ABSTRACT
In this study, we aimed to establish a rat liver micro-tissue evaluation system to evaluate the hepatotoxicity of the main monomers in Polygonum multiflorum. Rat primary hepatocytes were isolated and purified by two-step in situ perfusion method to prepare hepatic parenchymal cells. The ultra-low adsorption plate and the inverted model were used to establish an in vitro hepatotoxicity evaluation system. After the system was established, the main monomer components(monanthone with emodin type, rhein, emodin, emodin-8-O-β-D-glucopyranoside, physcion) of P. multiflorum were selected for in vitro hepatotoxicity evaluation. This study showed that the primary cells of the liver can form liver micro-tissues in the low adsorption plate method and the mold perfusion method, with good liver structure and function, which can be used to evaluate the hepatotoxicity of the drug to be tested after long-term administration. The five monomers to be tested in P. multiflorum can significantly affect the proliferation of primary liver micro-tissues in rats in a dose-and time-dependent manner. The hepatotoxic effects were as follows: monanthone with emodin type > rhein > emodin > emodin-8-O-β-D-glucopyranoside > physcion. The results suggested that the emodin-type monoterpene and rhein might be the potential hepatotoxic components, while the metabolites of emodin-8-O-β-D-glucoside and emodin methyl ether showed more toxic risks. The rat primary hepatocyte micro-tissue model system established in this experiment could be used to achieve long-term drug administration in vitro, which was consistent with the clinical features of liver injury caused by long-term use of P. multiflorum. The experimental results provided important information and reference on the clinical application and toxic component of P. multiflorum.
Subject(s)
Animals , Rats , Chemical and Drug Induced Liver Injury , Emodin , Fallopia multiflora , Glucosides , Plant Extracts , PolygonumABSTRACT
Objective@#To explore an open porous microtissue which is suitable for the growth of bone marrow mesenchymal stem cells (BMSCs) , and the optimal size of this microtissue.@*Methods@#Polymethyl methacrylate (PMMA) micro-stencil array chips with pore diameters of 1500 μm and 800 μm were constructed by laser drilling technique. The gelatin solution was chemically crosslinked and uniformly spread on the micro-stencil array chips. After freeze-drying for 16 h, the microgels in the chip were ejected by the ejector-pin array. SEM images were taken to observe the microstructure of microgels. Pore size and porosity of two kinds of microgels were calculated using Image J Pro software. The P3 BMSCs were seeded on microgels at the concentration of 5×106/ml. After 7 days culture in vitro, the microtissues were obtained. Thereafter, the viability of seeded BMSCs was assessed using live/dead cell assay. The growth of the cells on the microtissues and the secretion of ECM were observed by scanning electron microscopy (SEM).@*Results@#The SEM images showed that the open porous structured microgels were successfully fabricated using the micro-stencil array chip. The pore size and porosity of 1 500 μm microgels were about 1.5 times that of the 800 μm microgels. After 7 days culture in vitro, the number of cells 1 500 μm microgels were more compared to 800 μm microgels, in the live/dead cell assay. Fluorescent quantitative analyses showed that the viability of BMSCs on 1 500 μm microtissues was 4 times that of 800 μm microtissues. SEM images showed that the cells on 1 500 μm microtissues grew well and surrounded by ECM.@*Conclusions@#The micro-stencil array chips were successfully used to construct microgels with open porous, which could be further used to fabricate open porous microtissues. The 1 500 μm microtissues were more favorable for BMSCs, compared to 800 μm microtissues.
ABSTRACT
Despite the recent progress in physiological studies of acupuncture, few morphological studies from the viewpoint that acupuncture stimulation produces with a small tissue injury have been reported. In this study, we evaluated the mechanism of acupuncture stimulation by a histopathological approach. First, we confirmed the tissue structure from the skin to the muscle layer in a corpse. In the human body, free terminals (endings) of unmyelinated nerve fibers (1.5μmin diameter, Group III or Group IV) were present subcutaneously or in the endomysium of muscle tissues. Next, the extent of tissue injury caused by insertion of needles 0.20-0.95mm in diameter evaluated in human and animal tissues. The extent of the injury was in proportional to the diameter of the needle. These confirmed that acupuncture stimulation is clearly a small tissue injury. Also, the free terminals (endings) of unmyelinated nerves similar to those observe in the human tissue were obsurved in the endmysium near the injury site. These morphological findings suggest that acupuncture stimulation is related to Group III or Group IV fibers.