Identification of the Hub Genes and the Signaling Pathways in Human iPSC-Cardiomyocytes Infected by SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) is an enveloped single-stranded RNA virus that can lead to respiratory symptoms and damage many organs such as heart, kidney, intestine, brain and liver. It has not been clearly documented whether myocardial injury is caused by direct infection of cardiomyocytes, lung injury, or other unknown mechanisms. The gene expression profile of GSE150392 was obtained from the Gene Expression Omnibus (GEO) database. The processing of high-throughput sequencing data and the screening of differentially expressed genes (DEGs) were implemented by R software. The R software was employed to analyze the Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The protein-protein interaction (PPI) network of the DEGs was constructed by the STRING website. The Cytoscape software was applied for the visualization of PPI network and the identification of hub genes. The statistical analysis was performed by the GraphPad Prism software to verify the hub genes. A total of 516 up-regulated genes and 191 down-regulated genes were screened out. The top 1 enrichment items of GO in biological process (BP), Cellular Component (CC), and Molecular Function (MF) were type I interferon signaling pathway, sarcomere, and receptor ligand activity, respectively. The top 10 enrichment pathways, including TNF signaling pathway, were identified by KEGG enrichment analysis. A PPI network was established, consisting of 613 nodes and 3,993 edges. The 12 hub genes were confirmed as statistically significant, which was verified by GSE151879 dataset. In conclusion, the hub genes of human iPSC-cardiomyocytes infected with SARS-CoV-2 were identified through bioinformatics analysis, which may be used as biomarkers for further research.

Methyl 3,4-Dihydroxybenzoate Enhances Resistance to Oxidative Stressors and Lifespan in C. elegans Partially via daf-2/daf-16.

Genetic studies have elucidated mechanisms that regulate aging; however, there has been little progress in identifying drugs that retard ageing. Caenorhabditis elegans is among the classical model organisms in ageing research. Methyl 3,4-dihydroxybenzoate (MDHB) can prolong the life-span of C. elegans, but the underlying molecular mechanisms are not yet fully understood. Here, we report that MDHB prolongs the life-span of C. elegans and delays age-associated declines of physiological processes. Besides, MDHB can lengthen the life-span of eat-2 (ad1113) mutations, revealing that MDHB does not work via caloric restriction (CR). Surprisingly, the life-span⁻extending activity of MDHB is completely abolished in daf-2 (e1370) mutations, which suggests that daf-2 is crucial for a MDHB-induced pro-longevity effect in C. elegans. Moreover, MDHB enhances the nuclear localization of daf-16/FoxO, and then modulates the expressions of genes that positively correlate with defenses against stress and longevity in C. elegans. Therefore, our results indicate that MDHB at least partially acts as a modulator of the daf-2/daf-16 pathway to extend the lifespan of C. elegans, and MDHB might be a promising therapeutic agent for age-related diseases.

Neuroprotective Effects of Methyl 3,4-Dihydroxybenzoate against TBHP-Induced Oxidative Damage in SH-SY5Y Cells.

This study investigated the neuroprotective effects of methyl 3,4-dihydroxybenzoate (MDHB) against t-butyl hydroperoxide (TBHP) induced oxidative damage in SH-SY5Y (human neuroblastoma cells) and the underlying mechanisms. SH-SY5Y were cultured in DMEM + 10% FBS for 24 h and pretreated with different concentrations of MDHB or N-acetyl-l-cysteine (NAC) for 4 h prior to the addition of 40 µM TBHP for 24 h. Cell viability was analyzed using the methylthiazolyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays. An annexin V-FITC assay was used to detect cell apoptosis rates. The 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay was used to determine intracellular ROS levels. The activities of antioxidative enzymes (GSH-Px and SOD) were measured using commercially available kits. The oxidative DNA damage marker 8-OHdG was detected using ELISA. Western blotting was used to determine the expression of Bcl-2, Bax, caspase 3, p-Akt and Akt proteins in treated SH-SY5Y cells. Our results showed that MDHB is an effective neuroprotective compound that can mitigate oxidative stress and inhibit apoptosis in SH-SY5Y cells.

Neuroprotective effects of methyl 3,4-dihydroxybenzoate against H2O2-induced apoptosis in RGC-5 cells.

In the present study, we investigated the protective effect of methyl 3,4-dihydroxybenzoate (MDHB) against H2O2-induced apoptosis in RGC-5 cells. The RGC-5 cells were cultured in plates for 24 h, which were then pretreated with dimethyl sulfoxide, different concentrations of MDHB, or probucol for 12 h prior to addition of 300 µM H2O2 for 24 h. The cell viability was detected by MTT assay. The rate of apoptosis, level of lipid peroxidation, and mitochondrial membrane potential (MMP) were detected by flow cytometry. Western blot analysis was also used to measure the expression level of Bcl-2, Bax, caspase 9, and caspase 3 proteins in H2O2-treated RGC-5 cells. Our study showed that the cell viability of RGC-5 cells significantly decreased after treatment with 300 µM H2O2 for 24 h, but MDHB (8, 16, 32 µM) increased RGC-5 cell survival, suppressed the rate of apoptosis, scavenged reactive oxygen species, and restored MMP. MDHB also obstructed H2O2-induced apoptosis by regulating the expression of Bcl-2 and Bax, as well as suppressing the activation of caspase 9 and caspase 3. Our results showed that MDHB is an effective neuroprotective compound that mitigates oxidative stress and inhibits apoptosis in RGC-5 cells.

[The research progress of metals correlated to Alzheimer's disease].

Alzheimer's disease (AD) is a kind of neurodegenerative diseases, the most common cause of dementia. Although AD has been studied more than, 100 years and the Aß and tau theory are most widely accepted among the theories achieved, yet it is not really clear what the mechanism related to AD works up to now. However, it is certain that AD is a kind of diseases resulting from multi-causes. Except for causes correlated with heredity, aging and life habits, environmental role is worth taking into consideration as well. Some metals, such as copper, aluminum, zinc and iron et al, can also have close relationship with AD. Now, we make an overview on the correlative researches in the field.

Methyl 3,4-dihydroxybenzoate protects primary cortical neurons against Aß25₋35-induced neurotoxicity through mitochondria pathway.

Amyloid-ß peptides (Aß), which can aggregate into oligomers or fibrils in neurons, play a critical role in the pathogenesis of Alzheimer's disease (AD). Methyl 3,4-dihydroxybenzoate (MDHB), a phenolic acid compound, has been reported to have antioxidative and neurotrophic effects. The present study investigated the neuroprotective effects of MDHB against Aß-induced apoptosis in rat primary cortical neutons. The primary cortical neurons were pretreated with different concentrations of MDHB for 24 hr, then incubated with 10 µM Aß25-35 for 24 hr. The results showed that Aß25-35 could induce neurotoxicity as evidenced by the decreased cell viability and the increased apoptotic rate. In parallel, Aß25-35 significantly increased the reactive oxygen species accumulation and decreased mitochondrial membrane potential. However, pretreatment of the primary cortical neurons with MDHB could effectively suppress these cellular events caused by Aß25-35 exposure. In addition, MDHB could increase the level of Bcl-2, decrease the level of Bax, and inhibit the activation of caspase-9 and caspase-3 in Aß25-35 -treated primary cortical neurons. All these results were beneficial in their protective effect against Aß-induced neurotoxicity. Our results suggest that MDHB has a neuroprotective effect that provides a pharmacological basis for its clinical use in the treatment of AD.

[Neurotrophic effects of senegenin].

OBJECTIVE: To study the neurotrophic effects of senegenin on the expression of MAP2 mRNA and BDNF mRNA in cultured cerebral cortical neurons. METHODS: The newborn rat cerebral cortex neurons were cultured in vitro. LDH assay was used to investigate the effect of senegenin on the neuronal viability and reverse transcription polymerase chain reaction (RT-PCR) was carried out to determine the expression level of MAP2 mRNA and BDNF mRNA. RESULTS: LDH assay showed that senegenin at the concentration of 0. 5 micromol/L,1 micromol/L and 2 micromol/L could obviously enhance the survival of cells and the survival rates were in dose-dependent manner to some extent. Moreover, the low, medium and high concentrations of senegenin significantly increased the expression of MAP2 mRNA and BDNF mRNA. CONCLUSION: The study suggests that suitable dose of senegenin can increase the expression of MAP2 mRNA and BDNF mRNA in cultured cerebral cortical neurons, and its mechanism needs further study.

Neurotrophic effects of magnesium fructose 1, 6-diphosphate on cortical neurons.

In this study, we evaluated the neurotrophic effects of magnesium fructose 1, 6-diphosphate (FDP-Mg) on cortical neurons. The results demonstrated that FDP-Mg promoted dendrite outgrowth and neuronal survival in a dose-dependent manner. In order to investigate the associated mechanisms, we determined adenosine triphosphate (ATP) levels and brain-derived neurotrophic factor (BDNF) mRNA expression in cortical neurons. Treatment with FDP-Mg significantly increased ATP levels and BDNF mRNA expression in cortical neurons. These data suggest that FDP-Mg can exert neurotrophic effects on cortical neurons. The increases in BDNF mRNA expression and cellular ATP levels are involved in the neurotrophic effects produced by FDP-Mg.

[The inhibition effect of protocatechuic acid on the mRNA expression of APP on M146L cell].

OBJECTIVE: To investigate the effect of protocatechuic acid on the mRNA expression of APP in double transfected (human APP gene and presenlin-1 gene) Chinese hamster ovary (CHO) cells (M146L). METHODS: Abeta42 overexpressing cell model was established in vitro by culturing Chinese hamster ovary cells stably expressing amyloid beta-protein precursor and mutant presenilin (M146L). The MTT assay was used to test cytotoxicity of protocatechuic acid, and reverse transcription polymerase chain reaction (RT-PCR) was carried out to determine the mRNA expression level of APP. RESULTS: The MTT assay showed that protocatechuic acid at suitable concentrations didn't have cytotoxicity on M146L cell survival. Protocatechuic acid at the concentration of 0.25 mmol/L, 0.5 mmol/L and 1.0 mmol/L significantly inhibited the mRNA expression of APP. CONCLUSION: The study suggests that the suitable dose of protocatechuic acid could inhibit the mRNA expression of APP in M146L cell,and its mechanism needs further study.

[Effects of ferulic acid on bFGF-treated PC12 cells].

OBJECTIVE: To explore the effect and mechanism of ferulic acid on differentiation in bFGF-treated PC12 cells. METHODS: The length of neurite outgrowth and the percentage of PC12 cells induced in the presence of 0 ng/mL or 1 ng/mL bFGF were assayed. RESULTS: Compared with that of control group,ferulic acid could enhance the differentiation effect of bFGF (1 ng/mL) in PC12 cells (P < 0.01) and the enhancing effect could be blocked by the specific MAPK kinase inhibitor, PD98059. CONCLUSION: Ferulic acid potentiates neurite outgrowth in bFGF-treated PC12 cells by MAPK-dependent signaling pathway.

[Neurotrophic effects of protocatechuic acid on neurite outgrowth and survival in cultured cerebral cortical neurons of newborn rat].

OBJECTIVE: To study the neurotrophic effects of protocatechuic acid on neurite outgrowth and survival in cultured cerebral cortical neurons. METHODS: The newborn rat cerebral cortex neurons were cultured in vitro. The convert phase microscope was used to count the survival neurons with neurites and measure the average length of neurites. MTT and LDH assay were carried out to investigate the effect of PCA on the neuronal viability. RESULTS: Compared with control group, different concentration of PCA could increase the number of survival neurons with neurites and the average length of neurites. MTT and LDH assay showed that PCA promoted neuron survival in a dose-dependent manner. CONCLUSION: PCA can enhance the survival of rat cortical neurons with neurites and promote the neurite outgrowth of rat cortical neurons.

Identification of potential biomarkers in dengue via integrated bioinformatic analysis.

Dengue fever virus (DENV) is a global health threat that is becoming increasingly critical. However, the pathogenesis of dengue has not yet been fully elucidated. In this study, we employed bioinformatics analysis to identify potential biomarkers related to dengue fever and clarify their underlying mechanisms. The results showed that there were 668, 1901, and 8283 differentially expressed genes between the dengue-infected samples and normal samples in the GSE28405, GSE38246, and GSE51808 datasets, respectively. Through overlapping, a total of 69 differentially expressed genes (DEGs) were identified, of which 51 were upregulated and 18 were downregulated. We identified twelve hub genes, including MX1, IFI44L, IFI44, IFI27, ISG15, STAT1, IFI35, OAS3, OAS2, OAS1, IFI6, and USP18. Except for IFI44 and STAT1, the others were statistically significant after validation. We predicted the related microRNAs (miRNAs) of these 12 target genes through the database miRTarBase, and finally obtained one important miRNA: has-mir-146a-5p. In addition, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were carried out, and a protein-protein interaction (PPI) network was constructed to gain insight into the actions of DEGs. In conclusion, our study displayed the effectiveness of bioinformatics analysis methods in screening potential pathogenic genes in dengue fever and their underlying mechanisms. Further, we successfully predicted IFI44L and IFI6, as potential biomarkers with DENV infection, providing promising targets for the treatment of dengue fever to a certain extent.

[Protective and therapeutic effects of schisandrol A on Abeta damaged PC12 cells].

OBJECTIVES: To observe the protective and therapeutic effect of schisandrol A on the Abeta damaged PC12 cells. METHODS: PC12 cells were damaged by Abeta in vitro. Morphological changes were observed and the number of cells with neurite was analyzed by phase contrast microscope. The cell viability of PC12 cells was determined by the MTT method. RESULTS: Many dispirited cells with atrophied or fragmented neurites in the Abeta damaged PC12 cells were observed under the microscope. More vital cells with longer neurites were observed in the schisandrol A treated PC12 cells and the number of cells with neurite increased. The difference of cell viability between the two groups was statistical significant. CONCLUSION: Schisandrol A can antagonize the neurotoxicity of Abeta and has protective and therapeutic efficacy on Abeta damaged PC12 cells.

Excretion, Metabolism and Cytochrome P450 Inhibition of Methyl 3,4-Dihydroxybenzoate (MDHB): A Potential Candidate to Treat Neurodegenerative Diseases.

BACKGROUND AND OBJECTIVES: Methyl 3,4-dihydroxybenzoate (MDHB) has the potential to prevent neurodegenerative diseases (NDDs). The present work investigated its excretion, metabolism, and cytochrome P450-based drug-drug interactions (DDIs). METHODS: After intragastric administration of MDHB, the parent drug was assayed in the urine and faeces of mice. Metabolites of MDHB in the urine, faeces, brain, plasma and liver were detected by liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF/MS). A cocktail approach was used to evaluate the inhibition of cytochrome P450 isoforms by MDHB. RESULTS: The cumulative excretion permille of MDHB in the urine and faeces were found to be 0.67 ± 0.31 and 0.49 ± 0.44‰, respectively. A total of 96 metabolites of MDHB were identified, and all IC50 (half-maximal inhibitory concentration) values of MDHB towards cytochrome P450 isoforms were > 100 µM. CONCLUSIONS: The results suggest that MDHB has a low parent drug cumulative excretion percentage and that MDHB has multiple metabolites and is mainly metabolized through the loss of -CH2 and -CO2, the loss of -CH2O, ester bond hydrolysis, the loss of -O and -CO2, isomerization, methylation, sulfate conjugation, the loss of -CH2O and -O and glycine conjugation, glycine conjugation, the loss of two -O groups and alanine conjugation, the loss of -CH2O and -O and glucose conjugation, glucuronidation, glucose conjugation, etc., in vivo. Finally, MDHB has a low probability of cytochrome P450-based DDIs.

The Immune System Drives Synapse Loss During Lipopolysaccharide-Induced Learning and Memory Impairment in Mice.

Although lipopolysaccharides (LPS) have been used to establish animal models of memory loss akin to what is observed in Alzheimer's disease (AD), the exact mechanisms involved have not been substantiated. In this study, we established an animal model of learning and memory impairment induced by LPS and explored the biological processes and pathways involved. Mice were continuously intraperitoneally injected with LPS for 7 days. Learning- and memory-related behavioral performance and the pathological processes involved were assessed using the Morris water maze test and immunostaining, respectively. We detected comprehensive expression of C1q, C3, microglia, and their regulatory cytokines in the hippocampus. After 7 days of LPS administration, we were able to observe LPS-induced learning and memory impairment in the mice, which was attributed to neural impairment and synapse loss in the hippocampus. We elucidated that the immune system was activated, with the classical complement pathway and microglial phagocytosis being involved in the synapse loss. This study demonstrates that an LPS-injected mouse can serve as an early memory impairment model for studies on anti-AD drugs.

Determination of the Pharmacokinetics and Tissue Distribution of Methyl 3,4-Dihydroxybenzoate (MDHB) in Mice Using Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND AND OBJECTIVES: Methyl 3,4-dihydroxybenzoate (MDHB) has the potential to prevent neurodegenerative diseases (NDDs). The present work aims to reveal the pharmacokinetics and tissue distribution characteristics of MDHB. METHODS: The pharmacokinetics and tissue distribution of MDHB were analyzed using LC-MS/MS after a single intragastric administration (50 to 450 mg/kg) in mice, and samples were collected from five animals at specific time points. RESULTS: Pharmacokinetic parameters of MDHB following intragastric administrations were: the time to peak concentration (Tmax) ranged from 0.033 to 0.07 h, the peak concentration (Cmax) ranged from 12,379.158 to 109798.712 µg/l, the elimination half-life (t1/2z) ranged from 0.153 to 1.291 h, the area under the curve (AUC0-∞) ranged from 640.654 to 20,241.081 µg/l × h, the mean residence time (MRT0-∞) ranged from 0.071 to 0.206 h, the apparent volume of distribution (Vz/F) ranged from 17.538 to 45.244 l/kg, and the systemic clearance (Clz/F) ranged from 22.541 to 80.807 l/h/kg. The oral bioavailability of MDHB was 23%. The maximum MDHB content was detected in the stomach, and the minimum content was observed in the testes; the peak content in the brain was 15,666.93 ng/g. CONCLUSIONS: The pharmacokinetic characteristics of MDHB include fast absorption, high systemic clearance, a short half-life and an oral bioavailability of 23%. Additionally, MDHB permeates the blood-brain barrier (BBB) and is rapidly distributed to all organs. The identification of the pharmacokinetics of MDHB following its oral administration will contribute to further preclinical and clinical studies of its effects.

Methyl 3,4-Dihydroxybenzoate Induces Neural Stem Cells to Differentiate Into Cholinergic Neurons in vitro.

Neural stem cells (NSCs) have been shown as a potential source for replacing degenerated neurons in neurodegenerative diseases. However, the therapeutic potential of these cells is limited by the lack of effective methodologies for controlling their differentiation. Inducing endogenous pools of NSCs by small molecule can be considered as a potential approach of generating the desired cell types in large numbers. Here, we reported the characterization of a small molecule (Methyl 3,4-dihydroxybenzoate; MDHB) that selectively induces hippocampal NSCs to differentiate into cholinergic motor neurons which expressed synapsin 1 (SYN1) and postsynaptic density protein 95 (PSD-95). Studies on the mechanisms revealed that MDHB induced the hippocampal NSCs differentiation into cholinergic motor neurons by inhibiting AKT phosphorylation and activating autophosphorylation of GSK3ß at tyrosine 216. Furthermore, we found that MDHB enhanced ß-catenin degradation and abolished its entering into the nucleus. Collectively, this report provides the strong evidence that MDHB promotes NSCs differentiation into cholinergic motor neurons by enhancing gene Isl1 expression and inhibiting cell cycle progression. It may provide a basis for pharmacological effects of MDHB directed on NSCs.

A novel effective chemical hemin for the treatment of acute carbon monoxide poisoning in mice.

There is no effective drug for the therapy of acute carbon monoxide (CO) poisoning. The purpose of the present study was to investigate the potential preventive and therapeutic effects of hemin on an animal model of acute CO poisoning and to provide a potential therapeutic candidate drug. A total of 80 Kunming mice were randomly divided into four groups, namely the air control, acute CO poisoning, hemin-treatment + CO and hemin-pretreatment + CO groups (n=20 each). Furthermore, the mortality rate of mice, blood carboxyhaemoglobin (HbCO) concentration and serum malondialdehyde (MDA) concentration were measured, and pathological changes of the hippocampal area were determined using histochemical staining. The mice with acute CO poisoning had a 50% mortality rate at 1 h, with an increase in blood HbCO, serum MDA levels and pathological impairments of the hippocampus. Furthermore, the mortality rate, blood HbCO and serum MDA levels of mice with pretreatment and treatment of hemin were decreased. Additionally, the pathological changes of the hippocampal area were improved in the hemin-treatment and hemin-pretreatment groups compared with the mice treated with CO. These results suggest that hemin is a novel effective chemical for the prevention and treatment of acute CO poisoning in mice. Therefore, the present study provides a novel method and experimental basis for the application of hemin in treating patients with acute CO poisoning.

[Gamma-Schisandrin inhibits production of amyloid beta-protein 42 in M146L cells].

AIM: To investigate the inhibition of amyloid beta-protein 42 (Abeta42) production in M146L cells by gamma-schisandrin. METHODS: M146L cells which can produce considerable Abeta42 in vitro were treated with gamma-schisandrin (1.67, 5.00 and 15.00 microg x mL(-1)), beta-secretase inhibitor (S4562, 100.00 microg x mL(-1)) and gamma-secretase inhibitor (S2188, 13.68 microg x mL(-1)), separately. Cell counting kit-8 (CCK-8) was used to assess cell viability. Enzyme-linked immunosorbent assay (ELISA) was carried out to determine the amount of Abeta42. Western blotting was used to examine C99, an intermediary product of APP cleaved by beta-secretase. beta-Secretase and gamma-secretase activities were assayed by commercial kits. RESULTS: The CCK-8 assay indicated that different concentrations of gamma-schisandrin had no neurotoxicity on the cultured M146L. And the ELISA test showed that the amount of Abeta42 secreted by M146L cells treated with gamma-schisandrin (5.00 and 15.00 microg x mL(-1)) decreased obviously as compared with solvent control. The results of Western blotting test indicated that there was no change of C99 contents and beta-secretase activity in gamma-schisandrin treated cells, while gamma-secretase activity decreased obviously. CONCLUSION: gamma-Schisandrin inhibited production of Abeta42 in M146L cells through inhibiting gamma-secretase.

[Deterimination of schisandrin in Kadsura heteroclita (Roxb.) Craib by RP-HPLC].

OBJECTIVE: To develop a method of RP-HPLC for determination of schisandrin content in Kadsura heteroclita (Roxb.) Craib. METHOD: RP-HPLC analysis was carried out by using Kromasil C18 column (5 microm, 250 mm x 4.6 mm) and methanol-water (65:35) as the mobile, and the detection wavelength was 250 nm. RESULTS: The method was linear in the range of 0.08-0.06 nicrog (r = 0.9998), and the average recovery was 100.24%, with the RSD of 2.09% (n=5). CONCLUSION: The method was simple, accurate, highly sensitive and reproducible. It may be used for the quantitative determination of schisandrin in extract of Kadsura heteroclita (Roxb.) Craib.