The gut microbiota metabolite butyrate mitigates MPTP/MPP+ -induced Parkinson's disease by inhibiting the JAK2/STAT3 signaling pathway.

Butyrate (BU), a gut microbiota-derived metabolite, has been reported to play a neuroprotective role in Parkinson's disease (PD). However, the specific molecular mechanism of BU has not been fully interpreted. This work aimed to verify the protective effects of BU against MPTP/MPP+ -induced neurotoxicity and explore the mechanisms involved. The results showed that BU protected against MPTP-induced motor dysfunction and decreased tyrosine hydroxylase (TH) and dopamine transporter (DAT) levels. Additionally, BU pretreatment improved PC12 cell viability and reduced MPP+ -induced PC12 cell apoptosis. BU treatment also attenuated MPP+ -stimulated oxidative stress and inflammatory response in PC12 cells. Furthermore, BU inhibited MPTP/MPP+ -induced hyperactivation of the JAK2/STAT3 signaling in mice and PC12 cells. Besides, a JAK2 agonist, Coumermycin A1 (C-A1), substantially reversed BU-mediated inhibition on JAK2/STAT3 phosphorylation in MPP+ -challenged PC12 cells and abated BU-induced repression on MPP+ -triggered apoptosis, oxidative stress, and inflammatory response in PC12 cells. To sum up, BU might exert neuroprotective effects against MPP+ /MPTP-induced neurotoxicity by inactivating the JAK2/STAT3 signaling.

The value of serum Lp-PLA2 combined with MPO in the diagnosis of cerebral infarction caused by large artery atherosclerosis.

OBJECTIVE: To explore the value of serum lipoprotein-associated phospholipase A2（Lp-PLA2）combined with myeloperoxidase（MPO）for the diagnosis of large artery atherosclerosis(LAA) cerebral infarction. METHODS: Baseline data were collected from patients with first-ever acute cerebral infarction, serum Lp-PLA2 and MPO levels were measured. The etiology of cerebral infarction was classified according to the Chinese Ischemic Stroke Subtype Classification Standard. The risk factors associated with LAA cerebral infarction were identified by univariate and multivariate regression analysis. The diagnostic value of serum Lp-PLA2 and MPO for LAA cerebral infarction was assessed by the area under the receiver-operating characteristic (ROC) curve. RESULTS: Overall 368 patients were involved, 148 patients (40.22 %) were LAA. The serum La-PLA2 and MPO levels were higher in the LAA group than those in non-LAA group (23.06 ± 3.39 ng/mL versus 17.48 ± 3.26 ng/mL; 93.60 ± 9.58 ng/mL versus 75.98 ± 15.53 ng/mL; P < 0.001 for both). Multivariate analysis showed that elevated levels of serum Lp-PLA2 (OR 1.742, 95 %CI 1.499-2.025; P < 0.001) and MPO (OR 1.060, 95 % CI 1.026-1.096; P = 0.001) were the independent risk factors of LAA cerebral infarction. The area under curve of the serum Lp-PLA2 combined with MPO for the diagnosis of LAA cerebral infarction was 0.896 [0.866 ∼ 0.927] (P < 0.001). CONCLUSION: Serum Lp-PLA2 combined with MPO could be valued as a predictor of acute cerebral infarction caused by large artery atherosclerosis.

Arteriolosclerosis CSVD: a common cause of dementia and stroke and its association with cognitive function and total MRI burden.

Objective: Arteriolosclerosis cerebral small vessel disease (CSVD) is a common type of CSVD. This study aimed to explore the factors associated with cognitive function and total MRI burden related to the disease. Methods: The demographic characteristics, clinical manifestations, cognitive function score, Barthel Index (BI), blood test index, and follow-up results of arteriolosclerosis CSVD patients treated for the first time in our hospital from January 2014 to August 2022 were collected. White matter hyperintensity (WMH) Fazekas score, total MRI burden, and cerebral atrophy grade were evaluated according to brain MRI findings. Factors associated with CSVD cognitive function were analyzed by binary logistic regression. The correlative factors related to the total MRI burden of CSVD were analyzed by ordered multiple logistic regression. Results: A total of 146 patients were included in this study, of which 132 cases (90.4%) had hypertension. There were 108 patients (74.0%) with cognitive dysfunction, 97 patients (66.4%) with balance and gait disorders, and 83 patients (56.8%) with moderate-to-severe dependence in daily life (BI ≤ 60 points). Of 146 patients, 79 (54.1%) completed clinical and imaging follow-ups for a median of 3 years. The number of patients with cognitive impairment and BI ≤ 60 points after follow-up significantly increased compared with the first admission (P < 0.001). There were also significant differences in total MRI burden (P = 0.001), WMH Fazekas score, and cerebral atrophy grade (P < 0.001). Mean age (P = 0.012), median deep WMH Fazekas score (P = 0.028), and median deep (P < 0.001) and superficial (P =0.002) cerebral atrophy grade of patients with cognitive impairment at first admission were all higher than those with non-cognitive impairment. Multivariate analysis showed that deep cerebral atrophy was independently and significantly associated with cognitive impairment of CSVD (P = 0.024), and hypertension was significantly and independently associated with total MRI burden (P = 0.001). Conclusion: The disease course of arteriolosclerosis CSVD may be related to cognitive function and total MRI burden. Deep cerebral atrophy was an independent risk factor for cognitive dysfunction in arteriolosclerosis CSVD, and hypertension was an independent risk factor for total MRI burden.

A retrospective study of individualized endovascular treatment for symptomatic intracranial atherosclerotic stenosis in patients with ischemic stroke/transient ischemic attack.

Background: Endovascular treatment (EVT) is one of the effective treatment procedure for the symptomatic intracranial atherosclerotic stenosis (sICAS). Aim and methods: We evaluated the efficacy and safety of individualized endovascular treatment for sICAS patients. Clinical and imaging follow-ups were carried out to collect the data of 29 sICAS patients after 6 months of individualized endovascular treatment. Different treatment strategies are selected based on arterial access and lesion morphology of patients. If standard surgical path, narrow artery straight, stenosis length ≤10 mm, then the appropriate specifications of balloon-mounted stent (BMS) treatment. the surgical path is tortuous, the narrow artery is curved, the angle is apparent, the diameter of the near and far ends is significantly different, or the length of the stenosis is >10 mm, self-expanding stent (SES) with appropriate specifications is selected for treatment. If the narrowed artery is hyper flexed and the surgeon deems stenting inappropriate, balloon dilation angioplasty (BDA) treatment is chosen. Results and conclusion: 31 lesions of 29 sICAS patients received endovascular treatment. The median age was 61 years (IQR 54-69 years). The median preoperative stenosis was 90% (IQR 80-95%), and the mean stenosis length was (8.10 ± 3.27) mm. The most commonly used surgical procedure was Balloon-Mounted Stent (BMS) in 19 cases (65.52%), Self-expanding Stent (SES) in seven cases (24.14%), Balloon Dilation Angioplasty (BDA) in three cases (10.34%). (11.86 + 1.46 mm) was greater than that in the BMS group (6.14 + 1.59 mm) (P < 0.001). The median stenosis was 90% (IQR 80-92.5%) in the BMS group, lower than 99% (IQR 95-100%) in the SES group (P < 0.001). The median post-operative residual stenosis was 20% (IQR 15-25%), significantly improved compared with preoperative (P < 0.001). The success rate of the surgical technique was 93.10% (27/29). One patient (3.45%) had IS recurrence within 48 h after surgery, and the restenosis rate within 6 months after surgery was 6.90% (2/29). No patient died or had recurrent IS. Our data demonstrated that individualized endovascular treatment method could be potentially significant and safe for sICAS patients. This study will provide an important reference for the endovascular treatment of sICAD.

High serum OX40 ligand correlates with severity and mortality in patients with massive cerebral infarction.

OX40 ligand (OX40L) is a member of tumor necrosis factors (TNF)/TNFR superfamily and is mainly expressed in activated T cells and participates in various inflammatory reactions. However, it remains unclear about the role of serum OX40L as a biomarker of cerebral infarction (CI). This study aimed to explore the possibility of serum OX40L as a meaningful predictor in mortality of CI. Severe CI patients were included to collect clinicopathological and laboratory data and measure serum OX40L level. Patients were followed up after discharge and 60-day survival rate was used as the study endpoint. The results showed that of all 294 patients, 123 (41.8%) died within 60 days after admission. Serum OX40L levels were significantly higher in patients with severe CI compared to healthy controls, and were significantly higher in nonsurvivors compared to survivors (P < .05). The levels of OX40L were correlated with Glasgow Coma Scale score, serum creatinine and high-sensitive C-reactive protein. Multivariate logistic regression analysis showed that serum OX40L level was an independent prognostic factor for 60-day mortality, after control of pulmonary infection, glasgow coma scale score and high-sensitive C-reactive protein (odds ratio = 1.089; 95% confidence interval = 1.053-1.126; P < .001). The receiver operating characteristic (ROC) curve was used to predict the best cut-off of serum OX40L for 60-day survival as 35.5 ng/mL. Patients with high serum OX40L levels (>35.5 ng/mL) had a significantly higher mortality within 60 days (hazard ratio = 2.885; 95% confidence interval = 1.901-4.378). In conclusion, OX40L is a serum biomarker of patients with CI and associated with severity and mortality of this disease.

[Relationship between PANTR1 and Imatinib Resistance of Chronic Myeloid Leukemia Cell Line K562 and Its Related Mechanisms].

OBJECTIVE: To investigate the relationship between long non-coding RNA (LncRNA) PANTR1 and imatinib resistance in chronic myeloid leukemia cell line K562 and its mechanism. METHODS: K562 control cells (Control) and K562 imatinib resistant cells (ImR) were cultured. Two siRNA vectors targeting PANTR1 and control vectors were transfected into K562-ImR cells by lentivirus as ImR-siPA#1, ImR-siPA#2 and ImR-siControl cells, respectively. Imatinib semi-inhibitory concentration (IC50) was detected by CCK-8 kit. The expression level of PANTR1, BCR/ABL, MDR, CD44 and CD133 mRNA were detected by fluorescence quantitative PCR（RT-qPCR）, and the expression level of BCR/ABL, MDR, CD44 and CD133 protein were detected by Western blot. RESULTS: Imatinib IC50 in ImR cells was significantly higher than that in control cells (P＜0.01), that of ImR-siPA#1 and ImR-siPA#2 cells were significantly lower than that in ImR-sicontrol cells (P＜0.01), but still significantly higher than that in control cells (P＜0.01). The mRNA expression level of PANTR1, BCR/ABL, MDR, CD44 and CD133 in ImR cells were significantly higher than those in control cells (P＜0.01). The mRNA expression level of PANTR1, MDR, CD44 and CD133 in ImR-siPA#1 and ImR-siPA#2 cells were significantly lower than those in ImR-siControl cells (P＜0.01), while the expression level of BCR/ABL mRNA was not significantly different (P＞0.05). The protein expression level of BCR/ABL, MDR, CD44 and CD133 in ImR cells were significantly higher than those in control cells, while the protein expression level of MDR, CD44 and CD133 in ImR-siPA#1 and ImR-siPA#2 were significantly lower than those in ImR-siControl cells. CONCLUSION: LncRNA PANTR1 can promote the expression of MDR and stem cell marker in chronic myeloid leukemia cell line K562, and mediate imatinib resistance.

Cestrum nocturnum Flower Extracts Attenuate Proliferation and Induce Apoptosis in Malignant Cells through Inducing DNA Damage and Inhibiting Topoisomerase II Activity.

Most of the existing chemotherapeutic drugs have plenty of side effects. Chinese herbal medicine has been used for pharmaceutical and dietary therapy for thousands of years with more effective and fewer side effects. Cestrum nocturnum (CN) has long been used to treat digestive diseases for centuries in China. Our previous study first proved that the n-butanol part isolated from the flowers of CN produced an inhibitory effect on the proliferation of malignant cells. However, the fractions responsible for the antiproliferation effect of n-butanol part from CN flowers and related mechanisms remain unknown. Thus, in this study, we extracted fractions C4 and C5 from n-butanol part of CN flowers and investigated their immune toxicity and antitumor activities. It was found that fractions C4 and C5 exhibited great cytotoxicity to cancer cell lines but had low immune toxicity towards T and B lymphocytes in vitro. The tested fractions also attenuated proliferation and induced apoptosis at G0/G1 and G2/M phases in Bel-7404 cells through inducing DNA damage and inhibiting topoisomerase II relaxation activity. These results suggest that fractions C4 and C5 may represent important sources of potential antitumor agents due to their pronounced antitumor effects and low immune toxicity.

Izalpinin from fruits of Alpinia oxyphylla with antagonistic activity against the rat bladder contractility.

BACKGROUND: Alpinia oxyphylla (Zingiberaceae), an herbaceous perennial plant, its capsular fruit is commonly used in traditional Chinese medicine for the treatment of different urinary incontinence symptoms including frequency, urgency and nocturia. These symptoms are similar to the overactive bladder syndrome. In our lab, we found that the 95% ethanol extract of the capsular fruits exhibited significant anti-muscarinic activity. Some constituents in capsular fruits including flavonoids (e.g., izalpinin and tectochrysin), diarylheptanoids (e.g., yakuchinone A and yakuchinone B) and sesquiterpenes (e.g., nootkatone), are regarded as representative chemicals with putative pharmacological activities. OBJECTIVE: This study aimed to evaluate the in vitro antagonistic actions of izalpinin on carbachol-induced contraction of the rat detrusor muscle. MATERIALS AND METHODS: In vitro inhibition of rat detrusor contractile response to carbachol was used to study the functional activity of izalpinin. The isolated detrusor strips of rats were mounted in organ baths containing oxygenated Krebs' solution. The cumulative consecutive concentration-response curves to carbachol-evoked contractions in strips of rat bladder were obtained. RESULTS: Carbachol induced concentration-dependent contractions of isolated rat bladder detrusor strips. The vehicle DMSO had no impact on the contraction response. The contraction effects were concentration-dependently antagonized by izalpinin, with a mean EC50 value of 0.35 µM. The corresponding cumulative agonist concentration-response curves shifted right-ward. CONCLUSIONS: Izalpinin exhibits inhibitory role of muscarinic receptor-related detrusor contractile activity, and it may be a promising lead compound to treat overactive bladder.

Iridovirus Bcl-2 protein inhibits apoptosis in the early stage of viral infection.

The grouper iridovirus (GIV) belongs to the family Iridoviridae, whose genome contains an antiapoptotic B-cell lymphoma (Bcl)-2-like gene. This study was carried-out to understand whether GIV blocks apoptosis in its host. UV-irradiated grouper kidney (GK) cells underwent apoptosis. However, a DNA fragmentation assay of UV-exposed GK cells after GIV infection revealed an inhibition of apoptosis. The UV- or heat-inactivated GIV failed to inhibit apoptosis, implying that a gene or protein of the viral particle might contribute to an apoptosis inhibitory function. The DNA ladder assay for GIV-infected GK cells after UV irradiation confirmed that apoptosis inhibition was an early process which occurred as early as 5 min post-infection. A GIV-Bcl sequence comparison showed distant sequence similarities to that of human and four viruses; however, all possessed the putative Bcl-2 homology (BH) domains of BH1, BH2, BH3, and BH4, as well as a transmembrane domain. Northern blot hybridization showed that GIV-Bcl transcription began at 2 h post-infection, and the mRNA level significantly increased in the presence of cycloheximide or aphidicolin, indicating that this GIV-Bcl is an immediate-early gene. This was consistent with the Western blot results, which also revealed that the virion carries the Bcl protein. We observed the localization of GIV-Bcl on the mitochondrial membrane and other defined intracellular areas. By immunostaining, it was proven that GIV-Bcl-expressing cells effectively inhibited apoptosis. Taken together, these results demonstrate that GIV inhibits the promotion of apoptosis by GK cells, which is mediated by the immediate early expressed viral Bcl gene.

Expression pattern of metallothionein, MTF-1 nuclear translocation, and its dna-binding activity in zebrafish (Danio rerio) induced by zinc and cadmium.

Metallothionein is a small (6-kDa), cysteine-rich protein expressed by a six-zinc finger protein called metal-responsive element-binding transcription factor-1 (MTF-1) in response to Zn and Cd. Our previous reports have shown the basal expression of metallothionein (mt) and MTF-I (mtf-1) genes in embryo and early larval stages of zebrafish (Danio rerio). In the present study, we investigated the mt expression in zebrafish early larvae induced by exposure to Cd and Zn (48, 72, 96, and 120 h postfertilization). Whole-mount in situ hybridization showed that Zn induced mt expression in the olfactory pit, cerebellum, ceratobranchials, liver, chloride cells, and neuromasts of the lateral line. Cadmium also induced mt expression in all the above regions except the cerebellum. Using fluorescence techniques, we have shown that Zn and Cd mediate cytoplasmic and nuclear translocation of MTF- 1-enhanced green fluorescent protein fusion protein in zebrafish liver cell line. The MTF-1 protein was produced recombinantly by inserting zebrafish mtf-1 cDNA (1.8 kb) into pET-20b(+) expression vector and expressing in Escherichia coli BL21 (DE3) pLysS host strain competent cell on induction with isopropyl-beta-D-thiogalactopyranoside. The protein was then purified by affinity chromatography on a nickel-nitrilotriacetic acid column. Electrophoretic mobility shift assay revealed binding of the recombinant MTF-1 in response to Zn and Cd at the putative metal-responsive elements (MREs) in the promoter region of the mt gene. Taken together, these results suggest that Zn and Cd are efficiently involved with mt expression induced in zebrafish embryos and with MTF-1 nuclear translocation and that this induction is achieved through the activation of MTF-1 binding at the MREs.

Expression of metallothionein gene during embryonic and early larval development in zebrafish.

Metallothionein (Mt) has been considered as a molecular marker of metal pollution in aquatic ecosystems. Less is known about the expression of mt gene during embryogenesis. Here, we report the cloning, sequencing, and the expression pattern of mt gene during developmental stages in zebrafish. The zebrafish embryogenesis when takes place in a medium containing a dosage of 1000 microM zinc resulted in high mortality, indicating the deleterious effect of zinc on development. The zebrafish mt gene consists of three exons encoding 60 amino acids with 20 conserved cysteine residues. RT-PCR result indicates the maternal contribution of Mt transcripts. Using digoxigenin (DIG)-labeled anti-sense RNA probe, whole-mount in situ hybridization was performed to observe the expression pattern of zebrafish mt gene during embryonic and early larval stages. Stronger as well as ubiquitous expression of mt gene during early embryonic stages narrowed to specific expression after hatching. The mt promoter region contains seven copies of putative metal-responsive elements (MREs), which are shown to be important for the high level activity by deletion analysis. The expression of mt gene during embryogenesis implies its significant role on development.

In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio).

Mammalian liver fatty acid binding protein (L-FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid trafficking and metabolism. To better understand its tissue-specific regulation during zebrafish hepatogenesis, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a green fluorescent protein (GFP) transgenic strategy to generate liver-specific transgenic zebrafish. The 2.8-kb 5'-flanking sequence of zebrafish L-FABP gene was sufficient to direct GFP expression in liver primordia, first observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42-51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp-1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development.

Molecular cloning and developmental expression of zinc finger transcription factor MTF-1 gene in zebrafish, Danio rerio.

Metal-responsive transcription factor, MTF-1 is a zinc finger protein, shown to be essential for embryonic development. Homozygous knockout mouse embryos for MTF-1 die in utero at day 14 of gestation, due to liver decay. In the present study, we report the complete nucleotide sequence of cDNA encoding zebrafish MTF-1 and the amino acid sequence similarity with that of mouse, human, fish and Drosophila. The size of the zebrafish MTF-1 cDNA is 3,379 bp and the coding region (1,779 bp) encodes a polypeptide of 593 amino acids. The putative zinc finger and transactivation domains comprised by zebrafish MTF-1 were also determined. The zebrafish MTF-1 shows high identity of 97, 93, 93 and 67% in the DNA binding zinc finger domain and 51, 44, 48 and 20% overall identity with fugu, human, mouse and Drosophila, respectively. RT-PCR results show the maternal expression of MTF-1 transcripts. The pattern of MTF-1 gene expression during embryonic and early larval development was studied by whole-mount in situ hybridization using DIG-labeled anti-sense RNA probe. Stronger and ubiquitous expression was observed during the embryonic stages whereas, specific expression, especially in the neural parts, was observed throughout the stages studied after hatching.