Effects and mechanisms of Xiaochaihu Tang against liver fibrosis: An integration of network pharmacology, molecular docking and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is a potentially harmful chronic liver disease caused by various etiologies. There is currently no specific drug for liver fibrosis. Xiaochaihu Tang (XCHT) is a traditional formula combined of seven herbs, which was first recorded in the Treatise on Febrile Diseases in Han Dynasty of ancient China. It is widely used in clinic to hepatic protection, analgesic, antipyretic and anti-inflammatory treatment. And it has been recommended for treating chronic hepatitis and chronic cholecystitis in the latest guidelines for the diagnosis and treatment of liver fibrosis with integrated traditional and western medicine. However, the underlying regulatory mechanisms remain elusive. AIM OF THE STUDY: This study aims to explore the therapeutic effects of XCHT on liver fibrosis and its underlying molecular mechanisms from the perspective of network pharmacology and experimental research. MATERIALS AND METHODS: Carbon tetrachloride (CCl4) induced and bile duct ligation (BDL) induced liver fibrosis models in mice were established to evaluate the anti-fibrosis effects of XCHT in vivo. Potential anti-fibrosis targets of XCHT were screened via network establishment. The underlying mechanisms were uncovered through GO and pathway enrichment analysis. Then, the core targets were identified from protein-protein interaction network by means of the Cytohubba plug-in of Cytoscape. Furthermore, two effective monomer components of XCHT were recognized by molecular docking. Moreover, the predicted components and pathways were verified by in vitro experiments. RESULTS: When treated with XCHT, liver fibrosis was alleviated in both mice models, showing as the improvement of liver function, the protection of hepatocytes, the inhibition of HSC activation and the reduction of hepatic collagen accumulation. 540 monomer components, 300 therapeutic targets, 109 signaling pathways, 246 GO biological processes, 77 GO cellular components, 107 GO molecular functions items and core targets were identified by network analysis. Then, 6-gingerol and baicalein were identified as the core components of anti-fibrosis effects of XCHT via leptin or Nrf2 signaling pathway. Furthermore, the experiment in vitro also validated the results. CONCLUSIONS: Our study suggests XCHT could alleviate liver fibrosis through multi-targets and multi-pathways; 6-gingerol and baicalein are its core components which may play an important role via leptin or Nrf2 signaling pathway.

Canonical transient receptor potential channel 1 aggravates myocardial ischemia-and-reperfusion injury by upregulating reactive oxygen species.

The canonical transient receptor potential channel (TRPC) proteins form Ca2+-permeable cation channels that are involved in various heart diseases. However, the roles of specific TRPC proteins in myocardial ischemia/reperfusion (I/R) injury remain poorly understood. We observed that TRPC1 and TRPC6 were highly expressed in the area at risk (AAR) in a coronary artery ligation induced I/R model. Trpc1-/- mice exhibited improved cardiac function, lower serum Troponin T and serum creatine kinase level, smaller infarct volume, less fibrotic scars, and fewer apoptotic cells after myocardial-I/R than wild-type or Trpc6-/- mice. Cardiomyocyte-specific knockdown of Trpc1 using adeno-associated virus 9 mitigated myocardial I/R injury. Furthermore, Trpc1 deficiency protected adult mouse ventricular myocytes (AMVMs) and HL-1 cells from death during hypoxia/reoxygenation (H/R) injury. RNA-sequencing-based transcriptome analysis revealed differential expression of genes related to reactive oxygen species (ROS) generation in Trpc1-/- cardiomyocytes. Among these genes, oxoglutarate dehydrogenase-like (Ogdhl) was markedly downregulated. Moreover, Trpc1 deficiency impaired the calcineurin (CaN)/nuclear factor-kappa B (NF-κB) signaling pathway in AMVMs. Suppression of this pathway inhibited Ogdhl upregulation and ROS generation in HL-1 cells under H/R conditions. Chromatin immunoprecipitation assays confirmed NF-κB binding to the Ogdhl promoter. The cardioprotective effect of Trpc1 deficiency was canceled out by overexpression of NF-κB and Ogdhl in cardiomyocytes. In conclusion, our findings reveal that TRPC1 is upregulated in the AAR following myocardial I/R, leading to increased Ca2+ influx into associated cardiomyocytes. Subsequently, this upregulates Ogdhl expression through the CaN/NF-κB signaling pathway, ultimately exacerbating ROS production and aggravating myocardial I/R injury.

TRPC1 contributes to endotoxemia-induced myocardial dysfunction via mediating myocardial apoptosis and autophagy.

Cardiac dysfunction is a vital complication of endotoxemia (ETM) with limited therapeutic options. Transient receptor potential canonical channel (TRPC)1 was involved in various heart diseases. While, the role of TRPC1 in ETM-induced cardiac dysfunction remains to be defined. In this study, we found that TRPC1 protein expression was significantly upregulated in hearts of lipopolysaccharide (LPS)-challenged mice. What's more, TRPC1 knockdown significantly alleviated LPS-induced cardiac dysfunction and injury. Further myocardial mRNA-sequencing analysis revealed that TRPC1 might participate in pathogenesis of ETM-induced cardiac dysfunction via mediating myocardial apoptosis and autophagy. Data showed that knockdown of TRPC1 significantly ameliorated LPS-induced myocardial apoptotic injury, cardiomyocytes autophagosome accumulation, and myocardial autophagic flux. Simultaneously, deletion of TRPC1 reversed LPS-induced molecular changes of apoptosis/autophagy signaling pathway in cardiomyocytes. Moreover, TRPC1 could promote LPS-triggered intracellular Ca2+ release, subsequent calpain activation and caveolin-1 degradation. Either blocking calpain by PD150606 or enhancing the amount of caveolin-1 scaffolding domain that interacts with TRPC1 by cell-permeable peptide cavtratin significantly alleviated the LPS-induced cardiac dysfunction and cardiomyocytes apoptosis/autophagy. Furthermore, cavtratin could inhibit LPS-induced calpain activation in cardiomyocytes. caveolin-1 could directly interact with calpain 2 both in vivo and in vitro. Importantly, cecal ligation and puncture-stimulated cardiac dysfunction and mortality were significantly alleviated in Trpc1-/- and cavtratin-treated mice, which further validated the contribution of TRPC1-caveolin-1 signaling axis in sepsis-induced pathological process. Overall, this study indicated that TRPC1 could promote LPS-triggered intracellular Ca2+ release, mediate caveolin-1 reduction, and in turn activates calpain to regulate myocardial apoptosis and autophagy, contributing to ETM-induced cardiac dysfunction of mice.

Amphipathic dendritic poly-peptides carrier to deliver antisense oligonucleotides against multi-drug resistant bacteria in vitro and in vivo.

BACKGROUND: Outbreaks of infection due to multidrug-resistant (MDR) bacteria, especially Gram-negative bacteria, have become a global health issue in both hospitals and communities. Antisense oligonucleotides (ASOs) based therapeutics hold a great promise for treating infections caused by MDR bacteria. However, ASOs therapeutics are strangled because of its low cell penetration efficiency caused by the high molecular weight and hydrophilicity. RESULTS: Here, we designed a series of dendritic poly-peptides (DPP1 to DPP12) to encapsulate ASOs to form DSPE-mPEG2000 decorated ASOs/DPP nanoparticles (DP-AD1 to DP-AD12) and observed that amphipathic DP-AD2, 3, 7 or 8 with a positive charge ≥ 8 showed great efficiency to deliver ASOs into bacteria, but only the two histidine residues contained DP-AD7 and DP-AD8 significantly inhibited the bacterial growth and the targeted gene expression of tested bacteria in vitro. DP-AD7anti-acpP remarkably increased the survival rate of septic mice infected by ESBLs-E. coli, exhibiting strong antibacterial effects in vivo. CONCLUSIONS: For the first time, we designed DPP as a potent carrier to deliver ASOs for combating MDR bacteria and demonstrated the essential features, namely, amphipathicity, 8-10 positive charges, and 2 histidine residues, that are required for efficient DPP based delivery, and provide a novel approach for the development and research of the antisense antibacterial strategy.

Old targets, new strategy: Apigenin-7-O-ß-d-(-6″-p-coumaroyl)-glucopyranoside prevents endothelial ferroptosis and alleviates intestinal ischemia-reperfusion injury through HO-1 and MAO-B inhibition.

With the increasing morbidity and mortality, intestinal ischemia/reperfusion injury (IIRI) has attracted more and more attention, but there is no efficient therapeutics at present. Apigenin-7-O-ß-D-(-6″-p-coumaroyl)-glucopyranoside (APG) is a new flavonoid glycoside isolated from Clematis tangutica that has strong antioxidant abilities in previous studies. However, the pharmacodynamic function and mechanism of APG on IIRI remain unknown. This study aimed to investigate the effects of APG on IIRI both in vivo and in vitro and identify the potential molecular mechanism. We found that APG could significantly improve intestinal edema and increase Chiu's score. MST analysis suggested that APG could specifically bind to heme oxygenase 1 (HO-1) and monoamine oxidase b (MAO-B). Simultaneously, APG could attenuate ROS generation and Fe2+ accumulation, maintain mitochondria function thus inhibit ferroptosis with a dose-dependent manner. Moreover, we used siRNA silencing technology to confirm that knocking down both HO-1 and MAO-B had a positive effect on intestine. In addition, we found the HO-1 and MAO-B inhibitors also could reduce endothelial cell loss and protect vascular endothelial after reperfusion. We demonstrate that APG plays a protective role on decreasing activation of HO-1 and MAO-B, attenuating IIRI-induced ROS generation and Fe2+ accumulation, maintaining mitochondria function thus inhibiting ferroptosis.

Coumarin Derivative N6 as a Novel anti-hantavirus Infection Agent Targeting AKT.

Hantaviruses are globally emerging zoonotic viruses that can cause hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, which is primarily caused by Hantaan virus (HTNV) infection, results in profound morbidity and mortality. However, no specific treatment is available for this disease. Coumarin derivatives have been reported as antiviral molecules, while studies about the bioactivity of coumarin derivatives against HTNV infection are limited. To study the potential antiviral activity of coumarin derivatives, 126 coumarin derivatives are synthesized, and their inhibitory activity against HTNV is analyzed in vitro. Among these compounds, N6 inhibits HTNV with relatively high selectivity index at 10.9, and the viral titer of HTNV is reduced significantly after 5, 10, and 20 µM N6 treatments. Furthermore, the administration of N6 at the early stage of HTNV infection can inhibit the replication and production of infectious HTNV in host cell, this therapeutic efficacy is confirmed in HTNV-infected newborn mice at the early stage of infection. The molecular docking results show that N6 forms interactions with the key amino acid residues at its active site, and reveals several molecular interactions responsible for the observed affinity, and the treatment of N6 can inhibit the expression of p (Ser473)Akt and HTNV nucleocapsid protein significantly. As such, these observations demonstrate that coumarin derivative N6 might be used as a potential agent against HTNV infection.

Association of ABCB1 polymorphisms with carbamazepine metabolism and resistance in epilepsy: A meta-analysis.

OBJECTIVE: ABCB1 polymorphisms were previously demonstrated to be associated with the metabolism and resistance of carbamazepine (CBZ) in epilepsy, but the results still remained controversial. Therefore, we performed this meta-analysis to further evaluate the impacts of ABCB1 polymorphisms on CBZ metabolism and resistance. METHODS: The PubMed, EMBASE, Cochrane library, Chinese National Knowledge Infrastructure, Chinese Science and Technique Journals Database and Wan Fang Database were searched for eligible publications up to 5 July 2021. The mean difference (MD), Odds ratio (OR) and 95 % confidence interval (CI) were calculated by Review Manager 5.3 software to assess the strength of the association. RESULTS: Twelve studies involving 2126 epilepsy patients were included in this meta-analysis. We found that the TC genotype (heterozygous model: TC vs. CC) of rs1045642 polymorphism was significantly connected with decreased CBZ concentration. Furthermore, this polymorphism was indicated to be associated with concentrations of carbamazepine-10, 11-epoxide (homozygote model: TT vs. CC; heterozygous model: TC vs CC; dominant model: TT + TC vs. CC; over-dominant model: TC vs. TT + CC) and carbamazepine-10, 11-trans dihydrodiol (heterozygous model: TC vs. CC; dominant model: TT + TC vs. CC). Moreover, the AG genotype of rs2032582 polymorphism was related to increased CBZ concentration in heterozygous (AG vs. GG), dominant (AA + AG vs. GG) and over-dominant (AG vs. AA + GG) models. Additionally, rs1128503 was associated with CBZ resistance in heterozygous model (TC vs. CC). CONCLUSIONS: ABCB1 rs1045642 and rs2032582 polymorphisms were associated with CBZ metabolism for epilepsy, and rs1128503 was related to CBZ resistance. These findings would contribute to improving individualized therapy of epileptic patients.

Canonical transient receptor potential channels and their modulators: biology, pharmacology and therapeutic potentials.

Canonical transient receptor potential channels (TRPCs) are nonselective, high calcium permeability cationic channels. The TRPCs family includes TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7. These channels are widely expressed in the cardiovascular and nervous systems and exist in many other human tissues and cell types, playing several crucial roles in the human physiological and pathological processes. Hence, the emergence of TRPCs modulators can help investigate these channels' applications in health and disease. It is worth noting that the TRPCs subfamilies have structural and functional similarities, which presents a significant difficulty in screening and discovering of TRPCs modulators. In the past few years, only a limited number of selective modulators of TRPCs were detected; thus, additional research on more potent and more selective TRPCs modulators is needed. The present review focuses on the striking desired therapeutic effects of TRPCs modulators, which provides intel on the structural modification of TRPCs modulators and further pharmacological research. Importantly, TRPCs modulators can significantly facilitate future studies of TRPCs and TRPCs related diseases.

Restoration of the Antibiotic Susceptibility of Methicillin-Resistant Staphylococcus aureus and Extended-Spectrum ß-Lactamases Escherichia coli Through Combination with Chelerythrine.

Multidrug resistance poses a severe threat to public health and urgently requires new solutions. The natural product chelerythrine (CHE) is a benzophenanthridine alkaloid with antimicrobial potential. In this study, CHE was effective against seven gram-positive bacterial strains, and the minimum inhibitory concentrations (MICs) ranged from 2 to 4 µg/mL. By contrast, CHE showed inferior antibacterial activities against 11 gram-negative strains, and the MICs varied from 16 to 256 µg/mL. We also determined the synergistic/additive effects of combining CHE with nine currently used antibiotics. CHE restored the antibacterial efficacy of the antibiotics against methicillin-resistant Staphylococcus aureus and extended-spectrum ß-lactamases producing Escherichia coli. This study suggests that the combination of CHE with conventional antibiotics may be a promising strategy to combat infections caused by multidrug-resistant organisms.

Loop-armed DNA tetrahedron nanoparticles for delivering antisense oligos into bacteria.

BACKGROUND: Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria. RESULTS: Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more effectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 µL/µg ratio to Td, but the same concentration of LP2000 had no apparent effect on linear DNA. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure. CONCLUSIONS: This study established an effective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics.

Identification of a prolonged action molecular GLP-1R agonist for the treatment of femoral defects.

Autografts are still regarded as the gold standard treatment for bone defects but they require additional surgery that causes pain for the patient. Thus, alternatives that can substitute for grafts are required. In the present study, a novel poly-GLP-1 molecule was developed using a polymeric pro-drug strategy which was found to accelerate bone healing in a mouse femoral defect model. Furthermore, the poly-GLP-1 molecule induced osteogenesis and inhibited adipogenesis in bone marrow-derived mesenchymal stem cells (BMSCs). The results demonstrate that poly-GLP-1 promoted M2 polarization of bone marrow-derived macrophages (BMDMs) and increased the levels of TGF-ß1 in the bone marrow, resulting in the migration of an increased number of CD29 + Sca-1 + BMSCs to the bone surface. Finally, we found that poly-GLP-1 facilitated the migration of BMSCs due to transduction of the Smad2 signaling pathway, causing increased numbers of CD31 + Endomucin + endothelial cells in bone marrow that promoted bone formation. These results support poly-GLP-1 as a potential bone-healing agent and suggest that it may play a promising role in the clinical treatment of fracture repair.

PAMP protects intestine from Enterohemorrhagic Escherichia coli infection through destroying cell membrane and inhibiting inflammatory response.

Proadrenomedullin N-terminal 20 peptide (PAMP) is elevated in sepsis, but the function and possible mechanism of PAMP in bacterial infection is elusive. This study is aim to evaluate the role of PAMP in the interaction between the Enterohemorrhagic E. coli (EHEC) and the host barrier. Our results showed that PAMP alleviated the EHEC-induced disruption of goblet cells and mucosal damage in the intestine, increased the expression of occludin in the colon of EHEC-infected mice, and reduced the proinflammatory cytokines level in serum significantly compared with the control group. Meanwhile, lipopolysaccharide (LPS) stimulation could dose-dependently induce the expression of preproADM, the precursor of PAMP, in human intestinal epithelial cell (HIEC) and human umbilical vein endothelial cell (HUVEC). In addition, PAMP inhibited the growth of EHEC O157:H7 and destroyed the inner and outer membrane. At low concentration, PAMP attenuated the EHEC virulence genes including hlyA and eaeA, which was also confirmed from reduced hemolysis to red cells and adhesion to HIEC. These results indicated that EHEC infection would modulate the expression of PAMP in intestinal epithelium or vascular endothelium, and in turn exerted a protective effect in EHEC induced infection by rupturing the bacterial cell membrane and attenuating the bacterial virulence.

Synthesis of novel cellulose- based antibacterial composites of Ag nanoparticles@ metal-organic frameworks@ carboxymethylated fibers.

A novel cellulose-based antibacterial material, namely silver nanoparticles@ metal-organic frameworks@ carboxymethylated fibers composites (Ag NPs@ HKUST-1@ CFs), was synthesized. The results showed that the metal-organic frameworks (HKUST-1) were uniformly anchored on the fiber's surfaces by virtue of complexation between copper ions in HKUST-1 and carboxyl groups on the carboxymethylated fibers (CFs). The silver nanoparticles (Ag NPs) were immobilized and well-dispersed into the pores and/or onto the surfaces of HKUST-1 via in situ microwave reduction, resulting in the formation of novel Ag NPs@ HKUST-1@ CFs composites. The antibacterial assays showed that the as-prepared composites exhibited a much higher antibacterial activity than Ag NPs@ CFs or HKUST-1@ CFs samples.

Heteropoly acid catalytic treatment for reactivity enhancement and viscosity control of dissolving pulp.

The reactivity enhancement and viscosity control are of practical importance during the manufacture of high-quality cellulose (also known as dissolving pulp). In the study, the concept of using phosphotungstic acid (HPW) for this purpose was demonstrated. The Fock reactivity of resultant pulp increased from 49.1% to 74.1% after the HPW catalytic treatment at a dosage of 86.4 mg HPW/g odp. The improved results can be attributed to the increased fiber accessibility, thanks to the favorable fiber morphologic changes, such as increased pore volume/size, water retention value and specific surface area. HPW can be readily recycled/reused by evaporating method, where maintaining 87.1% catalytic activity after six recycle times. The HPW catalytic treatment concept may provide a green alternative for the manufacture of high-quality dissolving pulp.

Advances in the delivery of antisense oligonucleotides for combating bacterial infectious diseases.

Discovery and development of new antibacterial drugs against multidrug resistant bacterial strains have become more and more urgent. Antisense oligonucleotides (ASOs) show immense potential to control the spread of resistant microbes due to its high specificity of action, little risk to human gene expression, and easy design and synthesis to target any possible gene. However, efficient delivery of ASOs to their action sites with enough concentration remains a major obstacle, which greatly hampers their clinical application. In this study, we reviewed current progress on delivery strategies of ASOs into bacteria, focused on various non-virus gene vectors, including cell penetrating peptides, lipid nanoparticles, bolaamphiphile-based nanoparticles, DNA nanostructures and Vitamin B12. The current review provided comprehensive understanding and novel perspective for the future application of ASOs in combating bacterial infections.

Exendin-4 Induces Bone Marrow Stromal Cells Migration Through Bone Marrow-Derived Macrophages Polarization via PKA-STAT3 Signaling Pathway.

BACKGROUND/AIMS: The synthesis and degradation processes involved in bone remodeling are critically regulated by osteoblasts and osteoclasts. The GLP-1 receptor agonist Exendin-4 is beneficial for osteoblast differentiation and increases the number of osteoblasts. METHODS: We constructed an ovariectomized model to evaluate the impact of Exendin-4 on bone formation in osteoporosis. A macrophage-depleted model was also created to investigate the effect of macrophages on bone formation. Thirty-two female WT C57BL/6 mice (aged 3 months) were randomly assigned to a normal control group and four ovariectomized (OVX) subgroups: OVX + vehicle group, OVX + Exendin-4 (4.2 µg/kg/day) group, OVX + chloride phosphate liposome group and OVX + chloride phosphate liposome + Exendin-4 group. RESULTS: In this study, we found that Exendin-4 not only increased the number of osteoblasts and decreased the number of osteoclasts, but also increased the number of bone marrow stromal cells (BMSCs) at the bone surface. Moreover, we found that OVX mice treated with Exendin-4 increased TGF-ß1 levels at the bone surface compared with that in OVX mice. Besides, Exendin-4 promoted the polarization of bone marrow-derived macrophages into M2 subtype and increased TGF-ß1 secretion by the M2 subtype. Finally, we found that Exendin-4 induced macrophage polarization via the cAMP-PKA-STAT3 signaling pathway. CONCLUSION: Exendin-4 promotes bone marrow-derived macrophage polarization to the M2 subtype and induces BMSC migration to the bone surface via PKA-STAT3 signaling.

The HRH4 rs11662595 mutation is associated with histamine H4 receptor dysfunction and with increased epithelial-to-mesenchymal transition progress in non-small cell lung cancer.

We previously demonstrated that histamine H4 receptor (HRH4) played important roles to suppress epithelial-to-mesenchymal transition (EMT) progress in non-small cell lung cancer (NSCLC). Furthermore, recent investigations suggested that genetic variations in HRH4 gene affected HRH4 function and eventually contributed to certain HRH4-related diseases. However, the relations between polymorphisms in HRH4 gene and NSCLC as well as their underlying mechanisms remain largely uninvestigated. This study aims to investigate the genetic effect of a nonsynonymous HRH4 polymorphism (rs11662595) on HRH4 function and its association with NSCLC both basically and clinically. For basic experiments, A549 cells were transfected with either wild type or rs11662595 mutated HRH4 clone and subjected to both in vitro and in vivo experiments. We showed that rs11662595 significantly decreased the ability of HRH4 to activate Gi protein, which resulted in facilitation of EMT progress, cell proliferation, and invasion behavior in vitro. Moreover, in vivo experiments also showed that rs11662595 attenuated the anti-EMT effects of HRH4 agonist in inoculated nu/nu mice. For clinical experiments, we performed a prospective cohort study among 624 NSCLC patients and further proved that rs11662595 was responsible for the prognosis, degree of malignancy and metastasis of NSCLC. In conclusion, these findings reveal that rs11662595 is a loss-of-function polymorphism that results in dysfunction of HRH4 and attenuates the anti-EMT function of HRH4 in NSCLC, which provides a promising biomarker for prognosis and therapy of NSCLC.

The Disulfide Bond of the Peptide Thanatin Is Dispensible for Its Antimicrobial Activity In Vivo and In Vitro.

Thanatin (THA) displays potent antibiotic activity, especially against extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli both in vitro and in vivo, with minimal hemolytic toxicity and satisfactory stability in plasma. However, the high cost of thanatin significantly limits its development and clinical application. To reduce the cost of peptide synthesis, a formulation of cyclic thanatin (C-thanatin) called linear thanatin (L-thanatin) was synthesized and its activity was evaluated in vivo and in vitro Results showed that C-thanatin and L-thanatin MICs did not differ against eight Gram-negative and two Gram-positive bacterial strains. Furthermore, the survival rates of ESBL-producing-E. coli-infected mice were consistent after C-thanatin or L-thanatin treatment at 5 or 10 mg/kg of body weight. Neither C-thanatin nor L-thanatin showed toxicity for human red blood cells (hRBCs) and human umbilical vein endothelial cells (HUVECs) at a concentration as high as 256 µg/ml. Results of circular dichroism spectroscopy indicated that the secondary structure of L-thanatin is extremely similar to that of C-thanatin. Membrane permeabilization and depolarization assays showed that C-thanatin and L-thanatin have similar abilities to permeabilize the outer and inner membranes and to induce membrane depolarization in ESBL-producing E. coli However, neither of them caused significant HUVEC membrane permeability. These findings indicate that the two peptides have similar effects on bacterial cell membranes and that the disulfide bond in thanatin is not essential for its antimicrobial activities in vivo and in vitro L-thanatin is thus a promising low-cost peptide candidate for treating ESBL-producing E. coli infections.

Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through ß-Catenin.

Glucagon-like peptide 1 (GLP-1) plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4) promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs), but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/ß-catenin and PKA/PI3K/AKT/GSK3ß signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.