Folic acid deficiency exacerbates the inflammatory response of astrocytes after ischemia‐reperfusion by enhancing the interaction between IL‐6 and JAK‐1/pSTAT3

Abstract Aim To demonstrate the role of IL‐6 and pSTAT3 in the inflammatory response to cerebral ischemia/reperfusion following folic acid deficiency (FD). Methods The middle cerebral artery occlusion/reperfusion (MCAO/R) model was established in adult male Sprague‐Dawley rats in vivo, and cultured primary astrocytes were exposed to oxygen‐glucose deprivation/reoxygenation (OGD/R) to emulate ischemia/reperfusion injury in vitro. Results Glial fibrillary acidic protein (GFAP) expression significantly increased in astrocytes of the brain cortex in the MCAO group compared to the SHAM group. Nevertheless, FD did not further promote GFAP expression in astrocytes of rat brain tissue after MCAO. This result was further confirmed in the OGD/R cellular model. In addition, FD did not promote the expressions of TNF‐α and IL‐1β but raised IL‐6 (Peak at 12 h after MCAO) and pSTAT3 (Peak at 24 h after MCAO) levels in the affected cortices of MCAO rats. In the in vitro model, the levels of IL‐6 and pSTAT3 in astrocytes were significantly reduced by treatment with Filgotinib (JAK‐1 inhibitor) but not AG490 (JAK‐2 inhibitor). Moreover, the suppression of IL‐6 expression reduced FD‐induced increases in pSTAT3 and pJAK‐1. In turn, inhibited pSTAT3 expression also depressed the FD‐mediated increase in IL‐6 expression. Conclusions FD led to the overproduction of IL‐6 and subsequently increased pSTAT3 levels via JAK‐1 but not JAK‐2, which further promoted increased IL‐6 expression, thereby exacerbating the inflammatory response of primary astrocytes.


| INTRODUC TI ON
Cerebral ischemia/reperfusion (I/R), caused by the restoration of blood supply to ischemic brain tissue, is a pathological injury that occurs during the treatment of ischemic stroke and is accompanied by high morbidity and mortality. 1 There are no specific drugs available to treat I/R injury. 2 Thus, in such a case, dietary supplements with low side effects may be considered to assist in promoting neurological recovery if supported by substantial scientific evidence. 3 Folic acid (FA), an essential nutrient in the regular human diet, is strongly associated with neuroinflammation. 4,5 Research has shown that folic acid deficiency (FD) triggers the activation of the neuroinflammatory cascade in Alzheimer's disease (AD). 6 In addition, Guest et al. observed a negative correlation between cerebrospinal fluid folate and levels of inflammation within the central nervous system (CNS) in the healthy population. 7 However, the exact mechanisms underlying the effects of FD on neuroinflammation following cerebral ischemia-reperfusion have not been fully elucidated. Our previous work suggests that FD may enhance the expression of inflammatory mediators following cerebral hypoxia-ischemia by activating microglia. 8 Although astrocytes and microglia are known to be critical regulators of the inflammatory response in the CNS, 9 the mechanisms by which astrocytes are involved in the effects of FD on stroke recovery require further investigation.
Astrocytes, the most common glial cells in the brain, are key regulators of the inflammatory response in the CNS. 10 For instance, in the early stages of AD, astrocytes become activated and release interleukins and nitric oxide, exacerbating the neuroinflammatory response. 11 In experimental autoimmune encephalomyelitis mice, astrocytes produce lactosylceramide, which promotes transcriptional levels of pro-inflammatory factors such as IL-1β and nitric oxide synthase in an autocrine manner. 12 Additionally, astrocyte proliferation is an important pathological feature of stroke. Reactive astrocytes can release pro-inflammatory cytokines in response to acute ischemia, especially IL-6, thereby triggering the production of secondary mediators, which may lead to persistent and neurotoxic effects. 13 Given that FD induces neuroinflammation in CNS disorders, FD may promote inflammatory responses in astrocytes following ischemia-reperfusion.
Interleukin-6 (IL-6)/signal transduction and transcription activator of 3 (STAT3) is an essential intracellular pathway that mediates inflammatory signaling and is a vital signaling component in reactive astrocytes. 14 As a core upstream regulator of the inflammatory response, IL-6 promotes inflammatory response waterfalls and simultaneously activates STAT3 via Janus kinases (JAKs). Subsequently, aberrant activation of STAT3 promotes transcriptions and expressions of many genes encoding pro-inflammatory mediators. 15 Here, we hypothesize that FD may exacerbate astrocyte injury through IL-6/pSTAT3 interactions.
In this present study, both the rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated primary astrocytes were used to observe FD's effects on astrocytes and further explore the underlying molecular mechanisms. The study shows for the first time that FD triggers an inflammatory response in astrocytes after ischemia-reperfusion through the IL-6/JAK-1/pSTAT3 pathway and exacerbates inflammation through the interaction between IL-6 and pSTAT3. This work will provide new insights into how FD leads to astrocyte injury after ischemic stroke.

| Surgical procedures
The MCAO rats were induced by the intraluminal filament technique, as described previously. 16 After 1 h of MCAO-induced focal cerebral ischemia, the line was carefully withdrawn to establish reperfusion. The rats were then allowed to recover from anesthesia at 37°C and were sacrificed at 12 h and 24 h after reperfusion for the following experiments.
To imitate the cerebral I/R model in vivo, the cells were induced by OGD/R. The normal medium (containing 10% FBS and 4.5 g/L glucose) was replaced by glucose-free DMEM (Gibco). Then, the cells were exposed to a three-gas incubator at 37°C containing 1.0% O 2 to initiate hypoxia for 1 h, followed by 3 h re-oxygenation in a normoxia incubator. Normal control cells were incubated in a regular cell culture incubator under normoxic conditions.

| Immunofluorescence
Immunofluorescence staining of the rat brain sections was performed as previously described. 22 In brief, the sections were dewaxed and hydrated to dispose of 3% H 2 O 2 for 10 min at room temperature, repaired by citric acid antigen, and blocked with goat serum for 1 h at 37°C. Then, they were incubated overnight 4°C with the primary antibodies (mouse anti-IL-6, rabbit anti-TNFα, rab-

| Western blot
Western blot was performed as previously described. 22  Then, the proteins were detected by chemiluminescence reagents (Millipore) and observed using a ChemiDoc™ XRS+ Imaging System (Bio-RAD, Hercules, USA). The protein levels were quantified by densitometry using Image J 1.4.3.67.

| Statistical analysis
SPSS V.20 and GraphPad Prism V.9.0 were used for the statistical analysis. All quantitative data were expressed as mean ± standard deviation (x±s). All data were tested for normality using the Shapiro-Wilk test. One-way ANOVA was used to assess the statistical significance of the differences among different experimental groups, followed by Student-Newman-Keuls multiple-range tests. p < 0.05 was assumed statistically significant.

| Folic acid deficiency does not further promote GFAP expression raised by ischemic injury in vitro and in vivo
Several lines of evidence support that in response to stroke, astrocytes convert to a reactive phenotype chiefly characterized by up-regulation of GFAP and cellular hypertrophy. 23 To determine the effect of FD on the reactive astrocytes, GFAP protein expression was detected in the MCAO rat brain and cultured primary astrocytes by immunohistochemical staining and western blot. The results showed an evident increase of GFAP expression at 12 h of reperfusion compared to the SHAM group, and further increased by 24 h (p < 0.05; Figure 1A, B). This result was further confirmed in in vitro OGD/R cellular model (p < 0.05; Figure 1C, D). However, FD did not significantly alter GFAP expression compared to the MCAO/R (or OGD/R) group.

| Folic acid deficiency promotes IL-6 but not TNFα and IL-1β expressions in astrocytes following ischemic injury
Astrocyte-derived neuroinflammation has been identified as a potential contributor to brain injury. 24 To determine whether FD could modulate astrocyte-mediated neuroinflammation, three proinflammatory cytokines, TNFα, IL-1β, and IL-6, were detected by immunofluorescence double-labeling and western blot analysis. As shown in Figure 2 In line with what was observed in vivo, FD promoted IL-6, but not TNFα or IL-1β levels in primary astrocytes exposed to OGD/R compared to the OGD/R alone (p < 0.05; Figure 2C, D).

| Folic acid deficiency results in an increase in pSTAT3 expression in the astrocytes following ischemic injury
Accumulated evidence suggested that activation of STAT3 plays an important role in IL-6-mediated inflammation. 25 The effect of FD on pSTAT3 expression in astrocytes was examined. The results indicated that pSTAT3 expression did not change significantly at 12 h after reperfusion but increased significantly after 24 h reperfusion compared to the SHAM group. FD further increased the number of GFAP/pSTAT3 double-positive cells in the ischemic brain compared with the MCAO/R group. Similarly, FD promoted pSTAT3 expression raised by OGD/R in primary astrocytes (p < 0.05; Figure 3 D-E).

| Folic acid deficiency increases the level of pSTAT3 through JAK-1 but not JAK-2
In inflammatory diseases, STAT3 is usually activated by phosphorylation through the activation of non-receptor protein tyrosine kinases JAKs. 15 To elucidate whether FD upregulated pSTAT3 expression in a JAK-dependent manner, the expression of pSTAT3 was detected.
As shown in Figure 4, Filgotinib administration significantly reduced the levels of IL-6 and pSTAT3, but AG490 treatment did not reveal any significant changes in the expression of IL-6 or pSTAT3. Our results proved that FD increased the level of pSTAT3 through JAK-1 instead of JAK-2.

| Interaction between IL-6 and pSTAT3 in hypoxic and glucose-deficient astrocytes after folic acid deficiency
STAT3, a key transcription factor, is involved in mediating acute inflammatory response activities located downstream of IL-6. 25 To explore the potential correlation between IL-6 and pJAK-1/pSTAT3, the cells were first treated with LMT-28. The Western blot results in Figure 5 showed that treatment with IL-6 inhibitor significantly inhibited both pSTAT3 and pJAK-1 expressions after OGD/R treatment in astrocytes (p < 0.05; Figure 5A-H). Then, whether pSTAT3 affected IL-6 expression was assessed by adding C188-9 to OGD/Rtreated astrocytes. As shown in Figure 5 I-M, the expression of IL-6 was also inhibited after adding STAT3 inhibitor (p < 0.05). Briefly, the results showed that inhibiting IL-6 expression reduces pSTAT3 levels, while pSTAT3 inhibition also decreases IL-6 expression, suggesting a positive feedback loop between these factors.

| DISCUSS ION
Inadequate levels of folic acid are associated with an increased risk of neurodegenerative diseases and cerebrovascular disease. 26 However, the exact mechanisms still need to be determined. Previous

F I G U R E 4
Folic acid deficiency regulates the expression of pSTAT3 in primary astrocytes exposed to hypoxia and glucose deficiency via the JAK-1 pathway. (A-E) The cells were harvested after incubating with Filgotinib (JAK-1 inhibitor). The protein expressions of IL-6 (A), pSTAT3, and STAT3 (B) were detected by western blot. Bar graphs show the relative levels of IL-6 (normalized to β-Actin) (C), pSTAT3 (normalized to STAT3) (D), and STAT3 (normalized to β-Actin) (E). (F-J) The cells were harvested after incubating with AG490 (JAK-2 inhibitor). The protein expressions of IL-6 (F), pSTAT3 and STAT3 (G) were detected by western blot. Bar graphs show the relative level of IL-6 (normalized to β-Actin) (H), pSTAT3 (normalized to STAT3) (I) and STAT3 (normalized to β-Actin) (J). Data are shown as mean ± SEM (n = 4). a p <0.05: Compared to OGD/R group. b p <0.05: Compared to FD + OGD/R group. astrocytes produce and release pro-inflammatory mediators, which may lead to neuronal death and infarct progression. 27 In the present study, we focused on astrocytes in order to gain insight into novel mechanisms by which FD affects neurological function. This is the first evidence that the IL-6/JAK-1/pSTAT3 pathway triggered the inflammatory response of astrocytes in the presence of FD. Notably, FD leads to the overproduction of IL-6 in the astrocytes, which next activates pSTAT3, leading to more IL-6 production and release. This interaction between IL-6 and pSTAT3 may amplify neuroinflammatory responses, leading to secondary brain damage.
There is strong experimental evidence that folic acid affects inflammation in the central nervous system; it also suggests intricate mechanisms by which this occurs. For instance, folic acid reduces hippocampal myeloperoxidase activity to alleviate neuroinflammation and improve memory impairment in sepsis-induced rats. 28 Another in vitro study indicated that lipopolysaccharide-activated microglia respond less inflammatory to folic acid because it inhibits the activation of NF-kB and JNK and upregulates p38 MAPK phosphorylation. 4 Besides, our previous work has shown that FD enhanced microglia immune responses via the Notch1/nuclear factor kappa B p65 pathway to increase brain injury. 8 The current study investigated the effect of FD on the astrocytes under ischemiareperfusion. We revealed that FD promoted the inflammatory response of astrocytes by exacerbating the interaction between IL-6 and JAK-1/pSTAT3. Multiple signaling molecules may be involved in FD's activation of neuroinflammation, which may vary depending on different cell types or disease conditions. Both JAK1 and JAK2 have been proven to be associated with the IL-6 activation of STAT3 pathway. 29 However, those two Janus kinases are known to each have different roles in different pathological and physiological processes. For instance, Yang et al. demonstrated that the release of IL-6 activated the JAK2/STAT3 pathway to aggravate neuronal degeneration in mice with Parkinson's disease. 30 Whereas, increased IL-6 expression exacerbates the inflammatory response of macrophages through the JAK1/STAT3 pathway in mouse models of ulcerative colitis. 31 To elucidate the exact pathway by which FD upregulates pSTAT3 expression, we blocked the activation of JAK-1 and JAK-2 using Filgotinib and AG490, respectively. The results demonstrate that FD-induced pSTAT3 expression was significantly inhibited in OGD/R-treated astrocytes after blocking the activation of JAK-1 but not JAK-2. Although different JAKs may have overlapping roles, each has an important role in mediating signaling. It has been shown that JAK1 is a central protein in the inflammatory response cytokine network and can produce pro-inflammatory activity. 32 Nevertheless, JAK-2 is mainly involved in processes such as mitotic reorganization and histone modification and is essential for bone marrow and platelet production. 33 These support our findings that FD exacerbates the inflammatory response in astrocytes via the IL-6/JAK-1/pSTAT3 pathway after ischemia-reperfusion.
There is a complex regulatory relationship between IL-6 and pSTAT3. As a transcription factor, STAT3 is involved in mediating the acute inflammatory response to the genes associated downstream of IL-6. 34 Binding of IL-6 to its receptor activates the phosphorylation of STAT3. pSTAT3 then binds to DNA and increases the expression of cytokine genes, resulting in the production of more interleukins.
This vicious cycle leads to persistent nervous system inflammation unless effectively controlled. 35 This is consistent with our results that there may be an interaction between IL-6 and pSTAT3 expressions in folic acid deficient OGD/R astrocytes and that the malignant feedback between them may play an essential role in FD-mediated astrocyte injury.
In general, STAT3 is a vital player in the proliferative response of reactive astrocytes. 23 Also, STAT3 is one of the transcription factors of GFAP and the increase of GFAP expression tends to be accompanied by STAT3 activation. 36 A noteworthy point to ponder is that FD promoted p-STAT3 expression but not GFAP activation in our study. This is possible because astrocyte activation is finely regulated by many intracellular and extracellular signaling molecules, such as TGFβ, NF-κB, and STAT3. [37][38][39] However, some regulatory factors, such as the FGF signaling pathway, inhibit the activation of astrocytes. 40 Therefore, we speculate that, in the case of FD, the activation of some inhibitory factors may be involved and thus FD did not further activate GFAP.
Additionally, Takumi Takizawa et al. proved that abnormal methylation of the STAT3 binding element in the GFAP promoter in astrocytes prevents the binding of STAT3, thereby inhibiting GFAP transcription. 41 Besides, the AP-1 transcription factor is essential for promoting the upregulation of GFAP genes in response to injury. 42 Folic acid is involved in DNA synthesis and methylation and thus plays a crucial role in maintaining genomic stability. 43  Firstly, the present study focused on the early molecular changes caused by FD at the onset of cerebral infarction. Considering that post-stroke neuroinflammation is a highly dynamic and complex adaptive process, 44 long-term FD intervention may be necessary for further behavioral observation and the exploration of molecular mechanisms at the later stage of disease in the future study.
Secondly, both astrocytes and microglia mediate inflammatory responses through related molecules in response to the stress of ischemic brain injury. 45 Further evidence supported that there are reciprocal interactions between microglia and astrocytes during neuroinflammation. 46 Our previous and present studies respectively verified that FD exacerbates the inflammatory response of microglia and astrocytes after ischemia-reperfusion. 8 However, in the light of the existing experiment data, we are unable to determine whether microglia or astrocytes play a more critical role during the regulation of FD on neuroinflammation, and whether FD affects the interaction between the two types of glials or not.
In conclusion, this study found that in the context of ischemiareperfusion, folic acid deficiency may trigger astrocytes' inflammatory response via the IL-6/JAK-1/pSTAT3 pathway. Furthermore, the interaction between IL-6 and pSTAT3 may amplify the | 1545 CHENG et al.
neuroinflammatory response, leading to secondary brain injury.
Therefore, specific inhibition of the IL-6/JAK-1/pSTAT3 pathway in astrocytes is a potential therapeutic approach to alleviate the progression of ischemic stroke caused by folic acid deficiency. This also suggests that folic acid supplementation is a potential preventive and therapeutic strategy to reduce brain damage in ischemic stroke.

ACK N O WLE D G E M ENTS
This work was supported by the National Natural Science Foundation of China (Grant numbers 82173519 and 81874262).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets and/or analyzed during the current study are available from the corresponding author on request.