TaoHe ChengQi decoction ameliorates sepsis-induced cardiac dysfunction through anti-ferroptosis via the Nrf2 pathway.

BACKGROUND: Sepsis-induced cardiac dysfunction (SICD) is a serious complication of sepsis that is associated with increased mortality. Ferroptosis has been reported in the SICD. TaoHe ChengQi decoction (THCQD), a classical traditional Chinese medicinal formula, has multiple beneficial pharmacological effects. The potential effects of THCQD on the SICD remain unknown. PURPOSE: To investigate the effect of THCQD on SICD and explore whether this effect is related to the regulation of myocardial ferroptosis through nuclear factor erythroid 2-related factor 2 (Nrf2) activation. METHODS: We induced sepsis in a mouse model using cecal ligation and puncture (CLP) and administered THCQD (2 and 4 g/kg) and dexamethasone (40 mg/kg). Mice mortality was recorded and survival curves were plotted. Echocardiography, hematoxylin and eosin staining, and analysis of serum myocardial injury markers and inflammatory factors were used to evaluate cardiac pathology. Myocardial ferroptosis was detected by quantifying specific biomarker content and protein levels. Through HPLC-Q-Exactive-MS analysis, we identified the components of the THCQD. Network pharmacology analysis and Cellular Thermal Shift Assay (CETSA) were utilized to predict the targets of THCQD for treating SICD. We detected the expression of Nrf2 using Western blotting or immunofluorescence. An RSL3-induced ferroptosis model was established using neonatal rat cardiomyocytes (NRCMs) to further explore the pharmacological mechanism of THCQD. In addition to measuring cell viability, we observed changes in NRCM mitochondria using electron microscopy and JC-1 staining. NRF2 inhibitor ML385 and Nrf2 knockout mice were used to validate whether THCQD exerted protective effects against SICD through Nrf2-mediated ferroptosis signaling. RESULTS: THCQD reduced mortality in septic mice, protected against CLP-induced myocardial injury, decreased systemic inflammatory response, and prevented myocardial ferroptosis. Network pharmacology analysis and CETSA experiments predicted that THCQD may protect against SICD by activating the Nrf2 signaling pathway. Western blotting and immunofluorescence showed that THCQD activated Nrf2 in cardiac tissue. THCQDs consistently mitigated RSL3-induced ferroptosis in NRCM, which is related to Nrf2. Furthermore, the pharmacological inhibition of Nrf2 and genetic Nrf2 knockout partially reversed the protective effects of THCQD on SICD and ferroptosis. CONCLUSION: The effect of THCQD on SICD was achieved by activating Nrf2 and its downstream pathways.

Ethyl ferulate suppresses post-myocardial infarction myocardial fibrosis by inhibiting transforming growth factor receptor 1.

BACKGROUND: With an increasing number of myocardial infarction (MI) patients, myocardial fibrosis is becoming a widespread health concern. It's becoming more and more urgent to conduct additional research and investigations into efficient treatments. Ethyl ferulate (EF) is a naturally occurring substance with cardioprotective properties. However, the extent of its impact and the underlying mechanism of its treatment for myocardial fibrosis after MI remain unknown. PURPOSE: The goal of this study was to look into how EF affected the signaling of the TGF-receptor 1 (TGFBR1) in myocardial fibrosis after MI. METHODS: Echocardiography, hematoxylin-eosin (HE) and Masson trichrome staining were employed to assess the impact of EF on heart structure and function in MI-affected mice in vivo. Cell proliferation assay (MTS), 5-Ethynyl-2'-deoxyuridine (EdU), and western blot techniques were employed to examine the influence of EF on native cardiac fibroblast (CFs) proliferation and collagen deposition. Molecular simulation and surface plasmon resonance imaging (SPRi) were utilized to explore TGFBR1 and EF interaction. Cardiac-specific Tgfbr1 knockout mice (Tgfbr1ΔMCK) were utilized to testify to the impact of EF. RESULTS: In vivo experiments revealed that EF alleviated myocardial fibrosis, improved cardiac dysfunction after MI and downregulated the TGFBR1 signaling in a dose-dependent manner. Moreover, in vitro experiments revealed that EF significantly inhibited CFs proliferation, collagen deposition and TGFBR1 signaling followed by TGF-ß1 stimulation. More specifically, molecular simulation, molecular dynamics, and SPRi collectively showed that EF directly targeted TGFBR1. Lastly, knocking down of Tgfbr1 partially reversed the inhibitory activity of EF on myocardial fibrosis in MI mice. CONCLUSION: EF attenuated myocardial fibrosis post-MI by directly suppressing TGFBR1 and its downstream signaling pathway.

Radix Glycyrrhizae extract and licochalcone a exert an anti-inflammatory action by direct suppression of toll like receptor 4.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Glycyrrhizae (GL), a herbal medicine that is widely available, has shown advantages for a variety of inflammatory diseases. Toll like receptor 4 (TLR4) pathway has been shown to play a key role in the progression of inflammation. AIM OF THE STUDY: The purpose of this study was to investigate the involvement of TLR4 in the anti-inflammatory mechanism of GL extract and its active constituent on acute lung injury (ALI). MATERIALS AND METHODS: A model of inflammation produced by lipopolysaccharide (LPS) was established in C57BL/6 mice and macrophages derived from THP-1. To screen the active components of GL, molecular docking was used. Molecular dynamics and surface plasmon resonance imaging (SPRi) were used to study the interaction of a specific drug with the TLR4-MD2 complex. TLR4 was overexpressed by adenovirus to confirm TLR4 involvement in the anti-inflammatory activities of GL and the chosen chemical. RESULTS: We observed that GL extract significantly reduced both LPS-induced ALI and the production of pro-inflammatory factors including TNF-α, IL-6 and IL-1ß. Additionally, GL inhibited the binding of Alexa 488-labeled LPS (LPS-488) to the membrane of THP-1 derived macrophages. GL drastically reduce on the expression of TLR4 and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-κB). Furthermore, molecular docking revealed that Licochalcone A (LicoA) docked into the LPS binding site of TLR4-MD2 complex. MD2-LicoA binding conformation was found to be stable using molecular dynamic simulations. SPRi indicated that LicoA bound to TLR4-MD2 recombinant protein with a KD of 3.87 × 10-7 M. LicoA dose-dependently reduced LPS-488 binding to the cell membrane. LicoA was found to significantly inhibit LPS-induced lung damage and inflammation. Furthermore, LicoA inhibited TLR4 expression, MAPK and NF-κB activation in a dose-dependent manner. The inhibitory effects of GL and LicoA on LPS-induced inflammation and TLR4 signaling activation were partly eliminated by TLR4 overexpression. CONCLUSION: Our findings imply that GL and LicoA exert inhibitory effects on inflammation by targeting the TLR4 directly.

Calycosin protects against oxidative stress-induced cardiomyocyte apoptosis by activating aldehyde dehydrogenase 2.

Myocardial infarction (MI) is the leading cause of death worldwide, and oxidative stress is part of the process that causes MI. Calycosin, a naturally occurring substance with cardioprotective properties, is one of the major active constituents in Radix Astragali. In this study, effect of Calycosin was investigated in vivo and in vitro to determine whether it could alleviate oxidative stress and oxidative stress-induced cardiac apoptosis in neonatal cardiomyocytes (NCMs) via activation of aldehyde dehydrogenase 2 (ALDH2). Calycosin protected against oxidative stress and oxidative stress-induced apoptosis in NCMs. Molecular docking revealed that the ALDH2-Calycosin complex had a binding energy of -9.885 kcal/mol. In addition, molecular docking simulations demonstrated that the ALDH2-Calycosin complex was stable. Using BLI assays, we confirmed that Calycosin could interact with ALDH2 (KD  = 1.9 × 10-4 M). Furthermore, an ALDH2 kinase activity test revealed that Calycosin increased ALDH2 activity, exhibiting an EC50 of 91.79 µM. Pre-incubation with ALDH2 inhibitor (CVT-10216 or disulfiram) reduced the cardio-protective properties Calycosin. In mice with MI, Calycosin therapy substantially reduced myocardial apoptosis, oxidative stress, and activated ALDH2. Collectively, our findings clearly suggest that Calycosin reduces oxidative stress and oxidative stress-induced apoptosis via the regulation of ALDH2 signaling, which supports potential therapeutic use in MI.

Acupuncture improves the symptoms, intestinal microbiota, and inflammation of patients with mild to moderate Crohn's disease: A randomized controlled trial.

BACKGROUND: The efficacy and mechanisms of acupuncture for Crohn's disease (CD) are not well understood. We investigated its effects on symptoms, intestinal microbiota, and circulating inflammatory markers in CD patients. METHODS: This 48-week, randomized, sham controlled, parallel-group clinical trial was performed at a tertiary outpatient clinic in China. From April 2015 to November 2019, 66 patients (mean age 40·4, 62·1% were male, all were Han Chinese) with mild to moderate active CD and unresponsive to drug treatment were enrolled and randomly assigned equally to an acupuncture group or a sham group. The treatment group received 3 sessions of acupuncture plus moxibustion per week for 12 weeks and a follow-up of 36 weeks. Clinicaltrials.gov: NCT02559037. FINDINGS: At week 12, the clinical remission rate (the primary outcome) and clinical response rate of acupuncture group were significantly higher than that of sham group, with a difference of 42·4% (95% CI: 20·1%-64·0%) and 45·5% (95% CI: 24·0%-66·9%), respectively, both of which maintained at week 48. The acupuncture group had significantly lower CD activity index and C-reactive protein level at week 12, which maintained at 36-week follow-up. The CD endoscopic index of severity, histopathological score, and recurrence rate at week 48 were significantly lower in acupuncture group. The number of operational taxonomic unit of intestinal microbiota and relative abundance of Faecalibacterium prausnitzii and Roseburia faecis were increased. Plasma diamine oxidase, lipopolysaccharide, and Th1/Th17 related cytokines were decreased in 12-week after acupuncture. INTERPRETATION: Acupuncture was effective in inducing and maintaining remission in patients with active CD, which was associated with increased abundance of intestinal anti-inflammatory bacteria, enhanced intestinal barrier, and regulation of circulating Th1/Th17-related cytokines. FUNDING: National Key Basic Research Program of China (2015CB554500 and 2009CB522900), Shanghai Rising-Star Program (19QA1408100).

Dihydrotanshinone I inhibits the growth of hepatoma cells by direct inhibition of Src.

BACKGROUND: Liver cancer is one of the leading causes of cancer-related death worldwide. Dihydrotanshinone I (DHI) was shown to inhibit the growth of several types of cancer. However, research related to hepatoma treatment using DHI is limited. PURPOSE: Here, we explored the inhibitory effect of DHI on the growth of hepatoma cells, and investigated the underlying molecular mechanisms. METHODS: The proliferation of Hep3B, SMCC-7721 and SK-Hep1 hepatoma cells was evaluated using the MTS and Edu staining assay. Hepatoma cell death was analyzed with a LIVE/DEAD Cell Imaging Kit. The relative expression and phosphorylation of proto-oncogene tyrosine-protein kinase Src (Src) and signal transducer and activator of transcription-3 (STAT3) proteins in hepatoma cells, as well as the expression of other protein components, were measured by western blotting. The structural interaction of DHI with Src proteins was evaluated by molecular docking, molecular dynamics simulation, surface plasmon resonance imaging and Src kinase inhibition assay. Src overexpression was achieved by infection with an adenovirus vector encoding human Src. Subsequently, the effects of DHI on tumor growth inhibition were further validated using mouse xenograft models of hepatoma. RESULTS: In vitro studies showed that treatment with DHI inhibited the proliferation and promoted cell death of Hep3B, SMCC-7721 and SK-Hep1 hepatoma cells. We further identified and verified Src as a direct target of DHI by using molecular stimulation, surface plasmon resonance image and Src kinase inhibition assay. Treatment with DHI reduced the in vitro phosphorylation levels of Src and STAT3, a transcription factor regulated by Src. In the xenograft mouse models, DHI dose-dependently suppressed tumor growth and Src and STAT3 phosphorylation. Moreover, Src overexpression partly abrogated the inhibitory effects of DHI on the proliferation and cell death in hepatoma cells. CONCLUSION: Our results suggest that DHI inhibits the growth of hepatoma cells by direct inhibition of Src.

Green tea protects against hippocampal neuronal apoptosis in diabetic encephalopathy by inhibiting JNK/MLCK signaling.

Although diabetic encephalopathy (DE) is a major late complication of diabetes, the pathophysiology of postural instability in DE remains poorly understood. Prior studies have suggested that neuronal apoptosis is closely associated with cognitive function, but the mechanism remains to be elucidated. Green tea, which is a non­fermented tea, contains a number of tea polyphenols, alkaloids, amino acids, polysaccharides and other components. Some studies have found that drinking green tea can reduce the incidence of neurodegenerative diseases and improve cognitive dysfunction. We previously found that myosin light chain kinase (MLCK) regulates apoptosis in high glucose­induced hippocampal neurons. In neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease, activation of the JNK signaling pathway promotes neuronal apoptosis. However, the relationship between JNK and MLCK remains to be elucidated. Green tea serum was obtained using seropharmacological methods and applied to hippocampal neurons. In addition, a type 1 diabetes rat model was established and green tea extract was administered, and the Morris water maze test, Cell Counting Kit­8 assays, flow cytometry, western blotting and terminal deoxynucleotidyl transferase­mediated dUTP nick end­labelling assays were used to examine the effects of green tea on hippocampal neuronal apoptosis in diabetic rats. The results demonstrated that green tea can protect against hippocampal neuronal apoptosis by inhibiting the JNK/MLCK pathway and ultimately improves cognitive function in diabetic rats. The present study provided novel insights into the neuroprotective effects of green tea.

Dauricine inhibits proliferation and promotes death of melanoma cells via inhibition of Src/STAT3 signaling.

Melanoma is the most common type of skin cancer. Signal transducer and activator of transcription 3 (STAT3) signaling has been demonstrated to be a therapeutic target for melanoma. Dauricine (Dau), an alkaloid compound isolated from the root of Menispermum dauricum DC., has shown tumor-suppressing effects in multiple human cancers, but its potential in melanoma remains unexplored. In this study, we demonstrated that Dau significantly inhibited the viability and proliferation of A375 and A2058 melanoma cells. Death of melanoma cells was also markedly promoted by Dau. Moreover, Dau inhibited phosphorylation-mediated activation of STAT3 and Src in a dose-dependent manner. Notably, constitutive activation of Src partially abolished the antiproliferative and cytotoxic activities of Dau on melanoma cells. Molecular docking showed that Dau could dock on the kinase domain of Src with a binding energy of -10.42 kcal/mol. Molecular dynamics simulations showed that Src-Dau binding was stable. Surface plasmon resonance imaging analysis also showed that Dau has a strong binding affinity to Src. In addition, Dau suppressed the growth of melanoma cells and downregulated the activation of Src/STAT3 in a xenograft model in vivo. These data demonstrated that Dau inhibits proliferation and promotes cell death in melanoma cells by inhibiting the Src/STAT3 pathways.

Taohong siwu decoction attenuates myocardial fibrosis by inhibiting fibrosis proliferation and collagen deposition via TGFBR1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Myocardial fibrosis after myocardial infarction (MI) leads to cardiac remodeling and loss of function. Taohong siwu decoction (THSWD), a well-known traditional Chinese medicinal prescription, has been clinically used to treat various cardiovascular and cerebrovascular diseases, but its potential functions in myocardial fibrosis after MI remain uncharacterized. AIM OF THE STUDY: The purpose of current study was to explore the potential mechanism action and anti-myocardial fibrosis effects of treatment with THSWD in vivo and in vitro. MATERIALS AND METHODS: Mouse underwent ligation of coronary artery to induce MI and divided equally into the sham group, model group and THSWD treatment groups. After 4 weeks, the effects of THSWD treatment on cardiac function were estimated by echocardiography. HE staining was used to detect the pathologic changes and Masson trichrome staining was used to estimate tissue fibrosis. To further explore the regulatory molecular mechanisms of THSWD, transcriptome analysis was performed. Furthermore, in vitro, we investigated the effect of THSWD on cell proliferation and collagen deposition in primary cardiac fibrosis cells and its possible mechanism of action. Overexpression of TGFBR1 was achieved by infection with an adenovirus vector encoding TGFBR1. RESULTS: Treatment with THSWD significantly decreased myocardial fibrosis and recovered cardiac function in the post-MI mouse. The transcriptomics data imply that the TGF-ß pathway might be a target in the anti-fibrosis effect of THSWD. THSWD inhibits TGF-ß1-induced proliferation of primary cardiac fibroblasts. THSWD decreased collagen expression and TGFBR1 and Smad2/3 phosphorylation. Moreover, the inhibitory effect of THSWD on CFs proliferation and collagen deposition, as well as TGFBR1 signaling pathway-associated proteins expression was partially abrogated by overexpression of TGFBR1. CONCLUSION: Collectively, the results implicate that THSWD attenuates myocardial fibrosis by inhibiting fibrosis proliferation and collagen deposition via inhibiting TGFBR1, and might be a potential therapeutic agent for treatment of myocardial fibrosis post-MI.

Arenobufagin Promoted Oxidative Stress-Associated Mitochondrial Pathway Apoptosis in A549 Non-Small-Cell Lung Cancer Cell Line.

Arenobufagin (ARE) has demonstrated potent anticancer activity in various types of tumor, but the role and mechanism of ARE for lung cancer remain unclear. Oxidative stress exists under normal conditions and is an inevitable state in the body. A variety of noxious stimuli can break the equilibrium state of oxidative stress and promote apoptosis. Here, we used a CCK-8 assay to examine cell viability. We determined oxidative stress damage by measuring levels of intracellular ROS and levels of GSH, SOD, and MDA. Annexin V-FITC/PI double staining assay, as well as the Hoechst 33258 staining, was used to detect ARE-induced apoptosis in A549 cell. Evaluation of the expression level of the specified molecule was indicated by Western blot and qRT-PCR. Loss of function experiment was carried out using NAC pretreatment. The experimental results show that ARE significantly declines in the viability of A549 cells and increases the apoptosis rate of A549 cells. As reflected in cell morphology, the A549 cells showed features of shrinkage and had incompletely packed membranes; the same phenomenon is manifested in Hoechst 33258 staining. Following ARE treatment, the ROS level in A549 cells was rising in a concentration-dependent manner, and so were MDA and GSH levels, while the SOD level was decreasing. Moreover, we found that ARE can decrease mitochondrial membrane potential (MMP), and a cascade of apoptotic processes can be triggered by decreased MMP. Importantly, we found significant changes in protein expression levels and mRNA levels of apoptosis-related proteins. Furthermore, when we used NAC to restrain oxidative stress, the expression levels of apoptosis-related proteins have also changed accordingly. Our data demonstrate that apoptosis in the non-small-cell lung cancer (NSCLC) cell line A549 is caused by oxidative stress due to ARE. Our research also shows that ARE may have the potential to become a targeted therapeutic for the treatment of NSCLC in the future.

Chinese Herbal Medicine Interventions in Neurological Disorder Therapeutics by Regulating Glutamate Signaling.

Glutamate is the major excitatory neurotransmitter in the central nervous system, and its signaling is critical for excitatory synaptic transmission. The well-established glutamate system involves glutamate synthesis, presynaptic glutamate release, glutamate actions on the ionotropic glutamate receptors (NMDA, AMPA, and kainate receptors) and metabotropic glutamate receptors, and glutamate uptake by glutamate transporters. When the glutamate system becomes dysfunctional, it contributes to the pathogenesis of neurodegenerative and neuropsychiatric diseases such as Alzheimer's disease, Parkinson's disease, depression, epilepsy, and ischemic stroke. In this review, based on regulating glutamate signaling, we summarize the effects and underlying mechanisms of natural constituents from Chinese herbal medicines on neurological disorders. Natural constituents from Chinese herbal medicine can prevent the glutamate-mediated excitotoxicity via suppressing presynaptic glutamate release, decreasing ionotropic and metabotropic glutamate receptors expression in the excitatory synapse, and promoting astroglial glutamate transporter expression to increase glutamate clearance from the synaptic cleft. However, some natural constituents from Chinese herbal medicine have the ability to restore the collapse of excitatory synapses by promoting presynaptic glutamate release and increasing ionotropic and metabotropic glutamate receptors expression. These regulatory processes involve various signaling pathways, which lead to different mechanistic routes of protection against neurological disorders. Hence, our review addresses the underlying mechanisms of natural constituents from Chinese herbal medicines that regulate glutamate systems and serve as promising agents for the treatment of the above-mentioned neurological disorders.

A traditional Chinese medicine therapy for coronary heart disease after percutaneous coronary intervention: a meta-analysis of randomized, double-blind, placebo-controlled trials.

Huoxue Huayu therapy (HXHY) has been widely used to treat cardiovascular diseases in traditional Chinese medicine (TCM) such as hypertension and coronary heart disease (CHD). The present study describes a meta-analysis of a series of prospective randomized, double-blind, placebo-controlled trials conducted to evaluate the effect of HXHY on patients with CHD after percutaneous coronary intervention (PCI). The Cochrane Library, PubMed, EMBASE, the China National Knowledge Infrastructure (CNKI), the Chinese Biomedical Literature database, and the Wanfang database were searched up until June 2018. A series of randomized controlled clinical trials were included and the subjects were patients with CHD who had undergone PCI. The experimental group was treated with HXHY therapy, and the control group was treated with placebo; meanwhile, all the patients accepted conventional Western medicine. Review Manager 5.3 software was used for the statistical analysis. Ten trials were included in the final study. The overall risk of bias assessment was low. HXHY had a greater beneficial effect on reducing the in-stent restenosis (ISR) rate (RR = 0.57, 95% confidence interval [CI] [0.40-0.80], P=0.001) and the degree of restenosis (MD = -8.89, 95% CI [-10.62 to -7.17], P<0.00001) compared with Placebo. Moreover, HXHY was determined to be more effective in improving Seattle Angina Questionnaires (SAQ) and the revascularization rate (RR = 0.54, 95% CI [0.32-0.90], P=0.02) compared with Placebo, whereas the rate of death and MI of patients treated with HXHY were no different from those treated with the placebo (P>0.05). Therefore, HXHY is an effective and safe therapy for CHD patients after PCI.

Different brain responses to electro-acupuncture and moxibustion treatment in patients with Crohn's disease.

This study aimed to investigate changes in resting state brain activity in remissive Crohn's Disease (CD) patients after electro-acupuncture or moxibustion treatment. Fifty-two CD patients and 36 healthy subjects were enrolled, and 36 patients were equally and randomly assigned to receive either electro-acupuncture or moxibustion treatment for twelve weeks. We used resting state functional magnetic resonance imaging to assess Regional Homogeneity (ReHo) levels, and Crohn's Disease Activity Index (CDAI) and Inflammatory Bowel Disease Questionnaire (IBDQ) scores to evaluate disease severity and quality of life. The results show that (i) The ReHo levels in CD patients were significantly increased in cortical but decreased in subcortical areas, and the coupling between them was declined. (ii) Both treatments decreased CDAI, increased IBDQ scores, and normalized the ReHo values of the cortical and subcortical regions. (iii) ReHo changes in multiple cortical regions were significantly correlated with CDAI score decreases. ReHo changes in several subcortical regions in the electro-acupuncture group, and those of several cortical regions in the moxibustion group, were correlated with reduced CDAI. These findings suggest that both treatments improved cortex-subcortical coupling in remissive CD patients, but electro-acupuncture regulated homeostatic afferent processing network, while moxibustion mainly regulated the default mode network of the brain.

Moxibustion treatment for diarrhea-predominant irritable bowel syndrome: study protocol for a randomized controlled trial.

BACKGROUND: Irritable bowel syndrome (IBS) is a worldwide disease with high morbidity. The effect of current treatment with Western medicine is not satisfactory. Although moxibustion treatment is widely used for gastrointestinal diseases, randomized controlled trials on the use of this treatment for IBS are limited. This study aims to evaluate the clinical efficacy and safety of moxibustion treatment in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). METHODS/DESIGN: A multi-center, randomized, single-blind and placebo-controlled trial is employed. 104 cases will be divided into two groups: (1) a mild-warm moxibustion group in which moxa stick is 3-5 cm away from acupuncture points and the skin temperature is maintained at 43 ± 1 °C; and (2) a placebo moxibustion group in which moxa stick is 8-10 cm away from acupuncture points and the skin temperature is maintained at 37 ± 1 °C. Moxibustion is performed on bilateral ST25 and ST36 in the two groups for 30 min each time, three times a week for 6 weeks. The patients are followed up at the 12th and 18th weeks. Adequate relief is used as a primary outcome measure; IBS symptom severity score, Bristol stool form scale, IBS quality-of-life questionnaire, and hospital anxiety and depression scale are used as secondary outcome measures. DISCUSSION: This study aims to demonstrate the safety and efficacy of moxibustion treatment for IBS-D, which may validate moxibustion as an effective therapy for treating IBS-D. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02421627 (8 April 2015).

Transgenic human alpha-hemoglobin stabilizing protein could partially relieve betaIVS-2-654-thalassemia syndrome in model mice.

Beta-thalassemia is an anemia caused by a relative excess of alpha-hemoglobin (alphaHb) due to absent or reduced beta-hemoglobin (betaHb) synthesis. In this study, we explore whether the introduction of alpha-hemoglobin stabilizing protein (AHSP), a chaperone protein for proper folding and stabilization of free alphaHb in red blood cells, thus aiding hemoglobin A (HbA) assembly, could relieve the pathogenic state of red blood cells in beta-thalassemia. For that, a human ahsp vector was constructed to generate transgenic human ahsp mice in a model of beta(IVS-2-654)-thalassemia by microinjecting the vector into fertilized eggs, resulting in the production of double heterozygous mice (h-ahsp(+)/beta(IVS-2-654+)). Real-time quantitative RT-PCR and Western blot analysis confirmed AHSP expression in three h-ahsp(+)/beta(IVS-2-654+) mice. Hematologic determination showed an improvement in the red blood cell indices of these h-ahsp(+)/beta(IVS-2-654+) mice. The red blood cell count and hemoglobin level were elevated to various extents as compared with their diseased siblings. A dramatic reduction in anisocytosis in the peripheral blood of h-ahsp(+)/beta(IVS-2-654+) mice was observed (16.2 +/- 4.6 vs. 30.0 +/- 5.2%). Few erythroid precursors appeared in the liver sinusoids of h-ahsp(+)/beta(IVS-2-654+) mice. Splenomegaly with extramedullary hematopoiesis was also ameliorated. Significantly, serum iron concentration was remarkably reduced as compared with that of h-ahsp(-)/beta(IVS-2-654+) mice (43.2 +/- 14.9 vs. 82.4 +/- 12.9 microM), and iron deposition in the liver was decreased in h-ahsp(+)/beta(IVS-2-654+) mice. All these results suggested amelioration of the anemia phenotype in h-ahsp(+)/beta(IVS-2-654+) mice after introduction of the ahsp gene. We therefore propose that an ahsp transgene could provide an adjuvant method for gene therapy of beta-thalassemia.