LPS priming-induced immune tolerance mitigates LPS-stimulated microglial activation and social avoidance behaviors in mice.

In this study, we investigated the regulatory mechanisms underlying the effects of LPS tolerance on the inflammatory homeostasis of immune cells. LPS priming-induced immune tolerance downregulated cyclooxygenase-2, and lowered the production of prostaglandin-E2 in microglial cells. In addition, LPS tolerance downregulated the expression of suppressor of cytokine signaling 3, and inducible nitric oxide synthase/nitric oxide; suppressed the LPS-mediated induction of tumor necrosis factor-α, interleukin (IL)-6, and IL-1; and reduced reactive oxygen species production in microglial cells. LPS stimulation increased the levels of the adaptive response-related proteins heme oxygenase-1 and superoxide dismutase 2, and the levels of heme oxygenase-1 (HO-1) enhanced after LPS priming. Systemic administration of low-dose LPS (0.5 mg/kg) to mice for 4 consecutive days attenuated high-dose LPS (5 mg/kg)-induced inflammatory response, microglial activation, and proinflammatory cytokine expression. Moreover, repeated exposure to low-dose LPS suppressed the recruitment of peripheral monocytes or macrophages to brain regions and downregulated the expression of proinflammatory cytokines. Notably, LPS-induced social avoidance behaviors in mice were mitigated by immune tolerance. In conclusion, immune tolerance may reduce proinflammatory cytokine expression and reactive oxygen species production. Our findings provide insights into the effects of endotoxin tolerance on innate immune cells and social behaviors.

Inhibitory Effects of Urolithins, Bioactive Gut Metabolites from Natural Polyphenols, against Glioblastoma Progression.

We previously reported that proinflammatory cytokines, particularly tumor necrosis factor (TNF)-α, promoted tumor migration, invasion, and proliferation, thus worsening the prognosis of glioblastoma (GBM). Urolithins, the potent metabolites produced by the gut from pomegranate polyphenols, have anticancer properties. To develop an effective therapy for GBM, this study aimed to study the effects of urolithins against GBM. Urolithin A and B significantly reduced GBM migration, reduced epithelial-mesenchymal transition, and inhibited tumor growth. Moreover, urolithin A and B inhibited TNF-α-induced vascular cell adhesion molecule (VCAM)-1 and programmed death ligand 1 (PD-L1) expression, thereby reducing human monocyte (HM) binding to GBM cells. Aryl hydrocarbon receptor (AhR) level had higher expression in patients with glioma than in healthy individuals. Urolithins are considered pharmacological antagonists of AhR. We demonstrated that the inhibition of AhR reduced TNF-α-stimulated VCAM-1 and PD-L1 expression. Furthermore, human macrophage condition medium enhanced expression of PD-L1 in human GBM cells. Administration of the AhR antagonist attenuated the enhancement of PD-L1, indicating the AhR modulation in GBM progression. The modulatory effects of urolithins in GBM involve inhibiting the Akt and epidermal growth factor receptor pathways. The present study suggests that urolithins can inhibit GBM progression and provide valuable information for anti-GBM strategy.

Machine learning-based dynamic prediction of lateral lymph node metastasis in patients with papillary thyroid cancer.

Objective: To develop a web-based machine learning server to predict lateral lymph node metastasis (LLNM) in papillary thyroid cancer (PTC) patients. Methods: Clinical data for PTC patients who underwent primary thyroidectomy at our hospital between January 2015 and December 2020, with pathologically confirmed presence or absence of any LLNM finding, were retrospectively reviewed. We built all models from a training set (80%) and assessed them in a test set (20%), using algorithms including decision tree, XGBoost, random forest, support vector machine, neural network, and K-nearest neighbor algorithm. Their performance was measured against a previously established nomogram using area under the receiver operating characteristic curve (AUC), decision curve analysis (DCA), precision, recall, accuracy, F1 score, specificity, and sensitivity. Interpretable machine learning was used for identifying potential relationships between variables and LLNM, and a web-based tool was created for use by clinicians. Results: A total of 1135 (62.53%) out of 1815 PTC patients enrolled in this study experienced LLNM episodes. In predicting LLNM, the best algorithm was random forest. In determining feature importance, the AUC reached 0.80, with an accuracy of 0.74, sensitivity of 0.89, and F1 score of 0.81. In addition, DCA showed that random forest held a higher clinical net benefit. Random forest identified tumor size, lymph node microcalcification, age, lymph node size, and tumor location as the most influentials in predicting LLNM. And the website tool is freely accessible at http://43.138.62.202/. Conclusion: The results showed that machine learning can be used to enable accurate prediction for LLNM in PTC patients, and that the web tool allowed for LLNM risk assessment at the individual level.

Paliperidone Inhibits Glioblastoma Growth in Mouse Brain Tumor Model and Reduces PD-L1 Expression.

A previous study from our group reported that monocyte adhesion to glioblastoma (GBM) promoted tumor growth and invasion activity and increased tumor-associated macrophages (TAMs) proliferation and inflammatory mediator secretion as well. The present study showed that prescribed psychotropic medicine paliperidone reduced GBM growth and immune checkpoint protein programmed death ligand (PD-L)1 expression and increased survival in an intracranial xenograft mouse model. An analysis of the database of patients with glioma showed that the levels of PD-L1 and dopamine receptor D (DRD)2 were higher in the GBM group than in the low grade astrocytoma and non-tumor groups. In addition, GFP expressing GBM (GBM-GFP) cells co-cultured with monocytes-differentiated macrophage enhanced PD-L1 expression in GBM cells. The enhancement of PD-L1 in GBM was antagonized by paliperidone and risperidone as well as DRD2 selective inhibitor L741426. The expression of CD206 (M2 phenotype marker) was observed to be markedly increased in bone marrow-derived macrophages (BMDMs) co-cultured with GBM. Importantly, treatment with paliperidone effectively decreased CD206 and also dramatically increased CD80 (M1 phenotype marker) in BMDMs. We have previously established a PD-L1 GBM-GFP cell line that stably expresses PD-L1. Experiments showed that the expressions of CD206 was increased and CD80 was mildly decreased in the BMDMs co-cultured with PD-L1 GBM-GFP cells. On the other hands, knockdown of DRD2 expression in GBM cells dramatically decreased the expression of CD206 but markedly increased CD80 expressions in BMDMs. The present study suggests that DRD2 may be involved in regulating the PD-L1 expression in GBM and the microenvironment of GBM. Our results provide a valuable therapeutic strategy and indicate that treatments combining DRD2 antagonist paliperidone with standard immunotherapy may be beneficial for GBM treatment.

Fenofibrate inhibits hypoxia-inducible factor-1 alpha and carbonic anhydrase expression through activation of AMP-activated protein kinase/HO-1/Sirt1 pathway in glioblastoma cells.

Cancer and its associated conditions have significant impacts on public health at many levels worldwide, and cancer is the leading cause of death among adults. Peroxisome proliferator-activated receptor α (PPARα)-specific agonists, fibrates, have been approved by the Food and Drug Administration for managing hyperlipidemia. PPARα-specific agonists exert anti-cancer effects in many human cancer types, including glioblastoma (GBM). Recently, we have reported that the hypoxic state in GBM stabilizes hypoxia-inducible factor-1 alpha (HIF-1α), thus contributing to tumor escape from immune surveillance by activating the expression of the pH-regulating protein carbonic anhydrase IX (CA9). In this study, we aimed to study the regulatory effects of the PPARα agonist fibrate on the regulation of HIF-1α expression and its downstream target protein in GBM. Our findings showed that fenofibrate is the high potency compound among the various fibrates that inhibit hypoxia-induced HIF-1α and CA9 expression in GBM. Moreover, fenofibrate-inhibited HIF-1α expression is mediated by HO-1 activation in GBM cells through the AMP-activated protein kinase (AMPK) pathway. In addition, fenofibrate-enhanced HO-1 upregulation activates SIRT1 and leads to subsequent accumulation of SIRT1 in the nucleus, which further promotes HIF-1α deacetylation and inhibits CA9 expression. Using a protein synthesis inhibitor, cycloheximide, we also observed that fenofibrate inhibited HIF-1α protein synthesis. In addition, the administration of the proteasome inhibitor MG132 showed that fenofibrate promoted HIF-1α protein degradation in GBM. Hence, our results indicate that fenofibrate is a useful anti-GBM agent that modulates hypoxia-induced HIF-1α expression through multiple cellular pathways.

Regulatory effects of IL-1ß in the interaction of GBM and tumor-associated monocyte through VCAM-1 and ICAM-1.

Glioblastoma (GBM) is the most common and lethal brain tumor with high inflammation. GBM cells infiltrate microglia and macrophages and are surrounded by pro-inflammatory cytokines. Interleukin (IL)-1ß, which is abundantly expressed in the tumor microenvironment, is involved in tumor progression. Intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 mediate cell-cell interactions, and these cell adhesion molecules (CAMs) can be regulated by cytokines in immune cells or cancer cells in the inflammatory tumor microenvironment. In this study, we found that ICAM-1 and VCAM-1 expression was induced when GBM cells were treated with IL-1ß, and that adhesive interaction between monocytes and GBM cells increased accordingly. The levels of soluble CAMs (sICAM-1 and sVCAM-1) were also increased in the supernatants induced by IL-1ß. Furthermore, the conditioned media contained sICAM-1 and sVCAM-1, which further promoted IL-6 and CCL2 expression in differentiated macrophages. IL-1ß downregulated Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1) in GBM. The expression of ICAM-1 and VCAM-1 was regulated by p38, AKT, and NF-κB signaling pathways, which were modulated by SHP-1 signaling. The present study suggests that IL-1ß-induced protein expression of ICAM-1 and VCAM-1 in GBM may modulate the adhesive interaction between GBM and monocytes. In addition, IL-1ß also induced the soluble form of ICAM-1 and VCAM-1 in GBM, which plays a key role in the regulation of tumor-associated monocyte/macrophage polarization.

SIRT1 activation by minocycline on regulation of microglial polarization homeostasis.

Sirtuin 1 (SIRT1) has been reported to be involved in the mechanisms underlying longevity and has also been indicated as a valuable regulator of age-related neurological disorders. Some natural products increase SIRT1 activity and stimulate deacetylation of various proteins. In the present study, SIRT1 overexpression by genetic modification or treatment with SIRT1 activators significantly inhibited the secretion of nitric oxide and expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory mediator-interleukin 1ß-in microglia. SIRT1 activation also decreased the levels of K379 acetyl-p53 and the protein inhibitor of activated Stat 1 expression in microglial cells. In addition, it dramatically promoted M2 polarization of microglia, which enhanced cell motility and altered phagocytic ability. We also used minocycline, a well-known inhibitor of microglial activation, to study the mechanism of SIRT1 signaling. Minocycline treatment decreased neuroinflammatory responses and promoted M2 polarization of microglia. It also reduced the acetyl-p53 level in the brain tissues in an inflammatory mouse model. Our findings demonstrated that SIRT1 participates in the maintenance of microglial polarization homeostasis and that minocycline exerts regulatory effects on SIRT1 activation. Therefore, our results indicate that SIRT1 activation may be a useful therapeutic target for the treatment of neuroinflammation-associated disorders.

CAIX Regulates GBM Motility and TAM Adhesion and Polarization through EGFR/STAT3 under Hypoxic Conditions.

Carbonic anhydrases (CAs) are acid-base regulatory proteins that modulate a variety of physiological functions. Recent findings have shown that CAIX is particularly upregulated in glioblastoma multiforme (GBM) and is associated with a poor patient outcome and survival rate. An analysis of the GSE4290 dataset of patients with gliomas showed that CAIX was highly expressed in GBM and was negatively associated with prognosis. The expression of CAIX under hypoxic conditions in GBM significantly increased in protein, mRNA, and transcriptional activity. Importantly, CAIX upregulation also regulated GBM motility, monocyte adhesion to GBM, and the polarization of tumor-associated monocytes/macrophages (TAM). Furthermore, the overexpression of CAIX was observed in intracranial GBM cells. Additionally, epidermal growth factor receptor/signal transducer and activator of transcription 3 regulated CAIX expression under hypoxic conditions by affecting the stability of hypoxia-inducible factor 1α. In contrast, the knockdown of CAIX dramatically abrogated the change in GBM motility and monocyte adhesion to GBM under hypoxic conditions. Our results provide a comprehensive understanding of the mechanisms of CAIX in the GBM microenvironment. Hence, novel therapeutic targets of GBM progression are possibly developed.

SUMO E3 ligase PIAS1 is a potential biomarker indicating stress susceptibility.

Prior studies suggest that individual differences in stress responses contribute to the pathogenesis of neuropsychiatric disorders. In the present study, we investigated the role of small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) in mediating stress responses to chronic social defeat stress (CSDS). We found that mRNA and protein levels of PIAS 1 were decreased in the hippocampus of high-susceptibility (HS) mice but not in low-susceptibility (LS) mice after CSDS. Local overexpression of PIAS1 in the hippocampus followed by CSDS exposure promoted stress resilience by attenuating social avoidance and improving anxiety-like behaviors. Viral-mediated gene transfer to generate a conditional knockdown of PIAS1 in the hippocampus promoted social avoidance and stress vulnerability after subthreshold microdefeat. HS mice displayed decreased levels of glucocorticoid receptor (GR) expression, and GR SUMOylation in the hippocampus was associated with stress vulnerability. Furthermore, cytokine/chemokine levels were changed predominantly in the hippocampus of HS mice. These results suggest that hippocampal PIAS1 plays a role in the regulation of stress susceptibility by post-translational modification of GRs.

Monocarboxylate Transporter 4 Regulates Glioblastoma Motility and Monocyte Binding Ability.

Glioblastoma (GBM) is characterized by severe hypoxic and acidic stress in an abnormal microenvironment. Monocarboxylate transporter (MCT)4, a pH-regulating protein, plays an important role in pH homeostasis of the glycolytic metabolic pathways in cancer cells. The present study showed that GBM exposure to hypoxic conditions increased MCT4 expression. We further analyzed the glioma patient database and found that MCT4 was significantly overexpressed in patients with GBM, and the MCT4 levels positively correlated with the clinico-pathological grades of gliomas. We further found that MCT4 knockdown abolished the hypoxia-enhanced of GBM cell motility and monocyte adhesion. However, the overexpression of MCT4 promoted GBM cell migration and monocyte adhesion activity. Our results also revealed that MCT4-regulated GBM cell motility and monocyte adhesion are mediated by activation of the serine/threonine-specific protein kinase (AKT), focal adhesion kinase (FAK), and epidermal growth factor receptor (EGFR) signaling pathways. Moreover, hypoxia mediated the acetylated signal transducer and activator of transcription (STAT)3 expression and regulated the transcriptional activity of hypoxia inducible factor (HIF)-1α in GBM cell lines. In a GBM mouse model, MCT4 was significantly increased in the tumor necrotic tissues. These findings raise the possibility for the development of novel therapeutic strategies targeting MCT4.

Melatonin Modulates the Microenvironment of Glioblastoma Multiforme by Targeting Sirtuin 1.

Natural products have historically been regarded as an important resource of therapeutic agents. Resveratrol and melatonin have been shown to increase SIRT1 activity and stimulate deacetylation. Glioblastoma multiforme (GBM) is the deadliest of malignant types of tumor in the central nervous system (CNS) and their biological features make treatment difficult. In the glioma microenvironment, infiltrating immune cells has been shown to possess beneficial effects for tumor progression. We analyzed SIRT1, CCL2, VCAM-1 and ICAM-1 in human glioma cell lines by immunoblotting. The correlation between those markers and clinico-pathological grade of glioma patients were assessed by the Gene Expression Omnibus (GEO) datasets analysis. We also used monocyte-binding assay to study the effects of melatonin on monocyte adhesion to GBM. Importantly, overexpression of SIRT1 by genetic modification or treatment of melatonin significantly downregulated the adhesion molecular VCAM-1 and ICAM-1 expression in GBM. CCL2-mediated monocyte adhesion and expression of VCAM-1 and ICAM-1 were regulated through SIRT1 signaling. SIRT1 is an important modulator of monocytes interaction with GBM that gives the possibility of improved therapies for GBM. Hence, this study provides a novel treatment strategy for the understanding of microenvironment changes in tumor progression.

Curcumin Promotes Connexin 43 Degradation and Temozolomide-Induced Apoptosis in Glioblastoma Cells.

Glioblastoma (GBM) is the most commonly occurring tumor in the cerebral hemispheres. Currently, temozolomide (TMZ), an alkylating agent that induces DNA strand breaks, is considered the frontline chemotherapeutic agent for GBM. Despite its frontline status, GBM patients commonly exhibit resistance to TMZ treatment. We have recently established and characterized TMZ-resistant human glioma cells. The aim of this study is to investigate whether curcumin modulates cell apoptosis through the alternation of the connexin 43 (Cx43) protein level in TMZ-resistant GBM. Overexpression of Cx43, but not ATP-binding cassette transporters (ABC transporters), was observed (approximately 2.2-fold) in TMZ-resistant GBM cells compared to the Cx43 levels in parental GBM cells. Furthermore, at a concentration of 10 µ M, curcumin significantly reduced Cx43 protein expression by about 40%. In addition, curcumin did not affect the expression of other connexins like Cx26 or epithelial-to-mesenchymal transition (EMT) proteins such as ß -catenin or α E-catenin. Curcumin treatment led to an increase in TMZ-induced cell apoptosis from 4% to 8%. Importantly, it did not affect the mRNA expression level of Cx43. Concomitant treatment with the translation inhibitor cycloheximide (CHX) exerted additional effects on Cx43 degradation. Treatment with the autophagy inhibitor 3-MA (methyladenine) did not affect the curcumin-induced Cx43 degradation. Interestingly, treatment with the proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) significantly negated the curcumin-induced Cx43 degradation, which suggests that curcumin-induced Cx43 degradation occurs through the ubiquitin-proteasome pathway.

Bradykinin B1 receptor contributes to interleukin-8 production and glioblastoma migration through interaction of STAT3 and SP-1.

Glioblastoma (GBM), the most aggressive brain tumor, has a poor prognosis due to the ease of migration to surrounding healthy brain tissue. Recent studies have shown that bradykinin receptors are involved in the progression of various cancers. However, the molecular mechanism and pathological role of bradykinin receptors remains unclear. We observed the expressions of two major bradykinin receptors, B1R and B2R, in two different human GBM cell lines, U87 and GBM8901. Cytokine array analysis showed that bradykinin increases the production of interleukin (IL)-8 in GBM via B1R. Higher B1R levels correlate with IL-8 expression in U87 and GBM8901. We observed increased levels of phosphorylated STAT3 and SP-1 in the nucleus as well. Using chromatin immunoprecipitation assay, we found that STAT3 and SP-1 mediate IL-8 expression, which gets abrogated by the inhibition of FAK and STAT3. We further demonstrated that IL-8 expression and cell migration are also regulated by the SP-1. In addition, expression levels of STAT3 and SP-1 positively correlate with clinicopathological grades of gliomas. Interestingly, our results found that inhibition of HDAC increases IL-8 expression. Moreover, stimulation with bradykinin caused increases in acetylated SP-1 and p300 complex formation, which are abrogated by inhibition of FAK and STAT3. Meanwhile, knockdown of SP-1 and p300 decreased the augmentation of bradykinin-induced IL-8 expression. These results indicate that bradykinin-induced IL-8 expression is dependent on B1R which causes phosphorylated STAT3 and acetylated SP-1 to translocate to the nucleus, hence resulting in GBM migration.

Regulatory Effects of Neuroinflammatory Responses Through Brain-Derived Neurotrophic Factor Signaling in Microglial Cells.

Inhibition of microglial over-activation is an important strategy to counter balance neurodegenerative progression. We previously demonstrated that the adenosine monophosphate-activated protein kinase (AMPK) may be a therapeutic target in mediating anti-neuroinflammatory responses in microglia. Brain-derived neurotrophic factor (BDNF) is one of the major neurotrophic factors produced by astrocytes to maintain the development and survival of neurons in the brain, and have recently been shown to modulate homeostasis of neuroinflammation. Therefore, the present study focused on BDNF-mediated neuroinflammatory responses and may provide an endogenous regulation of neuroinflammation. Among the tested neuroinflammation, epigallocatechin gallate (EGCG) and minocycline exerted BDNF upregulation to inhibit COX-2 and proinflammatory mediator expressions. Furthermore, both EGCG and minocycline upregulated BDNF expression in microglia through AMPK signaling. In addition, minocycline and EGCG also increased expressions of erythropoietin (EPO) and sonic hedgehog (Shh). In the endogenous modulation of neuroinflammation, astrocyte-conditioned medium (AgCM) also decreased the expression of COX-2 and upregulated BDNF expression in microglia. The anti-inflammatory effects of BDNF were mediated through EPO/Shh in microglia. Our results indicated that the BDNF-EPO-Shh novel-signaling pathway underlies the regulation of inflammatory responses and may be regarded as a potential therapeutic target in neurodegenerative diseases. This study also reveals a better understanding of an endogenous crosstalk between astrocytes and microglia to regulate anti-inflammatory actions, which could provide a novel strategy for the treatment of neuroinflammation and neurodegenerative diseases.

Differential Characterization of Temozolomide-Resistant Human Glioma Cells.

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. Temozolomide (TMZ) has been considered to be one of the most effective chemotherapeutic agents to prolong the survival of patients with glioblastoma. Many glioma cells develop drug-resistance against TMZ that is mediated by increasing O-6-methylguanine-DNA methyltransferase (MGMT) levels. The expression of connexin 43 was increased in the resistant U251 subline compared with the parental U251 cells. The expression of epithelial-mesenchymal transition (EMT)-associated regulators, including vimentin, N-cadherin, and ß-catenin, was reduced in the resistant U251 subline. In addition, the resistant U251 subline exhibited decreased cell migratory activity and monocyte adhesion ability compared to the parental U251 cells. Furthermore, the resistant U251 subline also expressed lower levels of vascular cell adhesion molecule (VCAM)-1 after treatment with recombinant tumor necrosis factor (TNF)-α. These findings suggest differential characteristics in the drug-resistant GBM from the parental glioma cells.

Interlukin-18 Is a Pivot Regulatory Factor on Matrix Metalloproteinase-13 Expression and Brain Astrocytic Migration.

The expression of matrix metalloproteinase-13 (MMP-13) has been shown to be elevated in some pathophysiological conditions and is involved in the degradation of extracellular matrix in astrocytes. In current study, the function of MMP-13 was further investigated. The conditioned medium (CM) collected from activated microglia increased interleukin (IL)-18 production and enhanced MMP-13 expression in astrocytes. Furthermore, treatment with recombinant IL-18 increased MMP-13 protein and mRNA levels in astrocytes. Recombinant IL-18 stimulation also increased the enzymatic activity of MMP-13 and the migratory activity of astrocytes, while administration of MMP-13 or pan-MMP inhibitors antagonized IL-18-induced migratory activity of astrocytes. In addition, administration of recombinant IL-18 to astrocytes led to the phosphorylation of JNK, Akt, or PKCδ, and treatment of astrocytes with JNK, PI3 kinase/Akt, or PKCδ inhibitors significantly decreased the IL-18-induced migratory activity. Taken together, the results suggest that IL-18-induced MMP-13 expression in astrocytes is regulated by JNK, PI3 kinase/Akt, and PKCδ signaling pathways. These findings also indicate that IL-18 is an important regulator leading to MMP-13 expression and cell migration in astrocytes.

Regulatory effects of caffeic acid phenethyl ester on neuroinflammation in microglial cells.

Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effects on microglia are therefore considered as a potential strategy to counter balance neurodegenerative progression. Caffeic acid phenethyl ester (CAPE), a natural phenol in honeybee propolis, is known to possess antioxidant, anti-inflammatory and anti-microbial properties. Accordingly, the current study intended to probe the effects of CAPE on microglia activation by using in vitro and in vivo models. Western blot and Griess reaction assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (NOS), cyclooxygenase (COX)-2 and the production of nitric oxide (NO). Administration of CAPE resulted in increased expressions of hemeoxygenase (HO)-1and erythropoietin (EPO) in microglia. The phosphorylated adenosine monophosphate-activated protein kinase (AMPK)-α was further found to regulate the anti-inflammatory effects of caffeic acid. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. The current study has evidenced several possible molecular determinants, AMPKα, EPO, and HO-1, in mediating anti-neuroinflammatory responses in microglial cells.