Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency.

OBJECTIVE: Melittin and its derivative have been developed to support effective gene delivery systems. Their ability to facilitate endosomal release enhances the delivery of nanoparticle-based gene therapy. Nevertheless, its potential application in the context of viral vectors has not received much attention. Therefore, we would like to optimize the rAAV vector by Melittin analog to improve the transduction efficiency of rAAV in liver cancer cells and explore the mechanism of Melittin analog on rAAV. METHODS: Various melittin-derived peptides were inserted into loop VIII of the capsid protein in recombinant adeno-associated virus vectors. These vectors carrying either gfp or fluc genes were subjected to quantitative polymerase chain reaction assays and transduction assays in human embryonic kidney 293 (HEK293T) cells to investigate the efficiency of vector production and gene delivery. In addition, the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice. Finally, the intricate details of the vector-mediated transduction mechanisms were explored by using pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle. RESULTS: A total of 76 melittin-related peptides were identified from existing literature. Among them, CMA-3, p5RHH and aAR3 were found to significantly inhibit transduction of rAAV2 vector crude lysate. The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV. Mechanistically, bafilomycin A1, a vacuolar endosome acidification inhibitor, completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors. Most importantly, p5RHH-rAAV8 vectors also increased hepatic transduction in vivo in C57BL/6 mice. CONCLUSION: The incorporation of melittin analogs into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression. While further modifications remain an area of interest, our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery. Please cite this article as: Meng J, He Y, Yang H, Zhou L, Wang S, Feng X, Al-shargi OY, Yu X, Zhu L, Ling, C. Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency. J Integr Med. 2024; 22(1): 72-82.

[Determination of eight active components of Bufei Huoxue Capsules in rat plasma and their pharmacokinetics by UHPLC-MS/MS].

An ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) method was established to investigate the pharmacokinetic behaviors of psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, psoralen, isopsoralen, methylnissolin, and neobavaisoflavone in rat plasma after oral administration of Bufei Huoxue Capsules. After SD rats were administered with Bufei Huoxue Capsules suspension by gavage, blood samples were collected from the inner canthus at different time points. After protein precipitation, plasma samples were separated on ACQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 µm). The mobile phase consisted of acetonitrile(A) and water(B) containing 0.1% formic acid in gradient elution. The positive and negative ions were measured simultaneously in the multi-reaction monitoring(MRM) mode. The pharmacokinetic parameters were calculated and fitted by DAS 3.2.8. Psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, psoralen, isopsoralen, methylnissolin, and neobavaisoflavone were detected in the rat plasma after drug administration, with AUC_(0-t) of(3 357±1 348),(3 555±1 696),(3.03±0.88),(2.21±0.33),(1 787±522),(2 295±539),(5.69±1.41) and(3.40±0.75) µg·L~(-1)·h, and T_(max) of(1.56±0.62),(1.40±0.70),(0.21±0.05),(0.25±0.12),(0.26±0.11),(0.34±0.29),(0.74±0.59), and 0.25 h. The method is proved specific and repeatable and is suitable for the determination of psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, pso-ralen, isopsoralen, methylnissolin, and neobavaisoflavone in the rat plasma, which can be applied to pharmacokinetic study.

Determination of eight active components of Bufei Huoxue Capsules in rat plasma and their pharmacokinetics by UHPLC-MS/MS / 中国中药杂志

An ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) method was established to investigate the pharmacokinetic behaviors of psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, psoralen, isopsoralen, methylnissolin, and neobavaisoflavone in rat plasma after oral administration of Bufei Huoxue Capsules. After SD rats were administered with Bufei Huoxue Capsules suspension by gavage, blood samples were collected from the inner canthus at different time points. After protein precipitation, plasma samples were separated on ACQUITY UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm). The mobile phase consisted of acetonitrile(A) and water(B) containing 0.1% formic acid in gradient elution. The positive and negative ions were measured simultaneously in the multi-reaction monitoring(MRM) mode. The pharmacokinetic parameters were calculated and fitted by DAS 3.2.8. Psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, psoralen, isopsoralen, methylnissolin, and neobavaisoflavone were detected in the rat plasma after drug administration, with AUC_(0-t) of(3 357±1 348),(3 555±1 696),(3.03±0.88),(2.21±0.33),(1 787±522),(2 295±539),(5.69±1.41) and(3.40±0.75) μg·L~(-1)·h, and T_(max) of(1.56±0.62),(1.40±0.70),(0.21±0.05),(0.25±0.12),(0.26±0.11),(0.34±0.29),(0.74±0.59), and 0.25 h. The method is proved specific and repeatable and is suitable for the determination of psoralenoside, isopsoralenoside, calycosin-7-glucoside, ononin, pso-ralen, isopsoralen, methylnissolin, and neobavaisoflavone in the rat plasma, which can be applied to pharmacokinetic study.

Metformin inhibits Aß25-35 -induced apoptotic cell death in SH-SY5Y cells.

Metformin, a first-line drug for type-2 diabetes, plays a potentially protective role in preventing Alzheimer's disease (AD), but its underlying mechanism is unclear. In this study, Aß25-35 -treated SH-SY5Y cells were used as a cell model of AD to investigate the neuroprotective effect of metformin, as well as its underlying mechanisms. We found that metformin decreased the cell apoptosis rate and death, ratio of Bcl-2/Bax, and expression of NR2A and NR2B, and increased the expression of LC3 in Aß25-35 -treated SH-SY5Y cells. Metformin also reduced intracellular and extracellular Glu concentrations, as well as the intracellular concentration of Ca2+ and ROS in Aß25-35 -treated SH-SY5Y cells. These findings suggest that metformin inhibits Aß25-35 -treated SH-SY5Y cell death by inhibiting apoptosis, decreasing intracellular Ca2+ and ROS by reducing neurotoxicity of excitatory amino acids, and by possibly reversing autophagy disorder via regulating autophagy process.

Genistein inhibits Aß25-35 -induced SH-SY5Y cell damage by modulating the expression of apoptosis-related proteins and Ca2+ influx through ionotropic glutamate receptors.

In this study, we investigated the protective effects of genistein against SH-SY5Y cell damage induced by ß-amyloid 25-35 peptide (Aß25-35 ) and the underlying mechanisms. Aß-induced neuronal death, apoptosis, glutamate receptor subunit expression, Ca2+ ion concentration, amino acid transmitter concentration, and apoptosis-related factor expression were evaluated to determine the effects of genistein on Aß-induced neuronal death and apoptosis. The results showed that genistein increased the survival of SH-SY5Y cells and decreased the level of apoptosis induced by Aß25-35 . In addition, genistein reversed the Aß25-35 -induced changes in amino acid transmitters, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, and N-methyl-d-aspartate (NMDA) receptor subunits in SH-SY5Y cells. Aß25-35 -induced changes in Ca2+ and B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax) protein and gene levels in cells were also reversed by genistein. Our data suggest that genistein protects against Aß25-35 -induced damage in SH-SY5Y cells, possibly by regulating the expression of apoptosis-related proteins and Ca2+ influx through ionotropic glutamate receptors.

[Treatment of Vascular Cognitive Impairment by "Huayu Tongluo" Moxibustion].

OBJECTIVE: To observe the therapeutic effect of "Huayu Tongluo"(blood stasis-removing and meridian-collateral-dredging) moxibustion for vascular cognitive impairment(VCI) patients and changes of insulin like growth factor -1(IGF-1) levels in serum after the treatment. METHODS: Sixty patients with VCI were randomly divided into medication (control) and moxibustion groups (n＝30 in each group). Cotton cloth-separated moxibustion was applied to Baihui (GV 20) and Shenting (GV 24), and conventional moxibustion applied to Dazhui (GV 14) and Yongquan (KI 1) for 30 min, once daily, 6 times a week and for 30 days. Patients of the control group were treated by oral administration of Donepezil hydrochloride at the dose of 5 mg/night for 30 days. The core symptoms of traditional Chinese medicine (TCM), mini-mental state examination(MMSE), activity of daily living(ADL) and Montreal cognitive assessment(MoCA) scales were used to assess the therapeutic effect after the treatment. The content of serum IGF-1 was determined by ELISA. RESULTS: Of the two 30 cases in the control and moxibustion groups, 9 and 17 experienced marked improvement, 13 and were effective, 8 and 3 ineffective, with the effective rates being 73.33% and 90.00%, respectively. The effective rate in the moxibustion group was obviously higher than that in the control group (P<0.05). After the treatment, the TCM symptom scores were significantly decreased, and the MMSE, ADL and MoCA scores considerably increased in both groups compared with those of their own individual pre-treatment (P<0.01). The TCM symptom score of the moxibustion group was significantly lower, and the MMSE and ADL scores were obviously higher than those of the control group (P<0.01). The serum IGF-1 content in both groups was significantly increased after the treatment relevant to that of their own individual pre-treatment (P<0.01), and was obviously higher in the moxibustion group than in the control group (P<0.01). No significant difference was found between the two groups in the MoCA score after the treatment (P>0.05)．. CONCLUSION: "Huayu Tongluo" moxibustion has a positive effect for patients with VCI, which may be associated with its effect in up-regulating serum IGF-1 level.

Zn2+ reduction induces neuronal death with changes in voltage-gated potassium and sodium channel currents.

In the present study, cultured rat primary neurons were exposed to a medium containing N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a specific cell membrane-permeant Zn2+ chelator, to establish a model of free Zn2+ deficiency in neurons. The effects of TPEN-mediated free Zn2+ ion reduction on neuronal viability and on the performance of voltage-gated sodium channels (VGSCs) and potassium channels (Kvs) were assessed. Free Zn2+ deficiency 1) markedly reduced the neuronal survival rate, 2) reduced the peak amplitude of INa, 3) shifted the INa activation curve towards depolarization, 4) modulated the sensitivity of sodium channel voltage-dependent inactivation to a depolarization voltage, and 5) increased the time course of recovery from sodium channel inactivation. In addition, free Zn2+ deficiency by TPEN notably enhanced the peak amplitude of transient outward K+ currents (IA) and delayed rectifier K+ currents (IK), as well as caused hyperpolarization and depolarization directional shifts in their steady-state activation curves, respectively. Zn2+ supplementation reversed the effects induced by TPEN. Our results indicate that free Zn2+ deficiency causes neuronal damage and alters the dynamic characteristics of VGSC and Kv currents. Thus, neuronal injury caused by free Zn2+ deficiency may correlate with its modulation of the electrophysiological properties of VGSCs and Kvs.

Lilium spp. pollen in China (Liliaceae): taxonomic and phylogenetic implications and pollen evolution related to environmental conditions.

Recent molecular and karyologic studies have significantly modified delimitation of Lilium. However, despite the importance of pollen evolution in the genus comprehensive studies with electron microscopy and evaluation of pollen evolution are lacking. Therefore, we studied pollen morphology in a sample of 65 individuals from 37 taxa covering all the sections distributed in the world, using scanning electron microscopy. Our collection of 49 individuals from 21 taxa covering all five sections in China was also included in the database. We found pollen tetrads in L. bakerianum. Based on present and previous studies, our results suggest that pollen from L. formosanum should be classified as a new type, Formosanum. Combined with morphological and molecular evidence, pollen sculpture patterns appear to reflect phylogenetic relationships and are useful for species or subsection delimitation. Based on a comprehensive survey and correlation with potential functional implications, we propose the following hypothesis: evolution of an exine sculpture shows pollen type trends from Martagon → Callose → Concolor → Formosanum. The evolutionary trend regarding pollen sculpture and size could be related to selective pressure to adapt to environmental conditions. Pollen size and shape showed a significantly positive correlation with annual precipitation, and smaller pollen grains appear to adapt better in habitats with extreme conditions. Evolution trends in exine sculpture do not appear to be definitively correlated with pollen size and shape.

Osthol regulates hepatic PPAR alpha-mediated lipogenic gene expression in alcoholic fatty liver murine.

Our previous studies found that osthol, an active constituent isolated from Cnidium monnieri (L.) Cusson (Apiaceae), could ameliorate the accumulation of lipids and decrease the lipid levels in serum and hepatic tissue in alcohol-induced fatty liver mice and rats. The objective of this study was to investigate its possible mechanism of the lipid-lowering effect. A mouse model with alcoholic fatty liver was induced by orally feeding 52% erguotou wine by gavage when they were simultaneously treated with osthol 10, 20, 40 mg/kg for 4 weeks. The BRL cells (rat hepatocyte line) were cultured and treated with osthol at 25, 50, 100, 200 microg/ml for 24h. The mRNA expressions of peroxisome proliferator-activated receptor (PPAR) alpha, diacylglycerol acyltransferase (DGAT), 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and cholesterol 7 alpha-hydroxylase (CYP7A) in mouse hepatic tissue or cultured hepatocytes were determined by reverse transcription polymerase chain reaction (RT-PCR). After treatment with osthol, the PPAR alpha mRNA expression in mouse liver and cultured hepatocytes was increased in dose dependent manner, while its related target genes for mRNA expression, e.g., DGAT and HMG-CoA reductase, were decreased, the CYP7A was inversely increased. And osthol-regulated mRNA expressions of DGAT, HMG-CoA reductase and CYP7A in the cultured hepatocytes were abrogated after pretreatment with specific inhibitor of PPAR alpha, MK886. It was concluded that osthol might regulate the gene expressions of DGAT, HMG-CoA reductase and CYP7A via increasing the PPAR alpha mRNA expression.

Two new N-oxide Lycopodium alkaloids from Huperzia serrata.

Two new Lycopodium alkaloids, N-oxidehuperzine E (1) and N-oxidehuperzine F (2), along with two known alkaloids, huperzines E (3) and F (4), were isolated from Huperzia serrata (Thunb.) Trev. Their structures were elucidated by spectroscopic and chemical transformations.

[Effects of water stress on the growth of Lonicera japonica and quality of honeysuckle].

OBJECTIVE: Effects of water stress on the growth of Lonicera japonica and quality of honeysuckle were studied. METHODS: Different extent water stress treatment imposed to potted plants before the first florescence of honeysuckle, and the growth and related physiological indexes of Lonicera japonica were determined. RESULTS: Shoot grow slowly as well as the increases of specific weight of leaf and the decrease of the yield of honeysuckle under water stress. The contents of chlorophyl in the leaves treated water stress all increased, and the content of soluble suger had a rising stream. The content of soluble protein in leaves of plant imposed by minor stress treatment was obviously higher than that of CK and severe stress treatment. As the stress was hard, the content of proline in leaves increased significantly. With water stress severing, the content of chlorogenic acid in the flower bud decreased very significantly than CK. CONCLUSION: Water condition was a important factor affected the growth and development of shoots, leaves and flower buds as well as the quality of honeysuckle. In the cultivation of Loniccra japonica, irrigation timing and certain irrigation condition was necessary.

[Analysis on processing mechanism of calamine].

OBJECTIVE: To study processing method and mechanism of Calamine. METHOD: Thermogravimetry analysis method and nano-technology were adopted to analyze and synthesize the components in Calamine, Tetracycline was took as the comparison drug to determine the antibacterial activity of Calamine and its components. RESULT: A part of zinc carbonate in Calamine was decomposed into zinc oxide when processing, and the particle size was smaller than before. The antibacterial activity of Calamine is decided by the content and particle size of zinc oxide, and has nothing with zinc carbonate. The more content and the smaller particle size of zinc oxide, the more powerful antibacterial activity of Calamine. CONCLUSION: The content and the particle size of zinc oxide can be the important targets in the processing of Calamine.

Analysis on processing mechanism of calamine / 中国中药杂志

<p><b>OBJECTIVE</b>To study processing method and mechanism of Calamine.</p><p><b>METHOD</b>Thermogravimetry analysis method and nano-technology were adopted to analyze and synthesize the components in Calamine, Tetracycline was took as the comparison drug to determine the antibacterial activity of Calamine and its components.</p><p><b>RESULT</b>A part of zinc carbonate in Calamine was decomposed into zinc oxide when processing, and the particle size was smaller than before. The antibacterial activity of Calamine is decided by the content and particle size of zinc oxide, and has nothing with zinc carbonate. The more content and the smaller particle size of zinc oxide, the more powerful antibacterial activity of Calamine.</p><p><b>CONCLUSION</b>The content and the particle size of zinc oxide can be the important targets in the processing of Calamine.</p>