Docosahexaenoic acid promotes M2 microglia phenotype via activating PPARγ-mediated ERK/AKT pathway against cerebral ischemia-reperfusion injury.

In ischemia-reperfusion stroke, microglia play a dual role in brain injury as well as brain repair, and promoting their switch from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype is considered to be a potential therapeutic strategy. Docosahexaenoic acid (DHA) is an essential long-chain omega-3 polyunsaturated fatty acid that exhibits potent anti-inflammatory properties in the acute phase of ischemic stroke, but its effect on microglia polarization is unknown. Thus, the objective of this study was to investigate the neuroprotective effects of DHA on rat brain following ischemia-reperfusion injury, and to investigate the mechanism by which DHA regulates microglia polarization. We administered DHA 5 mg/kg intraperitoneally daily for 3 d following a transient middle cerebral artery occlusion reperfusion model in rats. The protective effects of DHA on cerebral ischemia-reperfusion injury were detected by TTC staining, HE staining, Nissler staining, and TUNEL staining. Quantitative real-time PCR, immunofluorescence, western blot, and enzyme-linked immunosorbent assay were used to detect the expression of M1 and M2 microglia-associated markers and PPARγ-mediated ERK/AKT signaling pathway proteins. We found that DHA significantly improved brain injury by decreasing the expression of the M1 phenotypic marker (iNOS, CD16) and increasing the expression of the M2 phenotypic marker (Arg-1, CD206). DHA also increased the expression of peroxisome proliferator-activated receptor gamma (PPARγ) mRNA and protein, increased the expression of the pathway protein AKT, and decreased the expression of ERK1/2. In addition, DHA promoted the expression of anti-inflammatory factor IL-10 and decreased the expression of pro-inflammatory factors TNF-α and IL-1ß. However, the PPARγ antagonist GW9662 greatly blocked these beneficial effects. These results suggest that DHA may activate PPARγ to inhibit ERK and activate AKT signaling pathways to regulate microglia polarization, thereby reducing neuroinflammation and promoting neurological recovery to alleviate cerebral ischemia-reperfusion injury.

Neuroprotective effect of Dl-3-n-butylphthalide against ischemia-reperfusion injury is mediated by ferroptosis regulation via the SLC7A11/GSH/GPX4 pathway and the attenuation of blood-brain barrier disruption.

Background: Stroke is one of the most severe diseases worldwide, resulting in physical and mental problems. Dl-3-n-butylphthalide, a compound derived from celery seed, has been approved for treating ischemic stroke in China. No study has evaluated how Dl-3-n-butylphthalide affects the ferroptosis SLC7A11/GSH/GPX4 signal pathway and blood-brain barrier (BBB) PDGFRß/PI3K/Akt signal pathways in the rat middle cerebral artery occlusion/reperfusion (MCAO/R) model of ischemic stroke. Methods: Sprague-Dawley rats were used to develop the MCAO/R model. Our study used three incremental doses (10, 20, and 30) of Dl-3-n-butylphthalide injected intraperitoneally 24 h after MCAO/R surgery. The neuroprotective effect and success of the model were evaluated using the neurofunction score, brain water content determination, and triphenyl-tetrazolium chloride-determined infarction area changes. Pathological changes in the brain tissue and the degree of apoptosis were examined by hematoxylin and eosin, Nissl, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. In addition, pathway proteins and RNA expression levels were studied to verify the effects of Dl-3-n-butyphthalide on both pathways. At the same time, commercial kits were used to detect glutathione, reactive oxygen species, and malondialdehyde, to detect oxidative stress in brain tissues. Results: The middle dose of Dl-3-n-butylphthalide not only improved MCAO-induced brain dysfunction and alleviated pathological damage, brain inflammatory response, oxidative stress, and apoptosis but also protected against ferroptosis and reduced BBB damage. These changes resulted in improved neurological function in the cerebral cortex. Conclusion: We speculate that Dl-3-n-butylphthalide has a neuroprotective effect on focal cerebral ischemia/reperfusion, which may be mediated through ferroptosis-dependent SLC7A11/GSH/GPX4 signal pathway and PDGFRß/PI3/Akt signal pathway.

Kaemperfol Protects Dopaminergic Neurons by Promoting mTOR-Mediated Autophagy in Parkinson's Disease Models.

We previously showed that kaempferol (KAE) could exert neuroprotective effects against PD. It has been demonstrated that abnormal autophagy plays a key role in the development of PD. Mitochondrial dysfunction, involved in the development of PD, can damage dopaminergic neurons. Whether the protective effects of KAE were exerted via regulating autophagy remains largely undefined, however. This study aimed to investigate whether KAE could protect dopaminergic neurons via autophagy and the underlying mechanisms using a MPTP/MPP+-stimulated PD model. Cell viability was detected by cell counting kit-8 (CCK-8) assay, and protein levels of autophagy mediators along with mTOR signaling pathway molecules were investigated by immunohistochemistry and Western blot analyses. The results showed that KAE could ameliorate the behavioral impairments of mice, reduce the loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta, and reduce α-synuclein (α-syn) levels. Furthermore, KAE upregulated levels of autophagy effector protein of Beclin-1 and autophagy microtubule associated protein of light chain 3 (LC3) in the substantia nigra (SN) while rescuing mitochondrial integrity, and downregulated levels of ubiquitin binding protein p62 and cleaved caspase-3, probably by decreasing the mammalian target of rapamycin (mTOR) signaling pathway. Further in vitro experiments demonstrated similar results. In conclusion, KAE exerts neuroprotective effects against PD potentially by promoting autophagy via inhibiting the mTOR signaling pathway.

1,25-dihydroxyvitamin D3 ameliorates lupus nephritis through inhibiting the NF-κB and MAPK signalling pathways in MRL/lpr mice.

BACKGROUND: Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus (SLE). However, the aetiology and pathogenesis of LN remain unknown. 1,25-dihydroxyvitamin D3 [1,25-(OH)2-VitD3] is the active form of vitamin D, and it has been shown to perform important functions in inflammatory and immune-related diseases. In this study, we investigated the time-dependent effects of 1,25-dihydroxyvitamin D3 and explored the underlying mechanism in MRL/lpr mice, a well-studied animal model of LN. METHODS: Beginning at 8 weeks of age, 24-h urine samples were collected weekly to measure the levels of protein in the urine. We treated female MRL/lpr mice with 1,25-dihydroxyvitamin D3 (4 µg/kg) or 1% DMSO by intraperitoneal injection twice weekly for 3 weeks beginning at the age of 11 weeks. The mice were separately sacrificed, and serum and kidney samples were collected at the ages of 14, 16, 18, and 20 weeks to measure creatinine (Cr) levels, blood urea nitrogen (BUN) levels, histological damage, immunological marker (A-ds DNA, C1q, C3, IgG, IgM) levels, and inflammatory factor (TNF-α, IL-17, MCP-1) levels. Furthermore, the nuclear factor kappa B (NF-κB) and the mitogen-activated protein kinase (MAPK) signalling pathways were also assessed to elucidate the underlying mechanism. RESULTS: We found that MRL/lpr mice treated with 1,25-dihydroxyvitamin D3 displayed significantly attenuated LN. VitD3-treated mice exhibited significantly improved renal pathological damage and reduced proteinuria, BUN, SCr, A-ds DNA antibody and immune complex deposition levels (P < 0.05) compared with untreated MRL/lpr mice. Moreover, 1,25-dihydroxyvitamin D3 inhibited the complement cascade, inhibited the release of proinflammatory cytokines, such as TNF-α, IL-17, and MCP-1, and inhibited NF-κB and MAPK activation (P < 0.05). CONCLUSION: 1,25-dihydroxyvitamin D3 exerts a protective effect against LN by inhibiting the NF-κB and MAPK signalling pathways, providing a potential treatment strategy for LN. Interestingly, the NF-κB and MAPK signalling pathways are time-dependent mediators of LN and may be associated with lupus activity.

Kaemperfol alleviates pyroptosis and microglia-mediated neuroinflammation in Parkinson's disease via inhibiting p38MAPK/NF-κB signaling pathway.

The study aims to investigate whether kaemperfol (KAE) inhibits microglia pyroptosis and subsequent neuroinflammatory response to exert neuroprotective effects, along with the underlying mechanisms. The results showed KAE could ameliorate the behavioral deficits of Parkinson's disease (PD) rats, inhibit the activation of microglia and astrocytes, reduce the loss of TH-positive neurons, down-regulate levels of pyroptosis-related NOD-like receptor family pyrin domain containing 3 (NLRP3), GasderminD-N Term (GSDMD-NT), caspase1, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), interleukin (IL)-1ß, and IL-18, and decrease the levels of inflammatory molecules (inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)) and p38 mitogen-activated protein kinase/nuclear factor-kappaB (p38MAPK/NF-κB) signaling pathway molecules (p38MAPK, p-p38MAPK, NF-κB, and p-NF-κB) in the substantia nigra of PD rats. Further in vitro study indicated that KAE reversed the activation of BV2 cells and down-regulated the expressions of pyrolytic proteins, inflammatory mediators and key molecules in p38MAPK/NF-κB signaling pathway. Collectively, KAE inhibits the microglia pyroptosis and subsequent neuroinflammatory response to exert neuroprotective effects on 6-hydroxydopamine (6-OHDA)-induced PD rats and lipopolysaccharide (LPS)-induced BV2 inflammatory cells through inhibiting p38MAPK/NF-κB signaling pathway.

Polyphenols from Toona sinensiss Seeds Alleviate Neuroinflammation Induced by 6-Hydroxydopamine Through Suppressing p38 MAPK Signaling Pathway in a Rat Model of Parkinson's Disease.

Polyphenols from Toona sinensis seeds (PTSS) have demonstrated anti-inflammatory effects in various diseases, while the anti-neuroinflammatory effects still remain to be investigated. We aimed to investigate the effects of PTSS on Parkinson's disease and underlying mechanisms using a rat model. We employed 6-hydroxydopamine (6-OHDA) to male Sprague Dawley (SD) rats and PC12 cells to construct the in vivo and vitro models of PD and dopaminergic (DA) neuron injury, respectively. Cell viability was detected by cell counting kit-8 (CCK-8) assay and protein levels of inflammatory mediators and some p38 MAPK pathway molecules were investigated by immunohistochemistry and Western blot analyses. The results showed that 6-OHDA significantly increased protein levels of inflammatory mediators, such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and tumor necrosis factor α (TNF-α), which could be reversed by PTSS through suppressing the p38 MAPK pathway. The anti-inflammatory effects of PTSS were significantly enhanced by the specific p38 inhibitor of SB203580 in vitro. The present work suggests that PTSS can exert anti-inflammatory effects on PD models, which may be attributed to the suppression of p38 MAPK signaling pathway.

The lentiviral-mediated Nurr1 genetic engineering mesenchymal stem cells protect dopaminergic neurons in a rat model of Parkinson's disease.

Nuclear receptor-related factor 1 (Nurr1) has a crucial role in the development and maturation of mesencephalic dopamine (DA) neurons and also plays a protective role in maintenance of DA neurons by inhibiting the activation of microglia and astrocyte. Moreover, the mutations in Nurr1 gene are associated with familial Parkinson's disease (PD), suggested that Nurr1 modulation is a potential therapeutic target for PD. This study examines the therapeutic effects of transplantation of Nurr1 gene-modified bone marrow mesenchymal stem cells (MSCs) on 6-hydroxydopamine (6-OHDA)-induced PD rat models. MSCs were transduced with lentivirus expressing Nurr1 gene and then intrastriatally transplanted into PD rats. Our results showed that Nurr1 gene-modified MSCs overexpress and secrete Nurr1 protein in vitro and also survive and migrate in the brain. Four weeks after transplantation Nurr1 gene-modified MSCs dramatically ameliorated the abnormal behavior of PD rats and increased the numbers of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum, inhibited the activation of glial cells, and reduced the expression of inflammatory factors in the SN. Taken together, these findings suggest that intrastriatal transplantation of lentiviral vector mediated Nurr1 gene-modified MSCs has notable therapeutic effect for PD rats.

Nrf2-ARE signals mediated the anti-oxidative action of electroacupuncture in an MPTP mouse model of Parkinson's disease.

Oxidative stress and neuroinflammation are early events associated with dopaminergic neuronal degeneration in Parkinson's disease (PD). Previous studies indicated that electroacupuncture (EA) stimulation is effective in protecting dopaminergic neurons from degeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. In this study, we further characterized the effect of EA on MPTP-induced oxidative responses in the mouse dopamine system. We found that subacute administration of MPTP enhanced lipid and protein oxidation and reduced expression of endogenous antioxidant enzymes (such as superoxide dismutase and catalase) in the striatum. MPTP also reduced expression of an antioxidant transcription factor, nuclear factor-E2-related factor-2 (Nrf2), and Nrf2-regulated antioxidant enzymes (nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1 and heme oxygenase-1) in the striatum and/or midbrain. Using human placental alkaline phosphatase (hPAP) as a reporter of Nrf2-regulated gene expression in hPAP transgenic mice, we found that MPTP suppressed hPAP expression in the striatum and midbrain. Application of EA at an effective frequency (100 Hz) was sufficient to reverse these changes induced by MPTP. In addition, EA reduced microglia activation and astrogliosis in the striatum and midbrain, increased tyrosine hydroxylase levels in the striatum, and improved vertical movement in MPTP mice. These results provide further evidence supporting that EA produces a series of anti-oxidative effects that effectively counteract with the oxidative stress in the nigrostriatal dopamine system induced by MPTP in a mouse model of PD.

TGF-ß1 related inflammation in the posterior longitudinal ligament of cervical spondylotic myelopathy patients.

AIM: This study aimed to elucidate the pathogenesis of posterior longitudinal ligament (PLL) hypertrophy. METHODS: Cervical PLL specimens were collected from CSM patients during surgery (n = 30) and during routine autopsy (n = 14), and processed for histological examination (HE staining and Masson's Trichrome staining) and IHC (CD3, CD68, CD31, TGF-ß1 and collagen II). In addition, the mRNA expression of collagen I was detected in cervical PLL specimens from 16 CSM patients (n = 16) and from routine autopsy (n = 16) by RT-PCR. RESULTS: Obvious fibrosis, cartilage metaplasia and calcification were found in the cervical PLL of CSM patients. In the degenerated PLL, CD68(+) macrophages were frequently identified, CD3(+) T lymphocytes were occasionally found, and many newly generated small vessels were also present. In the degenerated PLL, of the number of TGF-ß1 positive cells increased markedly when compared with control group. IHC indicated TGF-ß1 was secreted by macrophages. RT-PCR showed a significantly lower mRNA expression of collagen I in the PLL of CSM patients as compared to control group. CONCLUSIONS: Macrophages are the major type of inflammatory cells involved in the cervical PLL degeneration, and TGF-ß1 is related to the cervical PLL degeneration. TGF-ß1 is mainly secreted by macrophages. Anti-inflammation may serve as an alternative non-surgical treatment and prophylactic strategy for PLL degeneration.

Electroacupuncture remediates glial dysfunction and ameliorates neurodegeneration in the astrocytic α-synuclein mutant mouse model.

BACKGROUND: The acupuncture or electroacupuncture (EA) shows the therapeutic effect on various neurodegenerative diseases. This effect was thought to be partially achieved by its ability to alleviate existing neuroinflammation and glial dysfunction. In this study, we systematically investigated the effect of EA on abnormal neurochemical changes and motor symptoms in a mouse neurodegenerative disease model. METHODS: The transgenic mouse which expresses a mutant α-synuclein (α-syn) protein, A53T α-syn, in brain astrocytic cells was used. These mice exhibit extensive neuroinflammatory and motor phenotypes of neurodegenerative disorders. In this study, the effects of EA on these phenotypic changes were examined in these mice. RESULTS: EA improved the movement detected in multiple motor tests in A53T mutant mice. At the cellular level, EA significantly reduced the activation of microglia and prevented the loss of dopaminergic neurons in the midbrain and motor neurons in the spinal cord. At the molecular level, EA suppressed the abnormal elevation of proinflammatory factors (tumor necrosis factor-α and interleukin-1ß) in the striatum and midbrain of A53T mice. In contrast, EA increased striatal and midbrain expression of a transcription factor, nuclear factor E2-related factor 2, and its downstream antioxidants (heme oxygenase-1 and glutamate-cysteine ligase modifier subunits). CONCLUSIONS: These results suggest that EA possesses the ability to ameliorate mutant α-syn-induced motor abnormalities. This ability may be due to that EA enhances both anti-inflammatory and antioxidant activities and suppresses aberrant glial activation in the diseased sites of brains.

Gene expression profiling analysis of locus coeruleus in idiopathic Parkinson's disease by bioinformatics.

This study aimed to explore the underlying molecular mechanisms of idiopathic Parkinson's disease (IPD) by bioinformatics analysis. Gene expression profile GSE34516 was downloaded from the Gene Expression Omnibus. Eight locus coeruleus post-mortem tissue samples derived from four IPD patients and four neurological healthy controls were used to identify the differentially expressed genes (DEGs) by paired t test. Based on the DEGs, principal components were analyzed. The Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes pathway analysis of the genome microarray data were then performed. Finally, protein-protein interaction (PPI) network of the DEGs was constructed. Total 261 DEGs including 195 up-regulated and 66 down-regulated DEGs were identified. Intracellular protein transport and RNA splicing via transesterification reactions were selected as the most two significantly enriched functions. Mismatch repair, N-glycan biosynthesis, spliceosome and nucleotide excision repair were the significantly enriched pathways. In the PPI network, CTSS, CD53, IGSF6, PTPRC and LAPTM5 were the hub nodes. Intracellular protein transport and RNA splicing via transesterification reactions were closely associated with IPD. The DEGs, such as CX3CR1, SLC5A7, CD53 and PTPRC may be the potential targets for IPD diagnosis and treatment.

An effective inducer of dopaminergic neuron-like differentiation.

Rat bone marrow-derived mesenchymal stem cells were cultured and passaged in vitro. After induction with basic fibroblast growth factor for 24 hours, passage 3 bone marrow-derived mesenchymal stem cells were additionally induced into dopaminergic neurons using three different combinations with basic fibroblast growth factor as follows: 20% Xiangdan injection; all-trans retinoic acid + glial-derived neurotrophic factor; or sonic hedgehog + fibroblast growth factor 8. Results suggest that the bone marrow-derived mesenchymal stem cells showed typical neuronal morphological characteristics after induction. In particular, after treatment with sonic hedgehog + fibroblast growth factor 8, the expressions of nestin, neuron-specific enolase, microtubuleassociated protein 2, tyrosine hydroxylase and vesicular monoamine transporter-2 in cells were significantly increased. Moreover, the levels of catecholamines in the culture supernatant were significantly increased. These findings indicate that Xiangdan injection, all-trans retinoic acid + glial-derived neurotrophic factor, and sonic hedgehog + fibroblast growth factor 8 can all induce dopaminergic neuronal differentiation from bone marrow-derived mesenchymal stem cells. In particular, the efficiency of sonic hedgehog + fibroblast growth factor 8 was highest.