Cystathionine γ-lyase inhibits mitochondrial oxidative stress by releasing H2S nearby through the AKT/NRF2 signaling pathway.

Cystathionine γ-lyase (CSE) is a major enzyme that produces hydrogen sulfide (H2S). Herein, we report how CSE plays a previously unknown role in regulating the antioxidant effects of the mitochondria in human umbilical vein endothelial cells by releasing H2S nearby under stress conditions. We found that H2S partially promoted angiogenesis in the endothelial cells through the AKT/nuclear factor erythroid 2-related factor 2 (AKT/NRF2) signaling pathway. H2S improved mitochondrial function by altering the expressions of the mitofusin2 and dynamin-1-like mitochondrial fission proteins to inhibit oxidative stress and enhance NRF2 nuclear translocation. CSE is located only in the cytoplasm and not in the mitochondria, but it is transported to the vicinity of the mitochondria to produce H2S, which plays an antioxidant role in human umbilical vein endothelial cells under stress. The CSE mutant (with mutated CSE activity center: CSED187A) partially decreased the effects on promoting angiogenesis, resisting oxidative stress, and entering the mitochondria. These results show that CSE translocation is a unique mechanism that promotes H2S production inside the mitochondria under stress stimulation. Therefore, the CSE mutant site (CSED187A) may be a potential target for drug therapy.

Comparative transcription profiling of mRNA and lncRNA in pulmonary arterial hypertension after C75 treatment.

OBJECTIVES: To investigate mRNA and long non-coding RNA (lncRNA) expression profiles in monocrotaline (MCT)- mice. MATERIALS AND METHODS: Lung tissues (Control-Vehicle, MCT-Vehicle, and MCT-C75) were examined by high-throughput sequencing (HTS). Aberrantly expressed mRNAs and lncRNAs were analyzed by bioinformatics. Cell proliferation and cell cycle analysis were performed to detect the potential protective effects of C75, an inhibitor of fatty acid synthase. The signaling pathways associated with inflammatory responses were verified by real time-PCR. RESULTS: RNA sequencing data reveals 285 differentially expressed genes (DEGs) and 147 lncRNAs in the MCT-Vehicle group compared to the control. After five-week of C75 treatment, 514 DEGs and 84 lncRNAs are aberrant compared to the MCT-Vehicle group. Analysis of DEGs and lncRNA target genes reveals that they were enriched in pathways related to cell cycle, cell division, and vascular smooth muscle contraction that contributes to the PAH pathological process. Subsequently, the expression of eight DEGs and three lncRNAs is verified using RT-PCR. Differentially expressed lncRNAs (ENSMUSG00000110393.2, Gm38850, ENSMUSG00000100465.1, ENSMUSG00000110399.1) may associate in PAH pathogenesis as suggested by co-expression network analysis. C75 can protect against MCT-induced PAH through its anti-inflammatory and anti-proliferation. CONCLUSIONS: These DEGs and lncRNAs can be considered as novel candidate regulators of PAH pathogenesis. We propose that C75 treatment can partially reverse PAH pathogenesis through modulating cell cycle, cell proliferation, and anti-inflammatory.

Case Report: Novel LIM domain-binding protein 3 (LDB3) mutations associated with hypertrophic cardiomyopathy family.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant cardiomyopathy, which is one of the most common reasons for cardiac arrest in children or adolescents. It is characterized by ventricular hypertrophy (usually left ventricle), small ventricular cavity, and reduced ventricular diastolic compliance found by echocardiography in the absence of abnormal load (such as hypertension or aortic stenosis). HCM is usually caused by mutations in genes encoding sarcomere or sarcomere-related genes. Whole exome sequencing (WES) is performed to identify probable causative genes. Through WES, we identified LIM domain-binding protein 3 (LDB3) mutations (R547Q and P323S) respectively in an 11-year-old HCM girl and a 6-year-old HCM boy. Neural network analyses showed that the LDB3 (R547Q and P323S) mutation decreased its protein stability, with confidence scores of -0.9211 and -0.8967. The STRUM server also confirmed that the mutation decreased its protein stability. Thus, LDB3 mutation may be associated with heritable HCM. To our knowledge, this is the first time to report LDB3 heterozygous variants (R547Q and P323S) responsible for heritable HCM.

TECRL deficiency results in aberrant mitochondrial function in cardiomyocytes.

Sudden cardiac death (SCD) caused by ventricular arrhythmias is the leading cause of mortality of cardiovascular disease. Mutation in TECRL, an endoplasmic reticulum protein, was first reported in catecholaminergic polymorphic ventricular tachycardia during which a patient succumbed to SCD. Using loss- and gain-of-function approaches, we investigated the role of TECRL in murine and human cardiomyocytes. Tecrl (knockout, KO) mouse shows significantly aggravated cardiac dysfunction, evidenced by the decrease of ejection fraction and fractional shortening. Mechanistically, TECRL deficiency impairs mitochondrial respiration, which is characterized by reduced adenosine triphosphate production, increased fatty acid synthase (FAS) and reactive oxygen species production, along with decreased MFN2, p-AKT (Ser473), and NRF2 expressions. Overexpression of TECRL induces mitochondrial respiration, in PI3K/AKT dependent manner. TECRL regulates mitochondrial function mainly through PI3K/AKT signaling and the mitochondrial fusion protein MFN2. Apoptosis inducing factor (AIF) and cytochrome C (Cyc) is released from the mitochondria into the cytoplasm after siTECRL infection, as demonstrated by immunofluorescent staining and western blotting. Herein, we propose a previously unrecognized TECRL mechanism in regulating CPVT and may provide possible support for therapeutic target in CPVT.

Identification and characterization of a novel ELN mutation in congenital heart disease with pulmonary artery stenosis.

Congenital heart defects, one of the most common birth defects, affect approximately 1% of live birth globally and remain the leading cause of infant mortality in developed countries. Utilizing the pathogenicity score and inheritance mode from whole exome sequencing results, a heterozygous mutation (NM_001278939.1: c.1939G>T, p.Gly647Ter) in elastin (ELN) was identified among 6,440 variants in a female proband born with an atrial septal defect accompanied by pulmonary artery stenosis. Results of RT-PCR showed that the mutation (NM_001278939.1: c.1939G>T, p.Gly647Ter) did not affect the expression levels of ELN mRNA but increased protein level. The content of ELN truncate (functional component) was significantly lower in both the intracellular and extracellular compartments after mutation. These results indicate that the ELN mutation (NM_001278939.1: c.1939G>T, p.Gly647Ter) affected the protein truncate, which may be a functional component of ELN and play crucial roles for this pedigree. Here we report of an ELN heterozygous variant associated with congenital heart disease accompanied with pulmonary artery stenosis, which is less common. Based on our results, we speculate that this may be the main molecular mechanism underlying the mutation-led functional changes, and propose that the decrease of ELN protein level may cause this pedigree vascular abnormality, especially pulmonary artery stenosis, and reinforce the view that ELN insufficiency is the primary cause of these vascular lesions. This may be the main molecular mechanism underlying the mutation-led functional changes. Thus, systematic analysis not only enables us to better understand the etiology of this disease but also contributes to clinical and prenatal diagnosis.

Generation of an induced pluripotent stem cell line from a Chinese Han child with arrhythmia.

CTNNA3, first reported in association with arrhythmogenic right ventricular cardiomyopathy in 2003, is an unique component of both desmosomes and adherens junctions. Using Sendaivirus-mediated reprogramming, we generated an induced pluripotent stem cell (iPSC) line from the peripheral blood mononuclear cells of a child with arrhythmia. The iPSCs exhibited stable amplification, expressed pluripotent markers, and differentiated spontaneously into three germ layers in vitro. Additionally, this iPSC line was found to maintain a normal karyotype and retain the pathogenic mutation in CTNNA3, facilitating a platform to study the disease mechanisms of arrhythmia and dysfunctions related to CTNNA3 mutations.

Targeted delivery of doxorubicin and vincristine to lymph cancer: evaluation of novel nanostructured lipid carriers in vitro and in vivo.

PURPOSE: Lymph cancers are heterogeneous malignancies of the hematopoietic and lymphoid tissues. Doxorubicin (DOX) and vincristine (VCR) are commonly used anti-cancer chemotherapeutic drugs, but their clinical uses are associated with dose-limiting systemic toxicity. METHODS: In the present study, DOX and VCR were encapsulated into nanostructured lipid carriers (NLCs) and used them to treat B-cell lymphoma cells through the targeted delivery of DOX and VCR to lymph cancer animal model. RESULTS: DOX and VCR encapsulated NLCs (DOX/VCR NLCs) demonstrated controlled drug release under physiological conditions. In addition, DOX/VCR NLCs exhibited the highest cytotoxicity and synergistic effect of two drugs in B-cell lymphoma cells and the best anti-tumor effect in vivo. CONCLUSION: DOX/VCR NLCs were proved to be more efficacious than the equivalent dose of free DOX and single drug (DOX or VCR) formulation in vitro and in vivo, and significantly reduced the drug-associated systemic toxicity.

Pharmacokinetics of timolol in aqueous humor sampled by microdialysis after topical administration of thermosetting gels.

In order to develop a thermosetting gel-based formulation, the ocular pharmacokinetics of timolol was studied utilizing microdialysis sampling technique after topical administration. A linear microdialysis probe was characterized and implanted in the anterior chamber of a rabbit. Dialysate samples collected from the aqueous humor (AH) were directly injected into the HPLC system without any pre-treatment and no interference was observed in the blank sample. The measured in vitro recovery of the probe was 57.67%; however, the in vivo recovery significantly decreased to 16.78% when assessed by the retrodialysis method, which was used to calculate the timolol concentration in AH. Although in the initial 15 min the drug concentrations in AH were comparable to that of the timolol solution, increased Cmax and significantly improved ocular bioavailability were obtained for the gel. When sodium deoxycholate (DC) was incorporated in the gel as a penetration enhancer, a 2-fold increment in the ocular bioavailability was achieved with an increased Cmax and significantly suspended Tmax. The results demonstrated that microdialysis coupled to HPLC is a powerful tool to investigate the ocular pharmacokinetic, and hence facilitates the design of ophthalmic formulations.

Effect of iontophoresis on skin permeation of defibrase.

AIM: To investigate the effect of iontophoresis on skin permeation of defibrase. METHODS: Iontophoresis was carried out in side-by-side chambers, excised rat skin membrane (RSM) or human epidermis membrane (HEM). The effects of electrode polarity, permeation medium pH and ionic strength were evaluated. RESULTS: Permeation of defibrase caused by anodal iontophoresis was more effective [the apparent permeability coefficient was (1.2 +/- 0.4) x 10(-4) cm x h(-1)] than that of cathodal iontophoresis [(4.3 +/- 1.4) x 10(-5) cm x h(-1)]. The amount of permeated defibrase caused by anodal iontophoresis in pH 7.4 medium was (25 +/- 5) x 10(-14) mol x cm(-2), which was higher than that of in pH 6. 4 permeation medium [(15 +/- 4) x 10(-14) mol x cm(-2)]. CONCLUSION: Iontophoresis could enhance skin permeation of defibrase. Electroosmotic flow effect played an important role.

[Diffusion behaviors of drugs in thermosensitive in situ gels].

AIM: To study the diffusion behaviors of drugs in thermosensitive in situ gels, and provide valuable information for designing such delivery systems. METHODS: A free diffusion model was used to evaluate the effects of concentration, the property of drugs, as well as the gel compositions on the diffusivity of drugs. RESULTS: Drug transport through the aqueous channels of the gel followed Fickian mechanism, and no significant influence on the diffusivity was observed when the drug concentration was lowered from 5% to 0.25%. The diffusion coefficients of propranolol, timolol maleate, and salbutamol sulfate were 0.91, 1.32, and 3.30 x 10(-6) cm2 x s(-1), respectively. The flux of hydrophilic drug was 3.6 fold faster than that of the lipophilic one implied the latter partitioned into the hydrophobic micellar core, and consequently the diffusion was retarded. The diffusivity was decreased with increased poloxamer and sodium hyaluronate concentration, due to the distorted aqueous channels and higher microviscosity. CONCLUSION: The result suggested that sustained release could be achieved for the thermosensitive in situ gel by incorporating lipophilic drug or increasing polymer concentration.

[Cutaneous permeation kinetics and pharmacodynamics of topical lidocaine gel in rat].

AIM: To study the cutaneous permeation kinetics and pharmacodynamics of lidocaine gel. METHODS: The concentration of lidocaine in dermis following topical application in rats was determined by the cutaneous microdialysis technique and related parameters were calculated; the pharmacodynamics of the gel was evaluated by electric stimulation method with EMLA (eutectic mixture of local anesthetics) cream as a control. RESULTS: The peak of percutaneous absorption kinetic profile of lidocaine gel across rat skin occurred at 1.25 h; the onset time of local anesthetic action of lidociane gel was similar to that of EMLA, but both the duration and depth of anesthetic effect were superior to EMLA cream. CONCLUSION: Lidocaine gel showed good anesthetic effect. The minimum effective concentration of lidocaine in dermis is 12 mg.L-1.

[Enhancement of gastrointestinal absorption of chitosan-coated insulin-loaded poly (lactic-co-glycolic acid) nanoparticles].

AIM: To study chitosan-coated poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on enhancing gastrointestinal absorption of insulin. METHODS: Insulin-loaded PLGA multiple emulsions were prepared by a double-emulsion method. Using chitosan as a stabilizer, chitosan-coated PLGA-NPs was prepared. The changes of the morphology, size distribution and Zeta potential of the NPs were examined. The encapsulation efficiency was determined by HPLC. The release behaviors in vitro were assessed, and the hypoglycemic effects were evaluated by monitoring the glucose levels in diabetic rats. RESULTS: Chitosan-coated PLGA-NPs showed a narrow size of distribution and regular surface with layer structure and their Zeta potential can be changed by chitosan. Chitosan-coating increased the encapsulation efficiency of insulin, reduced the initial burst and improved the release behavior of the NPs. About 14-16 h after intragastric administration of chitosan-coated INS-PLGA-NPs, the plasma glucose level decreased significantly compared with intragastric administration of same dose of non-coated NPs (P < or = 0.05), and the relative pharmacological availability was increased up to (15.4 +/- 1.2)%. CONCLUSION: Chitosan-coated PLGA-NPs could enhance gastrointestinal absorption of insulin.

In vitro evaluation of patch formulations for topical delivery of gentisic acid in rats.

Gentisic acid (GA) is used in cosmetics as a skin-whitening agent for the treatment of skin pigmentary disorders by influencing the synthesis of melanin through inhibition of melanosomal tyrosinase activity. In order to achieve effective topical delivery of GA to the active site in the skin, a matrix-type transdermal delivery system was developed. The in vitro skin permeation as well as skin deposition of GA was studied in rats. Among the five pressure-sensitive adhesives tested, DuroTak 87-2510 was the most effective to achieve the highest permeation rate of GA. Dodecylamine showed the most potent enhancement among the enhancers tested, and significantly increased the permeation rate of GA up to 112.99 (+/-30.12) microg/cm(2) per h at the concentration of 1%, when 6% GA was incorporated in DuroTak 87-2510. Moreover, a linear relationship was observed between the skin permeation rate of GA and the amount of the skin deposition after 12 h of permeation (r(2)=0.95). Thus, the in vitro skin permeation data may be useful to determine the amount of GA actually deposited in the skin.

Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo.

There are many ongoing investigations to improve the oral bioavailability of peptide and protein formulations. Bioadhesive polysaccharide chitosan nanoparticles (CS-NPs) would seem to further enhance intestinal absorption of them. In this study, Insulin-loaded CS-NPs were prepared by ionotropic gelation of CS with tripolyphosphate anions. Its particle size distribution and zeta potential were determined by photon correction spectroscopy and laser Dopper anemometry. The ability of CS-NPs to enhance intestinal absorption of insulin and increase the relative pharmacological bioavailability of insulin was investigated by monitoring the plasma glucose level of alloxan-induced diabetic rats after oral administration of various doses of insulin-loaded CS-NPs. CS-NPs had a particle size in the range of 250-400 nm and its polydispersity index was smaller than 0.1, positively charged, stable. Insulin association was found up to 80% and its in vitro release showed a great initial burst with a pH-sensitivity property. CS-NPs enhanced the intestinal absorption of insulin to a greater extent than the aqueous solution of CS in vivo. Above all, after administration of 21 I.U./kg insulin in the CS-NPs, the hypoglycemia was prolonged over 15 h and the average pharmacological bioavailability relative to SC injection of insulin solution was up to 14.9%.

Relationship between drug effects and particle size of insulin-loaded bioadhesive microspheres.

AIM: To formulate and characterize insulin-loaded adhesive microspheres (MP) and evaluate drug effects of MP with various sizes, 120, 350, and 1000 nm in diameter, in the alloxan-induced diabetic rats. METHODS: Insulin-loaded MP were formulated by an ionotropic gelation procedure. Particle size distributions were determined by photon correlation spectroscopy and optical microscopy. The factors that influenced the particle sizes and loading capacity were investigated, and the release properties were assessed in vitro. The hypoglycemic effect was investigated by monitoring the plasma glucose level of the alloxan-induced diabetic rats after oral administration. RESULTS: All the MPs with three sizes formulated were in the desired size range, and the loading capacity was 15.3 %+/-1.7 % (120 nm), 32.4 %+/-2.4 % (350 nm), and 53.3 %+/-2.7 % (1000 nm) respectively. The particle size also had an influence on the release property of the MPs. Half an hour later, 25 %+/-4 % (120 nm), 18.3 %+/-2.4 % (350 nm), and 8.6 %+/-1.3 % (1000 nm) of insulin were released. MP with different sizes had various degree of hypoglycemic effects after 10 h (P<0.05 vs control insulin solution). The plasma glucose level of 350 nm size particles remarkably decreased 15 h later (P<0.05 vs 120 nm) or 35 h later (P<0.01 vs others). The relative pharmacological availability was 10.2 %+/-0.5 % (120 nm), 14.9 %+/-1.3 % (350 nm), and 7.3 %+/-0.8 % (1000 nm) respectively. Particles of 350 nm showed a comparatively higher availability (P<0.05). CONCLUSION: Adhesive CS-MP were helpful in increasing the relative pharmacological bioavailability of insulin, and a distinct advantage of proper particle size helped to increase the drug effects.

[Effect of solution viscosity on polymer precorneal residence time evaluated by in vitro method].

AIM: To evaluate how solution viscosity affects the precorneal residence of five water-soluble polymers with different properties. METHODS: Captive bubble technique was used, with the consecutive change of contact angle interpreted as an indication of desorption process, to study the residence of those polymers in vitro on freshly enucleated rabbit eyes under physiological conditions. RESULTS: Carbopol and sodium hyaluronate (HA), which adsorbed to isolated ocular surface more than 15 min, showed the optimum precorneal retentive capabilities. When the solution viscosity increased from 12 mPa.s to 50 mPa.s, the residence time of carbopol and HA were prolonged 10 min and 7 min, respectively, but that of sodium carboxymethylcellulose was not affected. CONCLUSION: The result suggested that higher viscosity is beneficial to improve the ocular residence time of bio-adhesive polymers.

[Study on preparation and oral efficacy of insulin-loaded poly(lactic-co-glycolic acid) nanoparticles].

AIM: To investigate the possibility of poly(lactic-co-glycolic acid) as a carrier for the delivery of macromolecular. METHODS: Insulin-loaded poly (lactic-co-glycolic acid) nanoparticles (INS-PLGA-NPs) was prepared by a double-emulsion solvent evaporation method. The size distribution was examined by photo-correlation spectrometry. The entrapment efficiency was determined by HPLC and important factors that affected the entrapment efficiency were investigated. The loading mechanism of different size nanoparticles was assayed by radioimmunoassay (RIA). INS-PLGA-NPs release behavior in vitro was carried out under sink condition. After oral administration of the nanoparticles to alloxan-induced diabetic rats, its glucose level was determined by glucose oxidize method and the oral pharmacological bioavailability in contrast to s.c. of insulin solution was calculated according to the area over the curve. RESULTS: The INS-PLGA-NPs was prepared with poloxamer 188 as a emulsifier, the mean diameter was 149.6 nm and the polydispersity index was decreased to 0.09. While the entrapment efficiency was increased to 42.8%. Most of the insulin loaded was adsorbed on the surface of the nanoparticles. The release behavior in vitro showed an initial burst effect followed by a slower rate stage. After oral administration of 10 u.kg-1 INS-PLGA-NPs, the plasma glucose level decreased significantly after 4 h (P < 0.05), 10 h later the glucose level decreased to the lowest (52.4% +/- 10.2%, P < 0.01) and the relative pharmacological bioavailability is (10.3 +/- 0.8)%. CONCLUSION: PLGA-NPs might be used as a new oral carrier for protein drug delivery systems in the future.

[The enhancing effect of electroporation and iontophoresis on the permeation of insulin through human skin].

AIM: To study the enhancing effect of electroporation and iontophoresis on the permeation of insulin through human cadaver skin in vitro. METHODS: Using side by side two-chamber diffusion cells, the flux of insulin achieved with iontophoresis and electrophoration were compared. RESULTS: The application of high-voltage pulse combined with iontophoresis resulted in higher flux transdermal permeation of insulin than either one technique alone (P < 0.05). Pulsing at a higher voltage increased the flux of insulin more dramatically than pulsing at a lower voltage (P < 0.01). The transdermal transport of insulin by 90 pulse of 500 V (exponential pulse generater, pulse time: 20-24 ms, pulse frequency: 3 pulse.min-1) followed by iontophoresis led to a quick input and a high steady flux. CONCLUSION: Electroporation combined with iontophoresis can enhance the permeation of insulin significantly.