Inhibition of miR-141-3p attenuates apoptosis of neural stem cells via targeting PBX1 to regulate PROK2 transcription in MCAO mice.

MicroRNA-141-3p (miR-141-3p) has been found to be altered in the brain following a stroke. Herein, we investigate the impact of miR-141-3p on the apoptosis of neural stem cells (NSCs) in mice with middle cerebral artery occlusion (MCAO) and the potential mechanisms involved. Eight-week-old mice were injected intracerebroventricularly with miR-141-3p, antagomir-141-3p, or agomir negative control 2 h before MCAO, and animal behavior tests and infraction volume measurements were performed 24 h later. MCAO-mediated brain injury and NSCs apoptosis were observed by H&E, TTC, and TUNEL staining. The expression of cleaved caspase-3 and Ki67 was detected by western blotting. The luciferase reporter assay proved that miR-141-3p in combination with its target gene PBX homeobox 1 (PBX1). Exogenous miR-141-3p (agomir-141-3p) treatment increased infraction volume and brain edema and damaged neurological functions compared to control mice. Agomir-141-3p increased miR-141-3p expression in brain tissue of mice with MCAO and suppressed PBX1 expression. The effects of the agomir-141-3p-induced apoptosis in NSCs treated with oxygen-glucose deprivation (OGD)/reoxygenation (R) were abolished by PBX1 overexpression. The results from UCSC and JASPAR database showed that prokineticin 2 (PROK2) was a transcription factor of PBX1. The expression of PROK2 was transcriptionally regulated by PBX1 using RT-PCR and western blot assays. The effects of the apoptosis-promoting caused by PBX1 inhibition in NSCs treated with OGD/R were reversed by PROK2 inhibition. In conclusion, the miR-141-3p/PBX1/PROK2 axis might be a novel therapeutic target for the apoptosis of NSCs in MCAO.

YTHDF1 regulates endoplasmic reticulum stress, NF-κB, MAPK and PI3K-AKT signaling pathways in inflammatory osteoclastogenesis.

Abnormal increases in osteoclast differentiation and activity contribute to excessive bone resorption in inflammatory bone diseases. The specific m6A-binding protein YT521-B homology domain family 1 (YTHDF1) participates in many physiopathological processes by regulating mRNA stability or translation. However, whether YTHDF1 is involved in the regulation of inflammatory osteoclastogenesis remains a mystery. This study revealed that YTHDF1 expression was upregulated during lipopolysaccharide (LPS)-stimulated osteoclast differentiation. Knockdown of Ythdf1 inhibited osteoclast formation, bone resorption and the expression of osteoclast-related genes (Tnfrsf11a, Traf6, Mmp9 and Acp5). Analysis of RNA sequencing data showed that the genes downregulated by Ythdf1 knockdown were closely associated with endoplasmic reticulum (ER) stress and osteoclast differentiation. Western blotting confirmed that Ythdf1 depletion suppressed activation of the ER stress-related PERK, IRE1α and ATF6 signaling pathways. The ER stress activator tunicamycin (Tm) partially rescued the decreased expression of Mmp9 and Acp5 caused by Ythdf1 deficiency. Meanwhile, Ythdf1 depletion inhibited the phosphorylation levels of key proteins in the NF-κB, MAPK and PI3K-AKT signaling pathways and decreased the mRNA stability of Tnfrsf11a, which is the major upstream signaling molecule that mediates the activation of these pathways during osteoclast differentiation. In conclusion, our findings suggest that Ythdf1 knockdown inhibits inflammatory osteoclast differentiation and function by suppressing ER stress signaling pathways. Ythdf1 knockdown also inactivates the signaling pathways involved in osteoclast differentiation by inhibiting Tnfrsf11a mRNA stability. These findings will help shed light on the molecular mechanisms of m6A-mediated epigenetic regulation in inflammatory osteoclastogenesis.

Effect of Berberine Hydrochloride on the Diversity of Intestinal Flora in Parkinson's Disease Patients.

This study aimed to investigate the effect of berberine hydrochloride on the diversity of intestinal flora in patients with Parkinson's disease (PD). Prospectively selected 68 PD patients, admitted to our hospital from April 2021 to June 2021, were randomly assigned to an observation group and a control group (n = 34 per group). Patients in the control group were given conventional treatment in accordance with Parkinson's diagnosis and treatment guidelines. Patients in the observation group were administered berberine hydrochloride besides the treatment in the control group. After continuous treatment for 3 months, the enzyme-linked immunosorbent assay was applied to determine interleukin-8 (IL-8), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels. High-throughput sequencing technology was employed to perform DNA sequencing on the 16S rRNA genes of all bacteria in stool samples before and after treatment in the two groups to analyze the distribution of intestinal flora. After treatment, the levels of IL-8, IL-6, and TNF-α were lower in the observation group than those in the control group (P < 0.05). Regarding intestinal flora, the Chao index, Ace index, and Shannon index were higher in the observation group than those in the control group. The Simpson index was lower in the observation group than that of the control group (P < 0.05). The principal component analysis chart analysis exhibited that the overall structure of the intestinal flora was quite different between the observation and the control groups after treatment. Berberine hydrochloride can improve the disorder of intestinal flora in PD patients and suppress the expression of inflammatory factors.

AKR1C3 and Its Transcription Factor HOXB4 Are Promising Diagnostic Biomarkers for Acute Myocardial Infarction.

Background: A recent study disclosed that ferroptosis was an important myocyte death style in myocardial infarction (MI). However, the diagnostic value of ferroptosis regulators and correlated underlying mechanisms in acute myocardial infarction (AMI) remain unknown. Methods: Bioinformatical analyses were conducted to identify the candidate biomarkers for AMI, and the collected local samples were used to validate the findings via real-time quantitative PCR. Bioinformatical analysis and luciferase reporter assay were implemented to identify the transcriptional factor. Transient transfection and ferroptosis characteristic measurement, including glutathione peroxidase 4, malondialdehyde, iron, and glutathione, was performed to verify the ability of the candidate gene to regulate the ferroptosis of cardiomyocytes. A meta-analysis was conducted in multiple independent cohorts to clarify the diagnostic value. Results: A total of 121 ferroptosis regulators were extracted from previous studies, and aldo-keto reductase family 1 member C3 (AKR1C3) was significantly downregulated in the peripheral blood samples of AMI cases from the analysis of GSE48060 and GSE97320. HOXB4 served as a transcriptional activator for AKR1C3 and could suppress the ferroptosis of the H9C2 cells treated with erastin. Besides this, peripheral blood samples from 16 AMI patients and 16 patients without coronary atherosclerotic disease were collected, where AKR1C3 and HOXB4 both showed a high diagnostic ability. Furthermore, a nomogram including HOXB4 and AKR1C3 was established and successfully validated in six independent datasets. A clinical correlation analysis displayed that AKR1C3 and HOXB4 were correlated with smoking, CK, CK-MB, and N-terminal-pro-B-type natriuretic peptide. Conclusion: Taken together, this study demonstrates that AKR1C3 and HOXB4 are promising diagnostic biomarkers, providing novel insights into the ferroptosis mechanisms of AMI.

Circ_0062166 aggravates cerebral ischemia-reperfusion injury through targeting miR-526b-5p/BCL2L13 axis.

BACKGROUND: Cerebral ischemia-reperfusion (CIR) injury is a severe disease, which may cause serious dysfunction of the brain. Most circular RNAs (circRNAs) have been demonstrated to play a significant role in CIR injury. However, a novel circRNA, circ_0062166 (circ_BCL2L13) has not been investigated for CIR injury. Hence, we aim to disclose the role of circ_0062166 in CIR injury in this study. METHODS: Firstly, RT-qPCR was applied to examine the expression of circ_0062166 in oxygen-glucose deprivation and reoxygenation (OGD/R) cell model. Functional assays were conducted to detect the role of circ_0062166 in CIR injury. RNA pull down, RIP and luciferase reporter assays were implemented to probe into the regulatory mechanism of circ_0062166. RESULTS: Circ_0062166 was significantly up-regulated in neuro-2A (N2A) neuroblastoma cells following OGD/R. Functionally, the silencing of circ_0062166 inhibited cell proliferation and promoted cell apoptosis under OGD/R condition. From the perspective of mechanism, circ_0062166 functioned as a competing endogenous RNA (ceRNA) for microRNA-526b-5p (miR-526b-5p) and regulated BCL2 like 13 (BCL2L13). Eventually, the promoting role of the circ_0062166/miR-526b-5p/BCL2L13 axis in the CIR injury was verified. CONCLUSION: To sum up, the present study has demonstrated that circ_0062166/miR-526b-5p/BCL2L13 axis accelerated the progression of CIR injury, which might provide effective strategies for CIR injury therapy.

YTHDF2 mediates LPS-induced osteoclastogenesis and inflammatory response via the NF-κB and MAPK signaling pathways.

Aberrant elevation of osteoclast differentiation and function is responsible for disrupting bone homeostasis in various inflammatory bone diseases. YTH domain family 2 (YTHDF2) is a well-known m6A-binding protein that plays an essential role in regulating cell differentiation and inflammatory processes by mediating mRNA degradation. However, the regulatory role of YTHDF2 in inflammatory osteoclast differentiation remains unelucidated. Here, we detected the expression of m6A-related genes and found that YTHDF2 was upregulated in RANKL-primed osteoclast precursors stimulated with lipopolysaccharide (LPS). Ythdf2 knockdown in RAW264.7 cells and primary bone marrow-derived macrophages (BMMs) enhanced osteoclast formation and bone resorption, which was assessed by TRAP staining assay and pit formation assay. Ythdf2 depletion upregulated osteoclast-related gene expression and proinflammatory cytokine secretion. In contrast, overexpression of Ythdf2 produced the reverse effect. Furthermore, Ythdf2 knockdown enhanced the phosphorylation of IKKα/ß, IκBα, ERK, P38 and JNK. NF-κB and MAPK signaling pathway inhibitors effectively abrogated the enhanced expression of Nfact1, c-Fos, IL-1ß and TNF-α caused by Ythdf2 knockdown. Mechanistically, the mRNA stability assay revealed that Ythdf2 depletion led to stabilization of Tnfrsf11a, Traf6, Map4k4, Map2k3, Map2k4 and Nfatc1 mRNA. In summary, our findings demonstrated that YTHDF2 has a negative regulatory role in LPS-induced osteoclast differentiation and the inflammatory response via the NF-κB and MAPK signaling pathways.

A U-shaped association between serum betaine and incident risk of first ischemic stroke in hypertensive patients.

BACKGROUND & AIMS: Betaine (a micronutrient) has important biological functions (e.g., preventing premature apoptosis and serving as a methyl donor). We investigated the association between baseline serum betaine and the incident risk of first stroke in hypertensive patients. METHODS: We conducted a nested case-control study, including 622 patients with first stroke (including 502 ischemic stroke, 118 hemorrhagic stroke and 2 uncertain type of stroke) and 622 matched controls from the China Stroke Primary Prevention Trial (CSPPT). The study was conducted from May 2008 to August 2013. The study outcomes included first stroke and its subtypes: first ischemic and hemorrhagic stroke. RESULTS: There was a U-shaped association between baseline serum betaine and the risk of first ischemic stroke. The risk of first ischemic stroke decreased with the increment of betaine (per 10 µmol/L increase: OR, 0.87; 95%CI: 0.77-0.99) in patients with betaine <77.7 µmol/L, while the risk of first ischemic stroke increased with the betaine increment (OR, 1.17; 95%CI: 1.01-1.36) in patients with betaine ≥77.7 µmol/L. However, there was no significant association between serum betaine and risk of first hemorrhagic stroke (per 10 µmol/L increase: OR, 0.98; 95%CI: 0.82-1.17). CONCLUSIONS: There was a U-shaped association between baseline betaine levels and the risk of first ischemic stroke in hypertensive patients, with a turning point at about 77.7 µmol/L. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT00794885.

Effect of long-term low-dose folic acid supplementation on degree of total homocysteine-lowering: major effect modifiers.

We sought to examine the potential modifiers in the association between long-term low-dose folic acid supplementation and the reduction of serum total homocysteine (tHcy) among hypertensive patients, using data from the China Stroke Primary Prevention Trial (CSPPT). This analysis included 16 867 participants who had complete data on tHcy measurements at both the baseline and exit visit. After a median treatment period of 4·5 years, folic acid treatment significantly reduced the tHcy levels by 1·6 µmol/l (95 % CI 1·4, 1·8). More importantly, after adjustment for baseline tHcy and other important covariates, a greater degree of tHcy reduction was observed in certain subgroups: males, the methylenetetrahydrofolate reductase (MTHFR) 677TT genotype, higher baseline tHcy levels (≥12·5 (median) v. <12·5 µmol/l), lower folate levels (<8·0 (median) v. ≥8·0 ng/ml), estimated glomerular filtration rate (eGFR) <60 ml/min per 1·73 m2 (v. 60-<90 and ≥90 ml/min per 1·73 m2), ever smokers and concomitant use of diuretics (P for all interactions <0·05). The degree of tHcy reduction associated with long-term folic acid supplementation can be significantly affected by sex, MTHFR C677T genotypes, baseline folate, tHcy, eGFR levels and smoking status.

Effect of Smoking and Folate Levels on the Efficacy of Folic Acid Therapy in Prevention of Stroke in Hypertensive Men.

BACKGROUND AND PURPOSE: We aimed to examine whether the efficacy of folic acid therapy in the primary prevention of stroke is jointly affected by smoking status and baseline folate levels in a male population in a post hoc analysis of the CSPPT (China Stroke Primary Prevention Trial). METHODS: Eligible participants of the CSPPT were randomly assigned to a double-blind daily treatment of a combined enalapril 10-mg and folic acid 0.8-mg tablet or an enalapril 10-mg tablet alone. In total, 8384 male participants of the CSPPT were included in the current analyses. The primary outcome was first stroke. RESULTS: The median treatment duration was 4.5 years. In the enalapril-alone group, the first stroke risk varied by baseline folate levels and smoking status (never versus ever). Specifically, there was an inverse association between folate levels and first stroke in never smokers (P for linear trend=0.043). However, no such association was found in ever smokers. A test for interaction between baseline folate levels and smoking status on first stroke was significant (P=0.045). In the total sample, folic acid therapy significantly reduced the risk of first stroke in never smokers with folate deficiency (hazard risk, 0.36; 95% confidence interval, 0.16-0.83) and in ever smokers with normal folate levels (hazard risk, 0.69; 95% confidence interval, 0.48-0.99). CONCLUSIONS: Baseline folate levels and smoking status can interactively affect the risk of first stroke. Our data suggest that compared with never smokers, ever smokers may require a higher dosage of folic acid to achieve a greater beneficial effect on stroke. Our findings need to be confirmed by future randomized trials. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00794885.

Homocysteine and all-cause mortality in hypertensive adults without pre-existing cardiovascular conditions: Effect modification by MTHFR C677T polymorphism.

BACKGROUND: Previous studies support an association between elevated total homocysteine (tHcy) levels and increased all-cause mortality. However, few prospective studies have examined this association in hypertensive patients, and/or tested any effect modification by the methylene tetrahydrofolate reductase (MTHFR) C677T genotype. METHODS: This was a post hoc analysis of the China Stroke Primary Prevention Trial. Serum tHcy and folate were measured at baseline. Individual MTHFR C677T genotype (CC, CT, and TT) was determined. Evidence for death included death certificates or home visits. Cumulative hazards of all-cause mortality by tHcy quartiles were estimated using the Kaplan-Meier method, and group differences were compared by log-rank tests. Hazard ratios (HRs) and 95% confidence intervals were estimated by Cox proportional-hazard regression models, adjusting for age, sex, baseline folate, vitamin B12, blood pressure, body mass index, smoking and alcohol drinking status, study center, total cholesterol, triglycerides, high-density lipoprotein cholesterol, fasting glucose, creatinine, and treatment group. Potential effect modification by the MTHFR genotype on the relationship between tHcy and all-cause mortality was tested. RESULTS: The analyses included 20,424 hypertensive patients (41% males) without a history of myocardial infarction or stroke. Baseline mean age (SD) was 60 ±â€Š7.5 years and mean (SD) serum tHcy was 14.5 ±â€Š8.4 µmol/L. After a mean follow-up period of 4.5 years, there were 612 (3%) all-cause deaths. Kaplan-Meier survival curves revealed a graded relationship between tHcy quartiles and all-cause mortality. The HRs, using the lowest quartile as the reference, were 1.2, 1.2, and 1.5 in Q2, Q3, and Q4, respectively. A linear trend test, using natural log-transformed tHcy, resulted in an HR of 1.5 (95% confidence interval 1.2-1.9, P < .001) after adjustment for lifestyle and health-related variables. Whereas the MTHFR genotype alone had little effect on mortality, it significantly modified the tHcy-mortality association, which was much stronger in the CC/CT genotype than in the TT genotype (P for interaction < 0.05). CONCLUSIONS: Among Chinese hypertensive patients without cardiovascular comorbidities, elevated tHcy was a significant risk marker for death from all causes, and the association was subject to effect modification by MTHFR genotypes. If confirmed that tHcy and MTHFR genotypes may serve as useful biomarkers for mortality risk assessment and targeted intervention.

Prevalence of hyperhomocysteinaemia and its major determinants in rural Chinese hypertensive patients aged 45-75 years.

We aimed to investigate the prevalence of hyperhomocysteinaemia (total plasma homocysteine (tHcy) ≥ 10 µmol/l) and its major determinants in rural Chinese hypertensive patients. A cross-sectional investigation was carried out in Lianyungang of Jiangsu province, China. This analysis included 13 946 hypertensive adults. The prevalence of hyperhomocysteinaemia was 51.6 % (42.7 % in women and 65.6 % in men). The OR of hyperhomocysteinaemia were 1.52 (95 % CI 1.39, 1.67) and 2.32 (95 % CI 2.07, 2.61) for participants aged 55-65 and 65-75 v. 45-55 years; 1.27 (95 % CI 1.18, 1.37) for participants with a BMI ≥ 25 v. < 25 kg/m²; 1.14 (95 % CI 1.06, 1.23) for participants with v. without antihypertensive treatment; 1.09 (95 % CI 1.00, 1.18) for residents inland v. coastal; 0.89 (95 % CI 0.82, 0.97) and 0.83 (95 % CI 0.74, 0.92) for participants with moderate and high v. low physical activity levels; 1.54 (95 % CI 1.41, 1.68) and 2.47 (95 % CI 2.17, 2.81) for participants with a glomerular filtration rate 60-90 and < 60 v. ≥ 90 ml/min per 1.73 m²; and 1.20 (95 % CI 1.07, 1.35) and 3.81 (95 % CI 3.33, 4.36) for participants with CT and TT v. CC genotype at methylenetetrahydrofolate reductase 677C>T polymorphism, respectively. Furthermore, higher tHcy concentrations were observed in smokers of both sexes (men: geometric mean 12.1 (interquartile range (IQR) 9.2-14.5) v. 11.9 (IQR 9.-14.) µmol/l, P= 0.005; women: geometric mean 10·3 (IQR 8.3-13.0) v. 9.6 (IQR 7.8-11.6) µmol/l, P= 0.010), and only in males with hypertension grade 3 (v. grade 1 or controlled blood pressure) (geometric mean 12.1 (IQR 9.2-14.4) v. 11.7 (IQR 9.2-14.0), P= 0.016) and in male non-drinkers (yes v. no) (geometric mean 12.3 (IQR 9.4-14.8) v. 11.7 (IQR 9.1-13.9), P= 0.014). In conclusion, there was a high prevalence of hyperhomocysteinaemia in Chinese hypertensive adults, particularly in the inlanders, who may benefit greatly from tHcy-lowering strategies, such as folic acid supplementation and lifestyle change.