Empagliflozin and liraglutide ameliorate HFpEF in mice via augmenting the Erbb4 signaling pathway.

Heart failure with preserved ejection fraction (HFpEF) is closely associated with metabolic derangement. Sodium glucose cotransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) exert anti-HFpEF effects, but the underlying mechanisms remain unclear. In this study, we explored the anti-HFpEF effects of empagliflozin and liraglutide and the underlying molecular mechanisms in a mouse model of HFpEF. This model was established by high-fat diet (HFD) feeding plus Nω-nitro-L-arginine methyl ester (L-NAME) treatment. The mice were treated with empagliflozin (20 mg·kg-1·d-1, i.g.) or liraglutide (0.3 mg·kg-1·d-1, i.p.) or their combination for 4 weeks. At the end of the experimental protocol, cardiac function was measured using ultrasound, then mice were euthanized and heart, liver, and kidney tissues were collected. Nuclei were isolated from frozen mouse ventricular tissue for single-nucleus RNA-sequencing (snRNA-seq). We showed that administration of empagliflozin or liraglutide alone or in combination significantly improved diastolic function, ameliorated cardiomyocyte hypertrophy and cardiac fibrosis, as well as exercise tolerance but no synergism was observed in the combination group. Furthermore, empagliflozin and/or liraglutide lowered body weight, improved glucose metabolism, lowered blood pressure, and improved liver and kidney function. After the withdrawal of empagliflozin or liraglutide for 1 week, these beneficial effects tended to diminish. The snRNA-seq analysis revealed a subcluster of myocytes, in which Erbb4 expression was down-regulated under HFpEF conditions, and restored by empagliflozin or liraglutide. Pseudo-time trajectory analysis and cell-to-cell communication studies confirmed that the Erbb4 pathway was a prominent pathway essential for both drug actions. In the HFpEF mouse model, both empagliflozin and liraglutide reversed Erbb4 down-regulation. In rat h9c2 cells, we showed that palmitic acid- or high glucose-induced changes in PKCα and/or ERK1/2 phosphorylation at least in part through Erbb4. Collectively, the single-cell atlas reveals the anti-HFpEF mechanism of empagliflozin and liraglutide, suggesting that Erbb4 pathway represents a new therapeutic target for HFpEF. Effects and mechanisms of action of empagliflozin and liraglutide in HFpEF mice. HFpEF was induced with a high-fat diet and L-NAME for 15 weeks, and treatment with empagliflozin and liraglutide improved the HFpEF phenotype. Single nucleus RNA sequencing (snRNA-seq) was used to reveal the underlying mechanism of action of empagliflozin and liraglutide.

Corrosion Inhibition Studies of 8-Hydroxyquinoline Derivatives for N80 Steel in a 1.0 M HCl Solution: Experimental, Computational Chemistry, and Quantitative Structure-Activity Relationship Studies.

Twelve kinds of 8-hydroxyquinoline derivatives were synthesized and characterized. The weight loss method was used to evaluate their inhibition efficiencies (IEs) in a 1.0 M HCl solution at 333 K. The results showed that the alkyl chain length, heteroatoms (S, N, and O), and number of benzene rings significantly affect the IE. Herein, the IE of 5-[(dodecylthio)methyl]-8-quinolinol reached 98.71%. Meanwhile, the potentiodynamic polarization results indicated that all 8-hydroxyquinoline derivatives were mixed-type inhibitors. Electrochemical impedance spectroscopy results revealed that 8-hydroxyquinoline derivatives can increase polarization resistance, supporting their adsorption on the N80 steel surface. Moreover, according to density functional theory (DFT), the frontier orbital distribution and quantum chemical parameters (EHOMO, ELUMO, dipole moment µ, etc.) were calculated, and the results confirmed that the substituents of protonated 8-hydroxyquinoline derivatives significantly influenced the frontier orbital distribution. Molecular dynamics simulation illustrated that all protonated 8-hydroxyquinoline derivatives were adsorbed parallel to the Fe(110) surface, and the interaction energy (Eint) evidenced that the molecular size would affect their strength of adsorption on the Fe(110) surface. The linear and nonlinear quantitative structure-activity relationship models were established by linear regression (LR) methods and BP neural networks (NN), respectively. The LR model was established by using Eint and µ, and the coefficient of determination (R2) was 0.934. In addition, the nonlinear NN model was obtained according to IE and all parameters (DFT parameters and Eint). Then, the two calculation inhibition efficiencies (IEcal) were obtained from the LR and NN models, and the R2 values of the linear correlation between the IEcal and the experimental IE were 0.940 and 0.951, respectively. In addition, the IE of the tested inhibitor was 51.86% and the IEcal values predicted by the LR and NN models were 52.68% and 53.06%, respectively. Our results demonstrate that both the LR and NN models have good fits and predictive ability.

Wogonin inhibits hepatoma cell proliferation by targeting miR-27b-5p/YWHAZ axis.

Wogonin (5,7-dihydroxy-8-methoxyflavone), a natural flavonoid compound in herbal plants, can suppress growth in hepatocellular carcinoma (HCC). However, the microRNA (miRNA) expression profiles that are influenced by wogonin have not been thoroughly described. To explore the novel miRNAs and the biological mechanism underlying the effect of wogonin on HCC cells. The effect of wogonin on Huh7 cell growth was assessed both in vitro and in vivo. The expression profiles of miRNAs were obtained by small RNA sequencing. Luciferase reporter experiment and bioinformatics analysis were conducted to determine whether tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) can bind to miR-27b-5p. Effects of the ectopic expression of YWHAZ and miR-27b-5p on Huh7 cells proliferation and apoptosis were evaluated. Furthermore, the cell cycle, apoptosis and multiple signaling pathway-related molecules were detected by Western blot analysis. Wogonin substantially inhibited the growth of Huh7 cells both in vitro and in vivo. Seventy miRNAs exhibited greater than twofold changes in wogonin-treated cells. Upregulation of miR-27b-5p inhibited Huh7 cell proliferation, and the anticancer effect of wogonin was reversed after miR-27b-5p knockdown. miR-27b-5p directly targeted YWHAZ in HCC cells. The proliferation-inhibiting effect of miR-27b-5p was revoked by YWHAZ overexpression. Meanwhile, inhibition of HCC growth was achieved by downregulating YWHAZ. Wogonin exerted antitumor activity through multiple signaling molecules, such as focal adhesion kinase, protein kinase B, mammalian target of rapamycin and molecules related to apoptosis and cell cycle by upregulating miR-27b-5p and downregulating YWHAZ. Our findings suggest that miR-27b-5p/YWHAZ axis contributes to the inhibitory effect of wogonin in HCC by targeting related genes and multiple signaling pathways.

[Network Meta-analysis of traditional Chinese medicine injections against anthracycline-induced cardiac injury].

This study aimed to systematically evaluate the effect of traditional Chinese medicine(TCM) injections on anthracycline-induced cardiac injury. The Cochrane Library, PubMed, EMbase, CNKI, and other databases were electronically retrieved to gather randomized controlled trials(RCTs) of TCM injections against anthracycline-induced cardiac injury from their inception to September 2021. After two research fellows independently screened the literature and extracted the data, the risk of bias of included RCTs was assessed and network Meta-analysis was performed by R 4.1.0 and Stata 15.1. A total of 50 RCTs were included, involving eight TCM injections. Network Meta-analysis showed that:(1)the combination of anthracyclines with Huachansu Injection might be the optimal treatment to reduce the abnormal electrocardiogram.(2)The combination with Shenfu Injection might be the optimum treatment to ameliorate the left ventricular ejection fraction(LVEF) decrease.(3)The combination with Shenqi Fuzheng Injection might reduce the incidence of cardiotoxicity most satisfactorily.(4)The combination with Xinmailong Injection might improve the elevated cardiac troponin I(cTnI) optimally.(5)The combination with Shenmai Injection might be optimal to control the rise of creatine kinase MB isoenzyme(CK-MB).(6)The combination with Kushen Injection might be the regimen with the lowest gastrointestinal reactions. TCM injections had desirable effect on anthracycline-induced cardiac injury, with low incidence of adverse reactions, and each TCM injection had its own unique advantages. Due to the limitations in quality and methodological conduct of the included studies, more high-level RCTs are needed to validate the conclusions.

Neuromodulatory Effects of Transcranial Direct Current Stimulation on Motor Excitability in Rats.

Transcranial direct current stimulation (tDCS) is a noninvasive technique for modulating neural plasticity and is considered to have therapeutic potential in neurological disorders. For the purpose of translational neuroscience research, a suitable animal model can be ideal for providing a stable condition for identifying mechanisms that can help to explore therapeutic strategies. Here, we developed a tDCS protocol for modulating motor excitability in anesthetized rats. To examine the responses of tDCS-elicited plasticity, the motor evoked potential (MEP) and MEP input-output (IO) curve elicited by epidural motor cortical electrical stimulus were evaluated at baseline and after 30 min of anodal tDCS or cathodal tDCS. Furthermore, a paired-pulse cortical electrical stimulus was applied to assess changes in the inhibitory network by measuring long-interval intracortical inhibition (LICI) before and after tDCS. In the results, analogous to those observed in humans, the present study demonstrates long-term potentiation- (LTP-) and long-term depression- (LTD-) like plasticity can be induced by tDCS protocol in anesthetized rats. We found that the MEPs were significantly enhanced immediately after anodal tDCS at 0.1 mA and 0.8 mA and remained enhanced for 30 min. Similarly, MEPs were suppressed immediately after cathodal tDCS at 0.8 mA and lasted for 30 min. No effect was noted on the MEP magnitude under sham tDCS stimulation. Furthermore, the IO curve slope was elevated following anodal tDCS and presented a trend toward diminished slope after cathodal tDCS. No significant differences in the LICI ratio of pre- to post-tDCS were observed. These results indicated that developed tDCS schemes can produce consistent, rapid, and controllable electrophysiological changes in corticomotor excitability in rats. This newly developed tDCS animal model could be useful to further explore mechanical insights and may serve as a translational platform bridging human and animal studies, establishing new therapeutic strategies for neurological disorders.

Relationship Between Modic Changes and Sagittal Balance Parameters in the Cervical Spine.

BACKGROUND We explored the possible relationship between Modic changes (MCs) and sagittal parameters of the cervical spine. MATERIAL AND METHODS We enrolled 150 patients with cervical MC on the magnetic resonance imaging (MRI) scans in the MC (+) group and divided them into 3 sub-groups with 50 patients each: the MC1 sub-group, the MC2 sub-group, and the MC3 sub-group. Another 150 healthy subjects receiving routine health examinations were also enrolled in the study as the MC (-) group. The sagittal parameters in the cervical spine were measured and compared and multiple logistic regression analysis was performed to analyze the risk factor for the occurrence of MC. RESULTS Four cervical sagittal parameters were measured and compared between all the enrolled groups, including neck tilt (NT), T1 slope (T1s), thoracic inlet angle (TIA), and Cobb C2-C7. The results confirmed that the parameter of Cobb C2-C7 was much smaller in the MC(+) group when compared with that in the MC(-) group (P<0.05), while no significant differences were detected between the MC(+) and MC(-) groups for the parameters of NT, T1 T1s, and TIA (P>0.05). Multiple logistic regression analysis showed that Cobb C2-C7 (less than 8.5°) could be regarded as the risk factor for the occurrence of MC, and the receiver operating characteristic (ROC) curve showed that moderate diagnostic significance was obtained with an area under curve (AUC) of 0.82. CONCLUSIONS The present study demonstrated that Cobb C2-C7 (less than 8.5°) is a potential risk factor for the development of MC.

Negative Pressure Therapy in the Regeneration of the Sciatic Nerve Using Vacuum - Assisted Closure in a Rabbit Model.

BACKGROUND The aim of this study was to investigate the effects of negative pressure therapy in the regeneration of the rabbit sciatic nerve using vacuum assisted closure (VAC). MATERIAL AND METHODS Thirty male New Zealand white rabbits underwent surgical injury of the sciatic nerve, followed by negative pressure therapy using vacuum assisted closure (VAC), in three treatment groups: Group A: 0 kPa; Group B: -20 kPa; Group C: -40 kPa. At 12 weeks following surgery, the following factors were studied: motor nerve conduction velocity (MNCV); the number of myelinated nerve fibers; the wet weight of the gastrocnemius muscle. Gastrocnemius muscle and sciatic nerve tissue samples were studied for the expression of S100, and brain-derived neurotrophic factor (BDNF) using Western blot. RESULTS At 12 weeks following VAC treatment, the MNCV, number of myelinated nerve fibers, and wet weight of the gastrocnemius muscle showed significant differences between the groups (p<0.05), in the following order: Group B >Group A >Group C. The sciatic nerve at 12 weeks following VAC in Group B and Group C showed a significant increase in expression of S100 and BDNF when compared with Group A; no significant differences were detected between Group B and Group C results from Western blot at 12 weeks. CONCLUSIONS The findings of this study, using negative pressure therapy in VAC in a rabbit model of sciatic nerve damage, have shown that moderate negative pressure was beneficial, but high values did not benefit sciatic nerve repair.

MAP4K4 promotes epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma.

Our previous study has reported that mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) regulates the growth and survival of hepatocellular carcinoma (HCC) cells. This study was undertaken to explore the roles of MAP4K4 in the epithelial-mesenchymal transition (EMT) and metastasis in HCC. Effects of overexpression and knockdown of MAP4K4 on the migration, invasion, and EMT of HCC cells were examined. The in vivo role of MAP4K4 in lung metastasis of HCC was determined in nude mice. The relationship between MAP4K4 expression and EMT in human HCC specimens was determined by immunohistochemistry. MAP4K4 overexpression significantly enhanced the migration and invasion of MHCC-97L HCC cells, whereas MAP4K4 silencing hindered the migration and invasion of MHCC-97H HCC cells. MAP4K4-overexpressing cells undergo EMT, which was accompanied by downregulation of E-cadherin and upregulation of vimentin. In contrast, MAP4K4 silencing caused a reversion from a spindle morphology to cobblestone-like morphology and induction of E-cadherin and reduction of vimentin. Pretreatment with chemical inhibitors of JNK and NF-κB abolished MAP4K4-mediated migration, invasion, and regulation of EMT markers in MHCC-97L cells. Ectopic expression of MAP4K4 promoted and knockdown of MAP4K4 inhibited lung metastasis of HCC, which was associated with regulation of JNK and NF-κB signaling and EMT markers. High MAP4K4 immunoreactivity was inversely correlated with E-cadherin and was positively correlated with vimentin, phospho-JNK, and phospho-NF-κB in HCC specimens. Taken together, MAP4K4 promotes the EMT and invasiveness of HCC cells largely via activation of JNK and NF-κB signaling.

α-Enolase plays a catalytically independent role in doxorubicin-induced cardiomyocyte apoptosis and mitochondrial dysfunction.

BACKGROUND: α-Enolase is a glycolytic enzyme with "second jobs" beyond its catalytic activity. However, its possible contribution to cardiac dysfunction remains to be determined. The present study aimed to investigate the role of α-enolase in doxorubicin (Dox)-induced cardiomyopathy as well as the underlying mechanisms. EXPERIMENTAL APPROACHES: The expression of α-enolase was detected in rat hearts and primary cultured rat cardiomyocytes with or without Dox administration. An adenovirus carrying short-hairpin interfering RNA targeting α-enolase was constructed and transduced specifically into the heart by intramyocardial injection. Heart function, cell apoptosis and mitochondrial function were measured following Dox administration. In addition, by using gain- and loss-of-function approaches to regulate α-enolase expression in primary cultured rat cardiomyocytes, we investigated the role of endogenous, wide type and catalytically inactive mutant α-enolase in cardiomyocyte apoptosis and ATP generation. Furthermore, the involvement of α-enolase in AMPK phosphorylation was also studied. KEY RESULTS: The mRNA and protein expression of cardiac α-enolase was significantly upregulated by Dox. Genetic silencing of α-enolase in rat hearts and cultured cardiomyocytes attenuated Dox-induced apoptosis and mitochondrial dysfunction. In contrast, overexpression of wide-type or catalytically inactive α-enolase in cardiomyocytes mimicked the detrimental role of Dox in inducing apoptosis and ATP reduction. AMPK dephosphorylation was further demonstrated to be involved in the proapoptotic and ATP-depriving effects of α-enolase. CONCLUSION: Our findings provided the evidence that α-enolase has a catalytically independent role in inducing cardiomyocyte apoptosis and mitochondrial dysfunction, which could be at least partially contributed to the inhibition of AMPK phosphorylation.

Downregulation of adipose triglyceride lipase promotes cardiomyocyte hypertrophy by triggering the accumulation of ceramides.

Adipose triglyceride lipase (ATGL), the rate-limiting enzyme of triglyceride (TG) hydrolysis, plays an important role in TG metabolism. ATGL knockout mice suffer from TG accumulation and die from heart failure. However, the mechanisms underlying cardiac hypertrophy caused by ATGL dysfunction remain unknown. In this study, we found that ATGL expression declined in pressure overload-induced cardiac hypertrophy in vivo and phenylephrine (PE)-induced cardiomyocyte hypertrophy in vitro. ATGL knockdown led to cardiomyocyte hypertrophy, while ATGL overexpression prevented PE-induced hypertrophy. In addition, ATGL downregulation increased but ATGL overexpression reduced the contents of ceramide, which has been proved to be closely associated with cardiac hypertrophy. Moreover, the accumulation of ceramide was due to elevation of free fatty acids in ATGL-knockdown cardiomyocytes, which could be explained by the reduced activity of peroxisome proliferator-activated receptor (PPAR) α leading to imbalance of fatty acid uptake and oxidation. These observations suggest that downregulation of ATGL causes the decreased PPARα activity which results in the imbalance of FA uptake and oxidation, elevating intracellular FFA contents to promote the accumulation of ceramides, and finally inducing cardiac hypertrophy. Upregulation of ATGL could be a strategy for ameliorating lipotoxic damage in cardiac hypertrophy.

Mitochondrial binding of α-enolase stabilizes mitochondrial membrane: its role in doxorubicin-induced cardiomyocyte apoptosis.

α-Enolase is a metabolic enzyme in the catabolic glycolytic pathway. In eukaryotic cells, the subcellular compartmentalization of α-enolase as well as its multifaceted functions has been identified. Here, we report that α-enolase is a regulator of cardiac mitochondria; it partially located in the mitochondria of rat cardiomyocytes. Doxorubicin treatment displaced α-enolase from mitochondria, accompanied by activation of mitochondrial cell death pathway. Furthermore, in isolated mitochondria, recombinant α-enolase significantly alleviated Ca(2+)-induced loss of membrane potential, swelling of matrix and permeabilization of membrane. In contrast, mitochondria from α-enolase knockdown H9c2 myoblasts underwent more severe membrane depolarization and swelling after Ca(2+) stimulation. In addition, α-enolase was further identified to interact with voltage dependent anion channel 1 in the outer membrane of mitochondria, which was weakened by doxorubicin. Collectively, the present study indicates that mitochondria-located α-enolase has a beneficial role in stabilizing mitochondrial membrane. In cardiomyocytes, the displacement of α-enolase from mitochondria by doxorubicin may involve in activation of the intrinsic cell death pathway.

Cold atmospheric plasma-activated saline alleviates secondary injury post-SCI by inhibiting extracellular matrix remodeling and infiltration of proinflammatory macrophages.

BACKGROUND: Cold atmospheric plasma (CAP) has been shown to improve the recovery of transected peripheral nerves. We determined the protective role of CAP-activated saline (CAP-AS) treatment in the acute and subacute stages of spinal cord injury (SCI) in mice. METHODS: C57BL/6 SCI mice were treated with CAP-AS for 14 days. Injury recovery was assessed weekly for four weeks by conducting motor function tests, including the Basso Mouse Scale (BMS) and footprint test. Transcriptome analysis was conducted on day 14 to elucidate potential mechanisms, which were further validated through immunofluorescence examinations of the injured spinal cord tissues on day 28 and the levels of proinflammatory cytokines produced by macrophages in vitro. RESULTS: Compared to the SCI group, the CAP-AS-treated groups presented significantly better hindlimb motor function after four weeks. The downregulated (SCI vs. SCI + CAP-AS, with CAP-AS activated for 20 min) differentially expressed genes (DEGs) were enriched in the extracellular region, extracellular matrix (ECM), and ECM-receptor interaction. In contrast, the upregulated DEGs were enriched in immune response-associated pathways. Histological changes in the CAP-AS-treated groups were observed to further validate the predicted mechanisms 28 days post-injury. The alleviation of secondary injury was confirmed by an increase in GFAP-positive and NFH-positive areas, and enhanced outgrowth of 5-HT-positive fibers. Inhibited ECM remodeling was confirmed by a decrease in the areas positive for PDGFRß, fibronectin, and laminin. A decrease in the infiltration of macrophages and activation of microglia was determined by a decrease in CD68-positive and F4/80-positive areas. The inhibitory effect of CAP-AS on inflammation was further supported by a decrease in the levels of the proinflammatory cytokines IL-1ß, IL-6, and TNF-α in CAP-AS-treated M1 macrophages. CONCLUSION: CAP-AS can alleviate secondary injury in SCI model mice by inhibiting ECM remodeling in injured tissues and reducing the infiltration or activation of proinflammatory macrophages/microglia.

SiRNA-targeted carboxypeptidase D inhibits hepatocellular carcinoma growth.

Carboxypeptidase D (CPD), a membrane-bound metallocarboxypeptidase that functions as a docking receptor for duck hepatitis B virus, is frequently overexpressed in human cancers. We have explored its expression pattern, clinical significance, and biological function of CPD in hepatocellular carcinoma (HCC). CPD expression was markedly elevated in HCCs relative to adjacent non-tumor liver tissues, as determined by quantitative real-time polymerase chain reaction and Western blot analysis. Immunohistochemistry showed that 164 of 400 (41%) HCCs had high expression of CPD. CPD overexpression was significantly associated with serum levels of hepatitis B surface antigen and hepatitis B e antigen, liver cirrhosis, pathological grade, and intrahepatic metastasis. Knockdown of endogenous CPD expression in Huh7 HCC cells by RNA interference reduced cell proliferation, blocked the cell cycle at G1 phase, and increased apoptosis. Many genes implicated in cell-cycle regulation, including P21waf1, P27 Kip1, SKP2, and CDC2, were deregulated by CPD downregulation. Thus CPD is frequently upregulated in HCC, and targeting CPD inhibits HCC cell proliferation through induction of G1 cell-cycle arrest and apoptosis, thereby providing a potential therapeutic target for this malignancy.

ß-Elemene induces apoptosis and autophagy in colorectal cancer cells through regulating the ROS/AMPK/mTOR pathway.

ß-Elemene is an effective anti-cancer ingredient extracted from the genus Curcuma (Zingiberaceae familiy). In the present study, we demonstrated that ß-elemene inhibited the proliferation of colorectal cancer cells and induced cell cycle arrest in the G2/M phase. In addition, ß-elemene induced nuclear chromatin condensation and cell membrane phosphatidylserine eversion, decreased cell mitochondrial membrane potential, and promoted the cleavage of caspase-3, caspase-9 and PARP proteins, indicating apoptosis in colorectal cancer cells. At the same time, ß-elemene induced autophagy response, and the treated cells showed autophagic vesicle bilayer membrane structure, which was accompanied by up-regulation of the expression of LC3B and SQSTM1. Furthermore, ß-elemene increased ROS levels in colorectal cancer cells, promoted phosphorylation of AMPK protein, and inhibited mTOR protein phosphorylation. In the experiments in vivo, ß-elemene inhibited the tumor size and induced apoptosis and autophagy in nude mice. In summary, ß-elemene inhibited the occurrence and development of colon cancer xenografts in nude mice, and significantly induced apoptosis and autophagy in colorectal cancer cells in vitro. These effects were associated with regulation of the ROS/AMPK/mTOR signaling. We offered a molecular basis for the development of ß-elemene as a promising anti-tumor drug candidate for colorectal cancer.

Identification of the neuronal effects of ethanol on C. elegans by in vivo fluorescence imaging on a microfluidic chip.

Caenorhabditis elegans (C. elegans) is a well-established model organism for investigating the correlations between behavioral and neuronal activities. Here, we demonstrated a microfluidic-based method that allowed stimulation-based neuronal analysis of immobilized C. elegans for identifying the neuronal effects of ethanol on the chemosensory responses of the right ASE (ASER) neuron. A one-piece microvalve was developed for the immobilization of C. elegans. Stimulations were realized by interface shifting of laminar flows. Well-fed transgenic worms expressing the calcium indicator G-CaMP in ASER neurons were used for in vivo fluorescence imaging. To evaluate the developed method, we first studied the effects of ethanol on the ASER neurons in response to a single NaCl stimulus. Results indicated that ethanol acutely suppressed the ON responses of ASER neurons to NaCl rather than the OFF response. Further studies of the adaptation of ASER neurons in response to NaCl and in the presence of ethanol suggested that ethanol interfered with the adaptation of neurons. The developed method exhibited the advantages of ease of operation and high throughput. We expect this new method to open up a new avenue for investigating the correlations between the behavioral and neuronal activities of C. elegans.

Apigenin Inhibits the Growth of Hepatocellular Carcinoma Cells by Affecting the Expression of microRNA Transcriptome.

BACKGROUND: Apigenin, as a natural flavonoid, has low intrinsic toxicity and has potential pharmacological effects against hepatocellular carcinoma (HCC). However, the molecular mechanisms involving microRNAs (miRNAs) and their target genes regulated by apigenin in the treatment of HCC have not been addressed. OBJECTIVE: In this study, the molecular mechanisms of apigenin involved in the prevention and treatment of HCC were explored in vivo and in vitro using miRNA transcriptomic sequencing to determine the basis for the clinical applications of apigenin in the treatment of HCC. METHODS: The effects of apigenin on the proliferation, cell cycle progression, apoptosis, and invasion of human hepatoma cell line Huh7 and Hep3B were studied in vitro, and the effects on the tumorigenicity of Huh7 cells were assessed in vivo. Then, a differential expression analysis of miRNAs regulated by apigenin in Huh7 cells was performed using next-generation RNA sequencing and further validated by qRT-PCR. The potential genes targeted by the differentially expressed miRNAs were identified using a curated miRTarBase miRNA database and their molecular functions were predicted using Gene Ontology and KEGG signaling pathway analysis. RESULTS: Compared with the control treatment group, apigenin significantly inhibited Huh7 cell proliferation, cell cycle, colony formation, and cell invasion in a concentration-dependent manner. Moreover, apigenin reduced tumor growth, promoted tumor cell necrosis, reduced the expression of Ki67, and increased the expression of Bax and Bcl-2 in the xenograft tumors of Huh7 cells. Bioinformatics analysis of the miRNA transcriptome showed that hsa-miR-24, hsa-miR-6769b-3p, hsa-miR-6836-3p, hsa-miR-199a-3p, hsa-miR-663a, hsa-miR-4739, hsa-miR-6892-3p, hsa-miR-7107-5p, hsa-miR-1273g-3p, hsa-miR-1343, and hsa-miR-6089 were the most significantly up-regulated miRNAs, and their key gene targets were MAPK1, PIK3CD, HRAS, CCND1, CDKN1A, E2F2, etc. The core regulatory pathways of the up-regulated miRNAs were associated with the hepatocellular carcinoma pathway. The down-regulated miRNAs were hsa-miR-181a-5p and hsa-miR-148a-3p, and the key target genes were MAPK1, HRAS, STAT3, FOS, BCL2, SMAD2, PPP3CA, IFNG, MET, and VAV2, with the core regulatory pathways identified as proteoglycans in cancer pathway. CONCLUSION: Apigenin can inhibit the growth of HCC cells, which may be mediated by up-regulation or down-regulation of miRNA molecules and their related target genes.

Scutellaria barbata D.Don and Oldenlandia diffusa (Willd.) Roxb crude extracts inhibit hepatitis-B-virus-associated hepatocellular carcinoma growth through regulating circRNA expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria barbata D.Don (SB) and Oldenlandia diffusa (Willd.) Roxb are commonly known as Ban Zhi Lian and Bai Hua She Cao in Chinese herbal medicines, respectively. As a pair of herbs, they have traditionally been used as ethnomedicines for clearing away heat and toxins, removing blood stasis, and promoting blood circulation, diuresis, and detumescence. AIM OF THE STUDY: The aim of the present study was to determine the active ingredients in SB and OD extracts and to investigate whether these extracts can inhibit the growth of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines (HepG2.2.15 and Hep3B) in vitro and in vivo, as well as to explore their mechanisms of action. MATERIALS AND METHODS: We determined the levels of total flavonoids, luteolin, and apigenin in SB and OD extracts via ultraviolet-visible spectrophotometry and high-performance liquid chromatography. The effects of SB and OD extracts on HBV-associated HCC cell growth were assessed by HepG2.2.15 and Hep3B cells phenotype and RNA sequencing of Hep3B cells in vitro, and xenograft models in vivo. RESULTS: The extracts of SB and OD contained total flavonoids. There were active ingredients of luteolin and apigenin in SB, but not in OD. The extracts of SB and OD significantly inhibited HCC growth, migration, invasion, and HBV activity in vitro and in vivo, as well as altered circRNA expression in Hep3B cells. Moreover, we constructed a circRNA-miRNA-mRNA co-expression network. CONCLUSIONS: The extracts of SB and OD may inhibit HCC cell growth and HBV activity in vitro and in vivo through altering circRNA-miRNA-gene expression and that the efficacies of these extracts may be related to the presence of luteolin and apigenin.

Three dimeric naphtho-γ-pyrones from the mangrove endophytic fungus Aspergillus tubingensis isolated from Pongamia pinnata.

Three new dimeric naphtho-γ-pyrones, named rubasperone A (1), rubasperone B (2), and rubasperone C (3), together with two known compounds, rubrofusarin (4) and rubrofusarin B (5), were isolated from the mangrove endophytic fungus Aspergillus tubingensis (GX1-5E). Their structures were determined by spectroscopic methods, including IR, MS, and 1D and 2D NMR. The structures of 1 and 2 were further confirmed by X-ray crystallography. In the bioactivity assays against tyrosinase and α-glucosidase, rubrofusarin (4) exhibited moderate tyrosinase inhibitory activity, with an IC(50) value of 65.6 µM, and rubasperone C (3) showed mild α-glucosidase inhibitory activity, with an IC(50) value of 97.3 µM.

Efficacy of comprehensive rehabilitation therapy for checkrein deformity: A case report.

BACKGROUND: In clinical practice, checkrein deformity is usually found in patients with calf injuries after ankle fracture or distal tibial fracture. The patients with checkrein deformity mainly report distending pain in toe tips, pain when walking or wearing shoes, and gait instability. Previous studies have mainly reported surgical treatments for checkrein deformity, while few studies have reported using comprehensive rehabilitation alone to improve the checkrein deformity. CASE SUMMARY: A 28-year-old woman was admitted to the hospital due to unstable gait caused by pain in the right hallux, for which she was unable to stretch for over three months. The patient had undergone "resection of ameloblastoma at the right mandible, mandibulectomy, and autogenous right fibula grafting". The patient's hallux toe, as well as the second and third toes of the right foot could not be stretched, with pain in all the toes during walking. Based on the medical records of the patient, as well as the results of physical and auxiliary examinations, the main diagnosis was checkrein deformity in the right foot. Since the patient refused surgical treatment, rehabilitation was the only treatment option. At discharge, the patient reported evident improvement in the pain in the toes, gait stability, as well as increased ability to climb up and downstairs. CONCLUSION: Comprehensive rehabilitation therapy could effectively alleviate the manifestations of checkrein deformity and improve the walking ability of the patients.

Xanthatin mediates G2/M cell cycle arrest, autophagy and apoptosis via ROS/XIAP signaling in human colon cancer cells.

Xanthatin is a natural plant bicyclic sesquiterpene lactone extracted from Xanthium plants (Asteraceae). In the present study, we demonstrated for the first time that Xanthatin inhibited cell proliferation and mediated G2/M phase arrest in human colon cancer cells. Xanthatin also activated caspase and mediated apoptosis in these cells. Concomitantly, Xanthatin triggered cell autophagic response. We found down-regulation of X-linked inhibitor of apoptosis protein (XIAP) contribute to the induction of apoptosis and autophagy. Moreover, reactive oxygen species (ROS) production was triggered upon exposure to Xanthatin in colon cancer cells. ROS inhibitor N-acetylcysteine (NAC) significantly reversed Xanthatin-mediated XIAP down-regulation, G2/M phase arrest, apoptosis and autophagosome accumulation. In summary, our findings demonstrated that Xanthatin caused G2/M phase arrest and mediated apoptosis and autophagy through ROS/XIAP in human colon cancer cells. We provided molecular bases for developing Xanthatin as a promising antitumor candidate for colon cancer therapy. AbbreviationsROSreactive oxygen speciesDMSOdimethyl sulfoxide5-FU5-Fluorouracil3-MA3-MethyladenineDCFH-DA2'7'-dichlorfluorescein-diacetateNACN-acetylcysteineXIAPX-linked inhibitor of apoptosis protein.