Ring Finger Protein 146-mediated Long-chain Fatty-acid-Coenzyme a Ligase 4 Ubiquitination Regulates Ferroptosis-induced Neuronal Damage in Ischemic Stroke.

Ischemic stroke (IS) is one of the leading causes of disability and death worldwide. Long-chain fatty-acid-coenzyme A ligase 4 (ACSL4) is a critical isozyme for ferroptosis that participates in the progression of IS. RING finger protein 146 (RNF146) is an E3 ligase predicted to interact with ACSL4 and regulated by activating transcription factor 3 (ATF3). The molecular mechanism of the RNF146/ACSL4 axis in IS is still unclear. Oxygen-glucose deprivation/reperfusion (OGD/R) treatment was used as the in vitro model, and middle cerebral artery occlusion (MCAO) mice were established for the in vivo model for IS. The protein level of ACSL4 was monitored by Western blot during ischemic injury. RNF146 was overexpressed in vitro and in vivo. The interaction of RNF146 and ACSL4 was determined by co-immunoprecipitation (Co-IP) assay. Chromatin immunoprecipitation (ChIP) assay and luciferase assay were utilized to determine the regulation of ATF3 on RNF146. Ferroptosis was evaluated by the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), Fe2+, and protein levels of related genes including ACSL4, SLC7A11, and GPX4. ACSL4 was downregulated upon OGD treatment and then increased by re-oxygenation. RNF146 was responsible for the ubiquitination and degradation of ACSL4 protein. RNF146 overexpression could prevent the stimulation of OGD/R-induced LDH, MDA, and Fe2+ levels and ferroptosis-related gene expression. ATF3 could activate the transcription and expression of RNF146, leading to the inhibition of OGD/R-induced neuron ferroptosis. The ATF3-mediated RNF146 could alleviate neuronal damage in IS by regulating ACSL4 ubiquitination and ferroptosis, providing a novel theoretical basis for exploring therapeutic targets and strategies.

Astragaloside IV alleviates neuronal ferroptosis in ischemic stroke by regulating fat mass and obesity-associated-N6-methyladenosine-acyl-CoA synthetase long-chain family member 4 axis.

Ischemic stroke (IS) is a detrimental neurological disease with limited treatment options. Astragaloside IV (As-IV) was a promising bioactive constituent in the treatment of IS. However, the functional mechanism remains unclear. Here, IS cell and mouse models were established by oxygen glucose deprivation/re-oxygenation (OGD/R) and middle cerebral artery occlusion (MCAO). Quantitative reverse transcription PCR (RT-qPCR), Western blotting, or Immunofluorescence staining measured related gene and protein expression of cells or mice brain tissues, and the results revealed altered expression of acyl-CoA synthetase long-chain family member 4 (Acsl4), fat mass and obesity-associated (Fto), and activation transcription factor 3 (Atf3) after treatment with As-IV. Then, increased N6 -methyladenosine (m6 A) levels caused OGD/R or MCAO were reduced by As-IV according to the data from methylated RNA immunoprecipitation (MeRIP)-qPCR and dot blot assays. Moreover, through a series of functional experiments such as observing mitochondrial changes under transmission electron microscopy (TEM), evaluating cell viability by cell counting kit-8 (CCK-8), analyzing infract area of brain tissues by 2,3,5-triphenyltetrazolium chloride (TTC) staining, measuring levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), Fe2+ , solute carrier family 7 member 11 (Slc7a11) and glutathione peroxidase 4 (Gpx4) and concentration of glutathione (GSH), we found that Fto knockdown, Acsl4 overexpression or Atf3 knockdown promoted the viability of OGD/R cells, inhibited cell ferroptosis, reduced infract size, while As-IV treatment or Fto overexpression reversed these changes. In mechanism, the interplays of YTH N6 -methyladenosine RNA-binding protein 3 (Ythdf3)/Acsl4 and Atf3/Fto were analyzed by RNA-pull down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay. Fto regulated the m6 A levels of Acsl4. Ythdf3 bound to Acsl4, and modulated its levels through m6 A modification. Atf3 bound to Fto and positively regulated its levels. Overall, As-IV promoted the transcription of Fto by upregulating Atf3, resulting in decreased m6 A levels of Acsl4, thus, improving neuronal injury in IS by inhibiting ferroptosis.

TaiChi and Qigong for Depressive Symptoms in Patients with Chronic Heart Failure: A Systematic Review with Meta-Analysis.

BACKGROUND: Depression is a debilitating comorbidity of heart failure (HF) that needs assessment and management. Along with mind-body exercise to deal with HF with depression, the use of TaiChi and/or Qigong practices (TQPs) has increased. Therefore, this systematic review assesses the effects of TQPs on depression among patients with HF. METHODS: Randomized controlled trials (RCTs) that examined the effect of TQPs on depression in patients with HF were searched by five databases (PubMed, Cochrane Central Register of Controlled Trials (CENTRAL), EMBASE, CINAHL, and China National Knowledge Infrastructure (CNKI)). With standardized mean difference (SMD) and 95% confidence intervals (95% CI), random-effects meta-analyses of the effect of TQPs on depressive symptoms were performed. RESULTS: Of eight included RCTs, seven (481 patients) provided data for the meta-analysis. The pooling revealed that TQPs contribute to depression remission in HF (SMD -0.66; 95% CI -0.98 to -0.33, P < 0.0001; I 2 = 64%). Its antidepressive effect was not influenced by intervention duration or exercise setting, but rather by ejection fraction subtype, depressive severity, and depression instruments. The beneficial effects were preserved when the study with the largest effect was removed. CONCLUSION: This study suggests that TQPs might be a good strategy for alleviating depressive symptoms in patients with HF. And rigorous-design RCTs, which focus on the identified research gaps, are needed to further establish the therapeutic effects of TQPs for depression in HF.

Paeonol inhibits the progression of intracerebral haemorrhage by mediating the HOTAIR/UPF1/ACSL4 axis.

Intracerebral haemorrhage (ICH) is a devastating subtype of stroke with high morbidity and mortality. It has been reported that paeonol (PAN) inhibits the progression of ICH. However, the mechanism by which paeonol mediates the progression of ICH remains unclear. To mimic ICH in vitro, neuronal cells were treated with hemin. An in vivo model of ICH was established to detect the effect of paeonol on ferroptosis in neurons during ICH. Cell viability was tested by MTT assay. Furthermore, cell injury was detected by GSH, MDA and ROS assays. Ferroptosis was examined by iron assay. RT-qPCR and western blotting were used to detect gene and protein expression, respectively. The correlation among HOTAIR, UPF1 and ACSL4 was explored by FISH, RNA pull-down and RIP assays. Paeonol significantly inhibited the ferroptosis of neurons in ICH mice. In addition, paeonol significantly reversed hemin-induced injury and ferroptosis in neurons, while this phenomenon was notably reversed by HOTAIR overexpression. Moreover, paeonol notably inhibited ferroptosis in hemin-treated neuronal cells via inhibition of ACSL4. Additionally, HOTAIR bound to UPF1, and UPF1 promoted the degradation of ACSL4 by binding to ACSL4. Furthermore, HOTAIR overexpression reversed paeonol-induced inhibition of ferroptosis by mediating the UPF1/ACSL4 axis. Paeonol inhibits the progression of ICH by mediating the HOTAIR/UPF1/ACSL4 axis. Therefore, paeonol might serve as a new agent for the treatment of ICH.

Chinese herbal formula Xuefu Zhuyu for primary dysmenorrhea patients (CheruPDYS): a study protocol for a randomized placebo-controlled trial.

BACKGROUND: Epidemiological studies have shown that young women often suffer from primary dysmenorrhea (PD) which is a common cause that affects their routine work and quality of life. Chinese herbal medicine has been widely used for PD in China. A systematic review found that Xuefu Zhuyu (XFZY) has a promising effect on PD management, yet there is a dearth of high-quality evidence in support of this claim. We want to conduct a randomized controlled trial to evaluate the efficacy and safety of XFZY for PD patients. METHODS: This is a protocol for a multicenter, randomized, placebo-controlled trial. A total of 248 participants with PD will be recruited at 6 centers and randomized into two groups-a herbal treatment group and a placebo group. The participants will receive either XFZY or placebo, three times per day, for 3 menstrual cycles, with a 12-week follow-up. The primary outcome will be the mean change in pain intensity as measured by VAS, while the change in menstrual pain duration, the change in peak pain intensity as measured by VAS, the Cox Menstrual Symptom Scale (CMSS), quality of life EQ-5D-5L, cumulative painkiller consumption, and health economics will be included as secondary outcomes. Adverse events will also be reported. DISCUSSION: This protocol describes a multicenter, double-blind, randomized, placebo-controlled trial that investigates the efficacy and safety of XFZY for primary dysmenorrhea. Validated evaluation tools will assess dysmenorrhea severity. We believe that this research will provide important evidence regarding the use of XFZY to treat dysmenorrhea. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR1900026819 . Registered on 23 October 2019.

Predictive value on diffusion weighted imaging scores for basilar artery occlusion after endovascular treatment.

PURPOSE: To assess the predictive value of three scoring systems based on diffusion weighted imaging in basilar artery occlusion patients after endovascular treatment. METHODS: We analyzed clinical and radiological data of patients with basilar artery occlusion from January 2010 to June 2019, with modified Rankin Scale of 0-2 and 3-6 defined as favorable outcome and unfavorable outcome at three months. Diffusion weighted imaging posterior circulation ASPECTS Score (DWI pc-ASPECT Score), Renard diffusion weighted imaging Score, and diffusion weighted imaging Brainstem Score were used to evaluate the early ischemic changes. RESULTS: There were a total of 88 basilar artery occlusion patients enrolled in the study after endovascular treatment, with 33 of them getting a favorable outcome. According to the analysis, the time from onset to puncture within 12 h (odds ratio: 4.34; 95% confidence interval: 1.55-12.16; P = 0.01), presence of collateral flow via PCoA (odds ratio: 0.31; 95%CI: 0.12-0.79; P = 0.01) or between PICA and SCA (odds ratio: 0.18; 95%CI: 0.07-0.47; P = 0.00), equal or less than 15 points on baseline NIHSS (area under the curve 0.79, 95% CI 0.69-0.89; sensitivity = 69.1%, specificity = 81.8%; P = 0.00), and equal or less than 1.5 points on diffusion weighted imaging Renard score (area under the curve 0.63, 95% CI 0.51-0.75; sensitivity = 83.6%, specificity = 39.4%; P = 0.046) were independently associated with favorable outcome. CONCLUSIONS: Renard diffusion weighted imaging score may be an independent predictor of functional outcome in basilar artery occlusion patients after endovascular treatment.

Chinese Herbal Formula Xuefu Zhuyu for Tension-Type Headache with Qi-Stagnation and Blood-Stasis Pattern (CheruXTH): Study Protocol for a Randomized Controlled Trial.

BACKGROUND: Tension-type headache (TTH) is the most common headache disorder. Current treatments for TTH have been reported to be associated with insufficient long-term benefits and unwanted adverse events (AEs). The Chinese herbal formula Xuefu Zhuyu (XFZY) has been utilized in TTH treatment, but the evidence supporting its efficacy remains unclear. This study will evaluate the efficacy and safety of XFZY for TTH. METHODS: This multicenter, double-blind, randomized, placebo-controlled trial will be undertaken in China. A total of 174 eligible participants will be randomly assigned to either an XFZY group or a placebo group (20 ml each dose, three times daily for 4 weeks) at a ratio of 1 : 1. The primary outcome is the change in mean headache intensity measured by a 10 cm visual analogue scale (VAS). Secondary outcomes include the area-under-the headache curve (AUC), headache frequency, rescue medication use, qi-stagnation and blood-stasis pattern measurement, quality of life measured by the EuroQol-5-Dimensions-5-Level (EQ-5D-5L), global evaluation of medication, and health economic indexes. Discussion. The results of the study are expected to provide evidence of high methodological and reporting quality on the efficacy and safety of XFZY for TTH. This trail is registered with ChiCTR1900026716 (registered on 19 October, 2019).

Chinese Herbal Formula Xuefu Zhuyu for Stable Angina (CheruSA): Study Protocol for a Multicenter Randomized Controlled Trial.

Introduction. Stable angina (SA) in coronary heart disease is a common ischemic heart disease endangering the patient's quality of life and longevity. Clinical trials have demonstrated that the Chinese herbal formula Xuefu Zhuyu (XFZY) has benefits for SA patients. However, there remains a lack of high-quality evidence to support clinical decision-making. Therefore, we designed a randomized controlled trial (RCT) to evaluate the efficacy and safety of XFZY for SA. Methods and Analysis. This multicenter, double-blinded RCT will be conducted in China. 152 eligible participants will be randomly assigned to either an XFZY group or a control group at a 1 : 1 ratio. Participants in the XFZY group will receive XFZY plus routine care, while those in the control group will receive placebo plus routine care. The study period is 26 weeks, including a 2-week run-in period, a 12-week treatment period, and a 12-week follow-up. The primary outcome is the change in visual analogue scale score for angina pain intensity from baseline to 12 weeks. The secondary outcomes are the angina attack frequency and duration, the nitroglycerin dosage consumed, the Canadian Cardiovascular Society grading of effort angina, the Seattle Angina Questionnaire, the EuroQol-5-Dimensions-5-Level, the incidence of major adverse cardiac events, health cost evaluation, and overall assessment for study drugs. Ethics and Dissemination. The study has been approved by the ethics committee of Guangdong Provincial Hospital of Chinese Medicine (approval no. BF2019-175-01). Results will be submitted for publication in peer-reviewed journals and disseminated at scientific conferences. This trial is registered with ChiCTR1900026899, registered on 26 October 2019.

Chinese herbal formula siwutang for treating primary dysmenorrhea: A systematic review and meta-analysis of randomized controlled trials.

INTRODUCTION: Primary dysmenorrhea (PD) is a common gynecological disorder that usually begins in adolescence, and affects patients' daily activities and quality of life. Non-steroidal anti-inflammatory drugs (NSAIDS) are considered the first-line treatment, and hormonal contraceptives are also recommended for PD, but both are prone to side-effects. The Chinese herbal formula Siwutang (SWT) and its derivative formulas are a common treatment for PD in China. This review assessed the efficacy and safety of SWT for the treatment of PD. METHODS: PubMed, EmBase, Cochrane CENTRAL, CNKI, Wanfang and CBM were searched. We included randomized controlled trials (RCTs) that investigated SWT for PD, compared with no intervention, placebo, or conventional Western medicine. The outcome measurements included pain intensity measured by visual analogue scale (VAS) or other validated scales, the Cox Menstrual Symptom Scale (CMSS), quality of life, response rate and adverse events. The Cochrane Collaboration's tool was used to assess the risk of bias. RevMan V.5.3 was used for data synthesis and meta-analysis. Risk ratio (RR) with 95 % confidence intervals (CIs) or mean difference (MD) with 95 % CIs was calculated for dichotomous data or continuous data, respectively. Heterogeneity among studies was evaluated using both a chi-square test and an I2 test. RESULTS: A total of 38 RCTs involving 3982 participants were identified. The methodological quality of the included trials was generally poor. Moreover, the results for SWT compared with placebo were unclear, as there was only 1 RCT. SWT improved pain intensity measured by VAS (3 RCTs, n = 220, MD:-2.61, 95 % CI:-3.72 to -1.51) when compared with conventional medicine, and these results were statistically significant. The meta-analysis showed the superior effect of SWT (including derivative formulas) on response rate (35 RCTs, n = 3,695, RR: 1.28, 95 % CI: 1.22-1.34) with medium heterogeneity (I2 = 48 %). Both original SWT and its derivative formula XFSWT had a higher response rate than conventional medicine (23 RCTs, n = 2,493, RR: 1.28, 95 % CI: 1.23-1.33) (11 RCTs, n = 1,076, RR: 1.36, 95 % CI: 1.20-1.53). These results were statistically significant. No trial reported on quality of life or CMSS. Adverse events were reported by 5 studies, and meta-analysis showed SWT may be safer than conventional medicine in terms of the incidence of adverse events (3 RCTs, n = 236, RR: 0.17, 95 % CI: 0.07-0.38, I2 = 0%). CONCLUSION: In conclusion, the included trials showed favorable effects of SWT for treating primary dysmenorrhea when compared with conventional medicine. SWT may be safer than conventional medicine, but insufficient data was reported. The level of evidence is low because of the high risk of bias. Thus, further well-designed clinical trials with large sample sizes are warranted. REGISTRATION NUMBER: CRD42019136230 in PROSPERO 2019.

The pro-algesic effect of γ-aminobutyric acid (GABA) injection into the masseter muscle of healthy men and women.

Background and aims Preclinical studies have reported that activation of peripheral γ-aminobutyric acid A (GABAA) receptors may result in analgesia. The current study was conducted in young healthy men (n = 30) and women (n = 28) to determine whether injections of GABA into the masseter muscle reduce pain in a sex-related manner. Methods The effect of injection of GABA alone, or in combination with the non-inflammatory algogen glutamate, was assessed in two separate studies. Lorazepam, a positive allosteric modulator of the GABAA-receptor, was co-injected with GABA in both studies to explore the role of this receptor in muscle pain responses of healthy human volunteers. Masticatory muscle mechanical pain intensity was recorded on an electronic visual analogue scale (VAS) while muscle pain sensitivity was assessed by determining the pressure pain threshold (PPT), tolerance and maximal jaw opening (MJO) of the subjects prior to, and again after the various intramuscular injections. Results Intramuscular injection of GABA alone was reported to be significantly more painful, in a concentration related manner, than saline control injections, and this pain was further increased by co-injection of lorazepam with GABA. Co-injection of GABA with glutamate was found to significantly increase glutamate-evoked masseter muscle pain in men, but not in women. There was no effect of injections of either GABA alone, or GABA with glutamate, on PPT, tolerance or maximum jaw opening. Conclusions Injection of GABA into the human masseter muscle appears to excite nociceptors to produce muscle pain without a longer term effect on mechanical pain sensitivity in the muscle. The findings suggest that GABA-mediated pain in humans is produced through peripheral GABAA receptor activation. The mechanism underlying the sex-related difference in the effect of GABA on glutamate-evoked muscle pain was speculated to be due to a methodological artifact. Implications This study was designed to detect analgesic rather than algesic effects of peripherally administered GABA, and as a result, the concentration of glutamate chosen for injection was close to the maximal pain response for healthy women, based on previously determined pain-concentration response relationships for glutamate. This may explain the finding of greater pain in men than women, when GABA and glutamate were co-injected. Overall, the findings suggest that activation of peripheral GABAA receptors in human masticatory muscle produces pain, possibly due to depolarization of the masticatory muscle afferent fibers.

First Report of Arg587Cys Mutation of Notch3 Gene in Two Chinese Families with CADASIL.

OBJECTIVE: To explore Notch3 mutation sites of Chinese patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). METHODS: Direct sequencing of all exons in Notch3 gene was performed on 12 unrelated suspected CADASIL cases from mainland China. RESULT: A missense p.Arg587Cys (1759C>T) mutation in exon 11 was identified in 2 patients through genetic analysis. CONCLUSION: Chinese patients with CADASIL of R587C mutation in exon 11 was firstly reported.

MicroRNA-190b inhibits tumor cell proliferation and induces apoptosis by regulating Bcl-2 in U2OS osteosarcoma cells.

Osteosarcoma (OS) is one of the most prevalent malignancies in bone with no established therapy so far. This study was aimed to clarify the role of miR-190b in tumor cell growth of OS. The miR-190b mimic, inhibitor and miR-control were transfected into human OS U2OS cells. Then U2OS cell proliferation was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) incorporation assay. The apoptotic U2OS cells were detected by flow cytometry. Additionally, cell-cycle regulators p27, p21 and apoptosis factors B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X (Bax), caspase-3 were examined by western blotting. Overexpressing miR-190b observably reduced cell viability, BrdU-positive cells (both P < 0.05) and caused strong accumulation of cell-cycle inhibitor p27 in U2OS cells compared with the miR-control, whereas the miR-190b inhibitor exerted opposite effects. Further, a marked increase of 18% rate of apoptotic cells by the overexpressing miR-190b (P < 0.01) and 4% decrease by miR-190b inhibitor (P < 0.05) were detected. The protein expressions of Bcl-2 were downregulated, Bax, pro-caspase-3 and active caspase-3 were upregulated by overexpressing miR-190b in U2OS cell line, while miR-190b inhibitor achieved opposite effects. The present study demonstrates that miR-190b inhibits tumor cell proliferation and induces apoptosis by regulating Bcl-2 in U2OS cells, which points to miR-190b as a novel oncosuppressor for OS. The identified tumor suppressive capacity of miR-190b provides novel avenues for achieving better OS therapy.