Deep Fusion of Intelligent Meridian Sensing Technology and Huoluo Xiaoling Pills in the Treatment of Knee Osteoarthritis.

Based on the deep fusion of intelligent meridian sensing technology and Huoluo Xiaoling Pill (HXP) in the treatment of knee osteoarthritis (KOA), firstly, the effective components and targets of Salvia miltiorrhiza, Angelica sinensis, frankincense, and myrrh were obtained by using TCMSP, SwissADME, and Swisstarget databases. Similarly, relevant targets of KOA were collected through GeneCards, OMIM, TTD, PharmGKB, and DrugBank databases. Next, the potential targets of ZXP in the treatment of KOA were obtained by intersection of drug and disease targets. Finally, Cytoscape 3.7.1 software was used to construct a "disease-drug-component-target" network, and Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Gnomes (KEGG) signaling pathway enrichment analysis were performed on the core targets through Metascape website. A total of 99 active components and 203 corresponding potential therapeutic targets were obtained from the components of HXP. And KOA has 2543 potential therapeutic targets, of which 120 cross targets correspond to 120 active compounds in HXP. Then, topology analysis displayed that the six targets form the core PPI network. In addition, GO and KEGG enrichment analyses showed that these core targets were mainly enriched in inflammatory response, apoptosis, oxidation reaction, and other related pathways.

Success rates and safety of a modified percutaneous PD catheter placement technique: Ultrasound-guided percutaneous placement of peritoneal dialysis catheters using a multifunctional bladder paracentesis trocar.

BACKGROUND: We modified the blind Seldinger technique by incorporating ultrasound guidance and the use of a multifunctional bladder paracentesis trocar for PD catheter (PDC) placement, which can be easily performed by a nephrologist and is a feasible technique. To compare success rates and safety of our modified percutaneous PD catheter placement technique to open surgery. METHODS: Two hundred and twelve stage-5 chronic kidney disease(CKD) patients receiving PD therapy from June 2016 to June 2019 were included, 105 patients treated by ultrasound-guided percutaneous placement of peritoneal dialysis catheters using a multifunctional bladder paracentesis trocar (Group A) and 107 patients receiving open surgical placement (Group B). Outcomes of patients via either catheter placement technique were retrospectively compared. The clinical success rate as defined by proper catheter drainage within 4 weeks after placement, complication rates (both technical complications and infections), and 1-year catheter survival were compared. RESULTS: There was no significant difference in sex ratio, age, or previous abdominal surgery history between groups (P > .05). Both surgical time and incision length were significantly shorter in Group A than in Group B (P < .05). Clinical success rate was also higher inGroup A (P < .05). Moreover, Group A demonstrated lower overall complication rates (P < .05) and lower incidence rates of early peritonitis, initial drainage disorder, and peritubular leakage (all P < .05). One-year catheter survival was also higher in Group A (P < .05). CONCLUSION: Percutaneous placement of PD catheters using our modified technique demonstrates superior success rates and safety compared to open surgery. In addition, our modified technique can be a better alternative to traditional Seldinger percutaneous catheterization for its higher success rate and safety, more accurate positioning.

Potent prophylactic and therapeutic efficacy of recombinant human ACE2-Fc against SARS-CoV-2 infection in vivo.

The current COVID-19 pandemic, caused by SARS-CoV-2, poses a serious public health threat. Effective therapeutic and prophylactic treatments are urgently needed. Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, which binds to the receptor binding domain (RBD) of SARS-CoV-2 spike protein. Here, we developed recombinant human ACE2-Fc fusion protein (hACE2-Fc) and a hACE2-Fc mutant with reduced catalytic activity. hACE2-Fc and the hACE2-Fc mutant both efficiently blocked entry of SARS-CoV-2, SARS-CoV, and HCoV-NL63 into hACE2-expressing cells and inhibited SARS-CoV-2 S protein-mediated cell-cell fusion. hACE2-Fc also neutralized various SARS-CoV-2 strains with enhanced infectivity including D614G and V367F mutations, as well as the emerging SARS-CoV-2 variants, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.1 (Kappa), and B.1.617.2 (Delta), demonstrating its potent and broad-spectrum antiviral effects. In addition, hACE2-Fc proteins protected HBE from SARS-CoV-2 infection. Unlike RBD-targeting neutralizing antibodies, hACE2-Fc treatment did not induce the development of escape mutants. Furthermore, both prophylactic and therapeutic hACE2-Fc treatments effectively protected mice from SARS-CoV-2 infection, as determined by reduced viral replication, weight loss, histological changes, and inflammation in the lungs. The protection provided by hACE2 showed obvious dose-dependent efficacy in vivo. Pharmacokinetic data indicated that hACE2-Fc has a relative long half-life in vivo compared to soluble ACE2, which makes it an excellent candidate for prophylaxis and therapy for COVID-19 as well as for SARS-CoV and HCoV-NL63 infections.

High performance polypyrrole/SWCNTs composite film as a promising organic thermoelectric material.

Conducting polymer thermoelectric (TE) materials have received great attention due to their unique properties. In this work, polypyrrole (PPy)/single-walled carbon nanotubes (SWCNTs) composite films with improved TE performance have been prepared by chemical interfacial polymerization at the cyclohexane/water interface under a controlled temperature. Attributed to the smooth surface, higher conjugation length and more ordered molecular structure of the interfacial polymerized PPy film, the electrical conductivity can be as high as ∼500 S cm-1. To further enhance the TE properties of PPy, SWCNT was added to construct a PPy/SWCNTs composite. Due to the synergistic effect between the two phases and the energy filtering effect at the interfaces between PPy and SWCNTs, the Seebeck coefficient of the composite enhanced significantly with the increase SWCNTs content. The composite shows an optimal power factor of 37.6 ± 2.3 µW mK-2 when the content of SWCNTs is 0.8 mg. This value is one of the largest values among the reported PPy based composites fabricated by the chemical polymerization method. The results indicate that interfacial polymerization under a controlled temperature is a promising way to improve the TE performance of conducting polymer based composite materials.

Palmitic Acid-Induced Podocyte Apoptosis via the Reactive Oxygen Species-Dependent Mitochondrial Pathway.

BACKGROUND/AIMS: Chronic kidney disease (CKD) is often accompanied by hyperlipidemia, which accelerates progression of the disease. Podocyte injury can lead to dysfunction of the glomerular filtration barrier, which is associated with proteinuria, a risk marker for the progression of CKD. Our previous studies demonstrated that palmitic acid (PA) can induce podocyte apoptosis; however, the underlying mechanisms are unclear. In the present study, we investigated the specific molecular mechanisms of PA-induced apoptosis in cultured podocytes. METHODS: We cultured mouse podocytes and treated them with PA. Then, cell viability was measured using the Cell Counting Kit-8 colorimetric assay, lipid uptake was assessed by Oil Red O staining and boron-dipyrromethene staining, apoptosis was measured by flow cytometry, mitochondrial injury was assessed by JC-1 staining and transmission electron microscopy, and mitochondrial production of reactive oxygen species (ROS) was evaluated by fluorescence microscopy using the MitoSOX Red reagent. The effects of PA on the mitochondria-mediated caspase activation pathway were investigated by examining the expression of caspase-8, cleaved caspase-9, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), Bax, Bid, cytochrome c, and Fas-associated protein with death domain (FADD) using western blotting. The translocation of Bax and cytochrome c were detected by immunofluorescence. RESULTS: PA treatment significantly increased lipid accumulation and induced podocyte apoptosis. We investigated whether the two primary apoptosis signaling pathways (death receptor-mediated pathway and mitochondria-mediated pathway) were involved in the execution of PA-induced podocyte apoptosis, and found that the levels of FADD, caspase-8, and Bid did not significantly change during this process. Meanwhile, PA treatment induced an increase in Bax protein expression and a decrease in Bcl-2 protein expression, with Bax translocation to the mitochondria. Furthermore, PA treatment induced mitochondrial impairment, and triggered the release of cytochrome c from the mitochondria to cytosol, with a concomitant dose-dependent increase in the levels of cleaved caspase-9, cleaved caspase-3, and PARP. Meanwhile, PA treatment increased mitochondrial production of ROS, and the mitochondria-targeted antioxidant mitoTEMPO significantly ameliorated PA-induced podocyte apoptosis. CONCLUSION: Our findings indicated that PA induced caspase-dependent podocyte apoptosis through the mitochondrial pathway, and mitochondrial ROS production participated in this process, thus potentially contributing to podocyte injury.

Berberine Protects Against Palmitate-Induced Apoptosis in Tubular Epithelial Cells by Promoting Fatty Acid Oxidation.

BACKGROUND Increased lipid accumulation in renal tubular epithelial cells (TECs) contributes to their injury and dysfunction and progression of tubulointerstitial fibrosis. Berberine (BBR), a natural plant alkaloid isolated from traditional medicine herbs, is effective in lowing serum lipid, and has a protective effect on chronic kidney disease (CKD) with dyslipidemia, including diabetic nephropathy. The aim of this study was to investigate the effect of BBR on palmitate (PA)-induced lipid accumulation and apoptosis in TECs. MATERIAL AND METHODS Human kidney proximal tubular epithelial cell line (HK-2) cells were treated with PA, BBR, and/or palmitoyltransferase 1A (CPT1A) inhibitor Etomoxir. Intracellular lipid content was assessed by Oil Red O and Nile Red staining. Cell apoptosis rate was evaluated by flow cytometry assay. The expression of apoptosis-related protein cleaved-caspase3 and fatty acid oxidation (FAO)-regulating proteins, including CPT1A, peroxisome proliferator-activated receptor α (PPARα), and PPARγ co-activator-1α (PGC1α), was measured by Western blot analysis and immunofluorescence. RESULTS In the present study, PA treatment increased intracellular lipid deposition accompanied by elevated apoptosis in TECs compared with control group, whereas the protein expression of CPT1A, PPARα, and PGC1α, did not correspondingly increase in TECs. BBR significantly up-regulated the protein expression of CPT1A, PPARα, and PGC1α in TECs treated with or without PA, and reversed PA-induced intracellular lipid accumulation and apoptosis. Moreover, the CPT1A inhibitor Etomoxir counteracted the protective effect of BBR in TECs. CONCLUSIONS These in vitro findings suggest that PA can induce intracellular lipid accumulation and apoptosis in TECs, and the mechanism may be associated with inducing defective FAO, whereas BBR can protect TECs against PA-induced intracellular lipid accumulation and apoptosis by promoting FAO.

Novel strategy using tryptic peptide immunoaffinity-based LC-MS/MS to quantify denosumab in monkey serum.

AIM: Denosumab is a recombinant fully human IgG2 that has a high affinity and specificity for human RANKL. Commercially available RANKL labeled with an Fc fragment cannot be used to establish an indirect ELISA. To characterize denosumab pharmacokinetic a robust and accuracy method should be developed urgently. RESULTS: In this study, an immunoaffinity enrichment method coupled with LC-MS/MS was established. The LC-MS/MS method acquired a linear range from 0.1 to 30 µg/ml. The intra- and inter-run precision (CV%) was within 11.5 and 10.5%, respectively. More importantly, the LC-MS/MS pharmacokinetic data were consistent with ELISA. CONCLUSION: This approach accelerated the quantification, reduced the costs and provided an alternative in case of lacking the special antigen to denosumab or a RANKL-biotinylated reagent.

[Study on High-yield Cultivation Measures for Arctii Fructus].

OBJECTIVE: To find out the high yield cultivation measures for Arctii Fructus. METHODS: Completely randomized block experiment design method was used in the field planting, to analyze the effect of different cultivation way on agronomic characters, phenological phase,quality and quantity of Arctii Fructus. RESULTS: Arctium lappa planted on August 28 had the best results of plant height, thousand seeds weight and yield. The highest yield of Arctii Fructus was got at the density of 1,482 plants/667 m2. Arctiin content was in an increase trend with the planting time delay and planting density increasing. The plant height, thousand seeds weight, yield and arctiin content by split application of fertilizer were significantly higher than that by one-time fertilization. Compared with open field Arctium lappa, plant height, yield, arctiin content and relative water content of plastic film mulching Arctium lappa was higher by 7.74%, 10.87%, 6.38% and 24.20%, respectively. In the topping Arctium lappa, the yield was increased by 11.09%, with 39. 89% less branching number. Early planting time and topping shortened the growth cycle of Arctium lappa plant. CONCLUSION: The high-yield cultivation measures of Arctii Fructus are: around August 28 to sowing, planting density of 1 482 plants/667 m2, split application of fertilizer for four times, covering film on surface of the soil and topping in bolting.

Optimization, validation and application of an assay for the activity of HMG-CoA reductase in vitro by LC-MS/MS.

A stable HMG-CoA reductase (HMGR) reaction in vitro was developed by a sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The optimized enzyme reaction condition contained 1.5 µg of HMGR, 20 nM of NADPH with 50 min of reaction time. The method was validated by several intra- and inter-day assays. The production transitions of m/z 147.0/59.1 and m/z 154.0/59.1 were used to detect and quantify mevalonolactone (MVAL) and MVAL-D7, respectively. The accuracy and precision of the method were evaluated over the concentration range of 0.005-1.000 µg/mL for MVAL and 0.010-0.500 µg/mL for lovastatin acid in three validation batch runs. The lower limit of quantitation was found to be 0.005 µg/mL for MVAL and 0.010 µg/mL for lovastatin acid. Intra-day and inter-day precision ranged from 0.95% to 2.39% and 2.26% to 3.38% for MVAL, 1.46% to 3.89% and 0.57% to 5.10% for lovastatin acid, respectively. The results showed that the active ingredients in Xuezhikang capsules were 12.2 and 14.5 mg/g, respectively. This assay method could be successfully applied to the quality control study of Xuezhikang capsule for the first time.

Ultra-high-performance liquid chromatography for the determination of exenatide in monkey plasma by tandem quadrupole mass spectrometry.

A highly sensitive ultra-high-performance liquid chromatographic-tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantification of the synthetic peptide drug of exenatide in monkey plasma. Sample preparation was carried out by solid-phase extraction (SPE), and bivalirudin was used as the internal standard (IS). An excellent chromatographic separation was obtained on a reversed-phase C18 column with a gradient elution. Detection utilized a Qtrap 5500 system operated in the positive ion mode with multiple reaction monitoring (MRM). The proposed method was validated by assessing the specificity, linearity, intra- and inter-day precision and accuracy, recovery, and stability. The method resulted in a linear calibration range of 0.10-30 ng/mL, extracting with only 50 µL monkey plasma aliquots. The intra- and inter-day precisions (as relative standard deviation) were less than 7.5% and 9.6%, respectively. The method could be successfully utilized for the pharmacokinetic study of exenatide in monkeys following a single subcutaneous injection of Byetta.

Quantitation of slow release triptorelin in beagle dog plasma by liquid chromatography-tandem mass spectrometry.

A sensitive method based on liquid chromatography-tandem mass spectrometry has been developed for the determination of triptorelin levels in beagle dog plasma. Plasma samples were applied to Oasis(®) HLB solid-phase extraction (SPE) cartridges. Extracted samples were evaporated under a stream of nitrogen and then reconstituted with 100 µl methanol:water:formic acid (60:40:0.08, v/v/v). The separation was achieved on a Venusil MP-C18 column (2.1 mm × 50 mm, 3 µm, Agela) with a gradient elution. Detection utilized a Qtrap5500 system operated in the positive ion mode with multiple reaction monitoring of the analyte at m/z 656.5→249.1 and of the I.S. at m/z 510.8→120.1. The proposed method was validated by assessing the specificity, linearity, precision and accuracy, recovery, matrix effects, and stability. Linear calibration curves were obtained in the concentration range of 0.01-10 ng/ml (the correlation coefficients were above 0.995). The lower limit of quantification (LLOQ) of the method was 0.01 ng/ml. The method was successfully applied to a pharmacokinetic study of a slow release triptorelin formulation in beagle dogs following a single intramuscular injection.

GABAA receptors in VTA mediate the morphine-induced release of ascorbic acid in rat nucleus accumbens.

Local perfusion of morphine produces increased levels of extracellular ascorbic acid (AA) in the nucleus accumbens (NAc) of freely moving rats. However, the pathways that regulate morphine-induced AA release in the NAc are unclear. In the present study, we used high performance liquid chromatography with electrochemical detection (HPLC-ECD) to examine the effects of intra-ventral tegmental area (VTA) administration of a GABA(A) agonist and antagonist on morphine-induced increases in AA of the NAc. Also, using high performance liquid chromatography with fluorescent detection (HPLC-FD) and HPLC-ECD, the releases of γ-aminobutyric acid (GABA) and dopamine (DA) in the NAc induced by intra-VTA administration of a GABA(A) agonist and antagonist were also investigated. The results obtained showed that morphine (1 mM), locally perfused into the NAc, significantly increased AA release in the NAc and also GABA release. Intra-VTA infusion of bicuculline (150 ng/rat), a GABA receptor antagonist, not only abolished the enhanced extracellular AA and GABA levels produced by local perfusion of morphine but also decreased the basal release of extracellular GABA and increased the basal release of extracellular DA in the NAc. Muscimol (100 ng/rat), a GABA receptor agonist, affected the basal release of GABA and DA, but not the basal AA levels, or the morphine-induced changes in AA and GABA levels. These findings suggest that the GABA(A) receptors in the VTA play an important role in the modulation of morphine-induced AA release in the NAc, and the effect of morphine on AA release in the NAc is partially regulated by the GABA(A) receptor-mediated action of DA afferents from the VTA.

Lesions of the medial prefrontal cortex prevent the acquisition but not reinstatement of morphine-induced conditioned place preference in mice.

In order to further investigate the role of the mPFC in morphine reward and drug priming induced relapse, the present study examined the effects of the mPFC lesions on the acquisition and morphine priming induced reinstatement of conditioned place preference (CPP). In the first experiment, mice received sham or bilateral kainic acid lesions of the mPFC and were subsequently tested for the acquisition of a morphine-induced CPP. In the second experiment, each mouse received lesions of mPFC following the establishment of morphine-induced CPP. Nine days later, a priming injection of morphine was given (2 mg/kg, i.p.) to reinstate the extinguished CPP. The results showed that pre-conditioning lesions of the mPFC blocked the acquisition of morphine-induced CPP, while post-conditioning lesions of the mPFC failed to prevent morphine priming induced reinstatement of CPP. These results provide the first direct evidence that the mPFC may be involved in the acquisition, but not morphine priming induced reinstatement of CPP.