalized cells in the tMCAO group was obviously increased compared with that in the sham group, indicating that cerebral ischemia can promote the polarization of astrocytes toward the A2 phenotype. Moreover, ICT treatment contributed significantly to this trend (Fig. 3A and B). Then, we further investigated the expression of A2 astrocyte-associated proteins. The results showed that the expression of Nrf2 was significantly lower in the tMCAO group than in the sham group; however, ICT treatment reversed this change (Fig. 3C and D).

These results demonstrate that ICT can markedly reduce the activation of astrocytes and their polarization toward the A1 phenotype and increase A2 polarization in tMCAO rats during the recovery period.

3.3 The effect of ICT on cytokines of A1, A2 astrocytes and neuroregenerative effects in tMCAO rats during recovery period

The results showed that compared with the sham group, the BDNF and TrkB levels were decreased, levels of TNF-α and IL-1β protein were significantly increased in the tMCAO group on day 28 after reperfusion. Furthermore, the ICT group had notably increased expression of BDNF and TrkB and decreased expression of TNF-α and IL-1β compared with the tMCAO group (Fig. 4A–C).

Immunofluorescence and Western bolt methods were used to detect the effects of ICT on neuronal regeneration and synapses. BrdU and NeuN proteins were co-labeled to verify the new neurons in the hippocampal DG region (Fig. 4E). The results showed that compared with the Sham group, the number of new neurons in the hippocampal DG region of rats in tMCAO group decreased. After 28 days of continuous ICT treatment, the number of new neurons in the hippocampal DG region of tMCAO + ICT group was significantly increased (Fig. 4F). The results of Western bolt showed that compared with Sham group, the expressions of Doublecortin, Synapsin-1 and PSD-95 in the ischemic cortex of rats in tMCAO group were significantly decreased. After ICT treatment, the protein expressions of Doublecortin, Synapsin-1 and PSD-95 in tMCAO + ICT group were significantly increased (Fig. 4G–I). These results suggest that ICT can inhibit the levels of factors secreted by A1 astrocytes and increase the levels of factors secreted by A2, and promote neuron regeneration in the DG region of the hippocampus and increase the expression of synapse-related proteins in the ischemic cortex of tMCAO rats.

3.4 G15 reversed the neuroprotective effect of ICT in tMCAO rats during recovery period

In order to investigate the mechanism of action of ICT, we conducted experiments using G15 intervention (an inhibitor of GPER). The experimental process is shown in the Fig. 5A, the 28-day survival rate of DMSO group was higher than that of G15 group (Fig. 5B). Compared with DMSO group, the volume of the right brain in G15 group was significantly reduced (Fig. 5C and D). G15 has no effect on the spontaneous alternation rate of Y maze experiment (Fig. 5E). However, G15 can affect the gait of rats, such as extension run duration, RH and LH support time (Fig. 5F–H). These results indicate that G15 reversed the effects of ICT on brain protection and gait improvement in tMCAO rats, suggesting that the effect of ICT on tMCAO rats during recovery period may be achieved via the GPER receptor.

3.5 G15 reversed the effect of ICT on the phenotypic polarization of astrocyte A1/A2 in tMCAO rats during recovery period.

As shown in the figure, the number of co-labeled cells of GFAP/GBP2 increased significantly after administration of G15, and the number of co-labeled cells of GFAP/S100A10 did not change significantly (Fig. 6A–E). The result show that the G15 group expression of GFAP, C3 and GBP2 in the ischemic cortex are increased (Fig. 6F–H), and the expression of Nrf2 is decreased (Fig. 6I). The results suggest that G15 can abolish the regulatory effect of ICT on astrocyte phenotypic polarization, indicating that ICT on astrocyte polarization may be regulated through GPER receptor.

3.6 G15 reversed the effect of ICT on cytokines of A1, A2 astrocytes in tMCAO rats during recovery period

After G15 intervention, the TNF-α、IL-1β and IL-6 protein expression significantly increased (Fig. 7A–C), while IL-10 and BDNF protein significantly decreased (Fig. 7D and E), the expression level of TrkB、Doublecortin and Synapsin-1 protein was also decreased (Fig. 7F–H). These results indicate that G15 intervention reverses the regulatory effect of ICT on A1/A2 astrocytes and inhibits the expression of proteins related to nerve regeneration, indicating that ICT on astrocyte polarization and promotes neurogenesis may achieved through GPER receptors.

We explored the effects of ICT on the recovery period after cerebral ischemia and on astrocyte activation using the tMCAO rat model in this study. We found that administration of ICT improved the prognosis of cerebral ischemia, as shown by the increase in the Garcia JH scale and the regain of weight in rats. In addition, ICT promoted the recovery of spatial memory after cerebral ischemia; moreover, ICT improved gait in tMCAO rats. Furthermore, ICT can regulate the activation and polarization of astrocytes during the recovery period after cerebral ischemia. ICT reduced astrocyte polarization toward the A1 phenotype and promoted astrocyte polarization toward the A2 phenotype in the ischemic cortices of tMCAO rats during the recovery period. Our study suggests that ICT promotes functional brain rehabilitation in ischemic stroke rats by regulating astrocyte activation and polarization.

Many behaviors of the animals were affected after cerebral ischemia. It has been reported that in tMCAO rats, behavioral deficits can last for up to three months after cerebral ischemic injury (Encarnacion et al., 2011). Our results showed that ICT can promote an increase in rat body weight and the Garcia JH scale, reduced defects in the right side of the brain after stroke, indicating that ICT is helpful for recovery after cerebral ischemic injury in rats. Learning and memory impairment is a common sequelae of stroke (Hong et al., 2000), and memory function, one of the most important cognitive abilities, can be evaluated by the Y maze test after cerebral ischemia (Li et al., 2020). Our findings showed that ICT treatment protected against learning and memory impairment. Cerebral ischemia leads to gait defects (Wang et al., 2008), and the CatWalk gait analysis system is a sensitive tool to detect the gait of rats after cerebral ischemia (Balkaya et al., 2013). Our results showed that ICT decreased the run duration and RH and LH stand time in the walkway, indicating that ICT can successfully ameliorate gait impairment in tMCAO rats during the recovery period. The above behavioral results show that ICT can promote neurological recovery during the recovery period of ischemic stroke.

Astrocytes exhibit various structural and functional changes after CNS damage and affect the prognosis of many diseases (Sofroniew and Vinters, 2010). After cerebral ischemia, astrocytes can be activated and polarized into A1/A2 phenotypes, A1 phenotype astrocytes release a variety of pro-inflammatory factors, which aggravate nerve injury (Qiu et al., 2016). A2 phenotype astrocytes release neurotrophic factors, which play a neuroprotective role (Buffo et al., 2010). It has been documented that ICT can inhibit lipopolysaccharide-induced activation of primary astrocytes, reduce the release of TNF-α and IL-1β to relieve neuroinflammation, and play a neuroprotective role (Zhang et al., 2021), suggest that ICT can regulate astrocytes. Our results show that after 28 days of continuous ICT treatment, the number of A1 phenotype astrocytes decreased, and the levels of proinflammatory factors TNF-α and IL-1β were decreased. On the other hand, the number of A2 phenotype astrocytes increased and the levels of neurotrophic factor and their receptors like BDNF and TrkB were up regulation. These results suggest that ICT can regulate the polarization of astrocytes and play a neuroprotective role.

The promotion of angiogenesis, synaptic regeneration and neuronal regeneration is an important strategy to promote cerebral ischemia rehabilitation. The hippocampus plays an important role in learning and memory, and can constantly generate new neurons. After cerebral ischemia, neuronal death occurs in both the infarct area and the hippocampus, resulting in abnormal cognitive function (Rudolph et al., 2021). Studies have shown that stimulating neuron regeneration in hippocampus after cerebral ischemia can improve cognitive dysfunction caused by stroke (Sun et al., 2020). The results show that ICT can promote neuron regeneration in DG region of hippocampus in tMCAO rats. Moreover, ICT can increase the expression of synaptic related proteins in ischemic cortex, indicating that ICT can protect the integrity of synapses, which also indicated the neuroprotective effect of ICT.

As an estrogen membrane receptor, GPER can be bind with soybean isoflavone plant estrogens such as genistein, daidzein and its metabolite s-equol to activate downstream transcription factors, playing an important role in neurological, immune, endocrine and cardiovascular diseases (Prossnitz and Barton, 2011). In addition, activation of GPER decreased levels of primary microglia proinflammatory cytokines IL-1β and TNF-α, while inhibition of GPER increased inflammatory responses (Zhao et al., 2016), suggesting that GPER has a protective role in cerebral ischemia by suppressing neuroinflammation. As a phytoestrogen, ICT may combine with GPER to play a brain protective role. Our experimental results showed that inhibition of GPER reversed the neuroprotective effects of ICT on tMCAO rats and the regulatory effects on astrocyte program, suggesting that ICT may act on GPER, regulate astrocyte polarization, and promote the rehabilitation of nerve function after cerebral ischemia.

There are some limitations to our current study. We only studied the possible effect of ICT on GPER, but the underlying mechanism remains unclear. In addition, we should make it clear that ICT does regulate astrocyte polarization through GPER at the in vitro level, which will be the direction of our follow-up work.

In summary, ICT has therapeutic advantages in alleviating dysfunction during the convalescent period of ischemic stroke. This effect may be achieved by acting on GPER to regulate the activation and polarization of astrocytes to different phenotypes and to reduce post-ischemic inflammation. This study confirmed the protective effect of phytoestrogens on the brain, providing new clues for the development of drugs to treat the recovery of nerve function after cerebral ischemia.

BDNF	Brain-derived neurotrophic factor
BrdU	5-bromodeoxyuridine
CNS	Central nervous system
C3	Complement-3
DG	Dentate gyrus
DMSO	Dimethyl sulfoxide
GBP2	Guanylate binding protein 2
GFAP	Glial fibrillary acidic protein
GPER	G protein-coupled estrogen receptor
ICT	Icaritin
IL-1β	Interleukin-1β
IL-6 IL-10	Interleukin-6 Interleukin-10
NeuN	Neuron specific nuclear protein
Nrf2	Nuclear factor erythroid 2-related factor 2
PSD-95	Post synaptic density 95
STAT3	Signal transducer and activator of transcription 3
tMCAO	Transient middle cerebral artery occlusion
TNF-α	Tumor necrosis factor-α
tPA	Tissue plasminogen activator
TrkB	Tyrosine kinase receptor B
S100A10	S100 calcium binding protein A10

Authors’ Contributions

Liangdong Li and Zhihua Huang participated in the design of this study, and they both performed the statistical analysis. Guangjun Su carried out the study and collected important background information. Guangjun Su drafted the manuscript with the help of Liangdong Li and Zhihua Huang. Zining Yu, Gaigai Liu, Limei Zhang, Guojian Zhao, Li Luo, Fangshi Cai, Qian Zhang, Shi Tang, Xinling Cao and Cheng Huang provided assistances for data acquisition, data analysis and statistical analysis. All of authors agree to be accountable for all aspects of work ensuring integrity and accuracy. All authors read and approved the final manuscript.

Funding

This work was supported by the grants from the National Natural Science Foundation of China (No. 31960190), Jiangxi Natural Science Foundation (No. 20161BAB205228, 20192BAB205117), Science and Technology Project of Bureau of Education of Jiangxi Province (No. GJJ2201427), the Open Project of Key Laboratory of Prevention and treatment of cardiovascular and cerebrovascular diseases, Ministry of Education (No. XN201811), Innovation Team Foundation of Gannan Medical University (No. TD201705, TD2021JC06) and Graduate Innovative Special Funds Project of Gannan Medical University (NO. YC2021-S805).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon request. All of data were generated in-house, and no paper mill was used.

Acknowledgement

The abstract diagram is supported by Figdraw with authorization unmber: WYIOlff654.

Ethics Approval

All animal procedures were performed following the guidelines of the China Council on Animal Care and were approved by the Gannan Medical Ethics Committee (Ganzhou China, ethical approval No. 2019140).

Consent For Publication

All the authors give consent.

Competing Interests

All of the authors declare that they have no conflict of interest and known competing financial interests.

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No competing interests reported.

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Icaritin Promotes Brain Functional Rehabilitation in Ischemic Stroke Rats by Regulating Astrocyte Activation and Polarization Via GPER

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Abstract

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Highlights

1. Introduction

2. Materials and methods

2.1 Experimental animals and drugs

2.2 Experimental protocol

2.3 tMCAO rat model

2.4 The survival and body weight of the rats were recorded.

2.5 Neurological test

2.6 Assessment of the right/left brain weight ratio

2.7 Y-maze test

2.8 Gait test

2.9 Immunofluorescence staining

2.10 Western blotting

2.11 Statistical analysis

3. Result

3.1 The protective effect of ICT in tMCAO rats during recovery period

3.4 G15 reversed the neuroprotective effect of ICT in tMCAO rats during recovery period

4. Discussion

5. Conclusion

Abbreviations

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References

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