OBJECTIVE: To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments. METHODS: A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed. RESULTS: FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05). CONCLUSION FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.
Mice ; Animals ; Mitogen-Activated Protein Kinase 14/metabolism* ; Wolfiporia ; Lipopolysaccharides/pharmacology* ; Sepsis/complications* ; Signal Transduction ; Inflammation/drug therapy* ; Oxygen Radioisotopes

1. INTRODUCTION: It is well known that a successful corneal graft depends primarily on the viability of corneal endothelium. And also corneal endothelial viability of donor eye is largely varied from the duration of storage time of enucleated eyes. It is generally agreed that penetrating corneal graft should be done within 48 hours when donor cornea was stored in moist chamber at 4 degrees C because of corneal endothelial viability. But there is some other opinion about the storage time as the school of Filatov insists that donor cornea could be storaged for 4~5 days before corneal graft. The permeability of the cornea is significant from several viewpoints, first, the nutrition of the cornea depends on the diffusion of oxygen and glucose and other substances from the surrounding fluids. Second, the transport of drugs or other substances across the cornea is determined by the permeability of the corneal layers. Present experiment involves the direct measurement for changes of tritiated water permeability, K trans, of rabbit corneal endothelium stored at 4 degrees C up to 5 days. 2. MATERIALS AND METHODS: Adult albino rabbits, weighing 2~3 kg. were killed by intravenous injection of air administered via the marginal vein of an ear. if more moist chamber bottles are prepared than needed for immediate use of enucleated eyes, they more stored at 4 degrees C in the refrigerator for periods of 24, 48, and 120 hrs. Preparation of endothelium was made simply by remove of the epithelium with gouze (Kim et ai, 1971). The remaining stroma was requisite as a mechanical support for th endothelium. And then a puncture incision was made through the sclera 1~2 mm peripheral to the limbus, and a circumferential cut was made at the same distance from the limbus. The excised tissue was then transferred immediately to a Petri dish containing Ringer's solution at 35 degrees C, and the lens and the iris were carefully removed together with any connective tissue that was attached to the sclera. Finally, the cornea with its scleral rim was mounted in a lucite chamber which was designed specially to hold the convex tissue(Fig. 1). After the tissue was mounted, the chambers were quickly filled with the experimental solution. The solutions on both sides of the tissue were stirred with Teflon-coated magnetic stirrer driven by horseshoe magnets rotating at 400 rev./min to reduce the rete-limiting effect of an unstirred layer on solute movement (Dainty, 1963). The composition of the experimental solution, based on normal (Krebs-bicarbonate) Ringer's solution, was presented in Table I (Green, 1965). A sufficient volume of solution for each experiment was brought to the required temperature(25 degrees C) immediately prior to the experiment. Radioisotopes 3H-labelled tritiated water(THO. specific activity, 5 mCi/ml; Molecular weight, 22) was obtained in solution form (Amersham Radiochemical Center, Buckinghamshire, England). A tracer amount of the radioactive substance to be studied, in normal Ringer's solution, was introduced into the chamber facing the endothelial surface and its rate of appearance on the other side was then determined. Samples (50 micro l) were taken with a micropipette (25 micro l) initially from both bathing solutions 1 hr. after addition of the solutions to the tissue, but thereafter only from the cold side at 60 minutes intervals for 3 hr duration. The samples were transferred to planchets which were then placed on sample spinner; a volume of methyl alcohol was added sufficient to cover the planchet and to allow even spreading of the sample which drying under on infrared lamp. The activity of the radioisotope samples were then assayed using a NMC Proportional Counter System, Model PC-3A, U.S.A. The permeability coefficient, K trans., for this radioactive substance, defined as the amount of the given substance crossing 1cm2 of membrane surface per second under a driving force of unit concentration gradient was calculated according to Maffly et al (1960). 3. RESULTS AND COMMENTS: The permeability coefficients, K trans., of fresh corneal endothelium as a control and the corneal endothelium stored AT 4 degrees C moist chamber in the refrigerator for different lengths of time were presented in Table 2, all these permeabilities were measured on highly swollen stromas since permeability determinations were not begun until 1 hr after exposure to the isotope solution. The rate of passage of tritiated water through the endothelium (plus stroma and Descemet's membrane) stored at 4 degrees C showed the linearity of the increasing count rate against time (Fig. 2). A rise in tritiated water permeability occurred in those stored for 48 hrs. and then followed by a fall in those stored for 120 hours, which there was no significant difference (p>0.3) in comparing with the permeability of fresh corneal endothelium used immediately after enucleation. It presumed that such a finding is due probably to the vitality change of the endothelium. Such a similar results were also obtained by a method of oxygen uptake by corneal endothelium of eyebank eyes stored at 4 degrees C for up to 6 days(Preziosi, 1971).
Adult ; Baths ; Connective Tissue ; Cornea ; Diffusion ; Ear ; Endothelium ; Endothelium, Corneal* ; Epithelium ; Glucose ; Humans ; Injections, Intravenous ; Iris ; Membranes ; Methanol ; Molecular Weight ; Oxygen ; Permeability* ; Polymethyl Methacrylate ; Punctures ; Rabbits ; Radioisotopes ; Sclera ; Tissue Donors ; Transplants ; Veins ; Water*

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it has a poor prognosis and few therapeutic options. Radiotherapy is one of the most effective forms of cancer treatment, and P53 protein is one of the key molecules determining how a cell responds to radiotherapy. The aim of this study was to determine the therapeutic efficacy of iodine-131 in three human HCC cell lines. METHODS: Western blotting was used to measure P53 expression. The effects of radiotherapy with iodine-131 were assessed by using the clonogenic assay to evaluate cell survival. Flow cytometry was carried out to examine the effects of iodine-131 on cell death, oxidative stress, reduced intracellular glutathione expression, the mitochondrial membrane potential, and the cell cycle. RESULTS: The P53 protein was not expressed in Hep3B2.1-7 cells, was expressed at normal levels in HepG2 cells, and was overexpressed in HuH7 cells. P53 expression in the HuH7 and HepG2 cell lines increased after internal and external irradiation with iodine-131. Irradiation induced a decrease in cell survival and led to a decrease in cell viability in all of the cell lines studied, accompanied by cell death via late apoptosis/necrosis and necrosis. Irradiation with 131-iodine induced mostly cell-cycle arrest in the G0/G1 phase. CONCLUSIONS: These results suggest that P53 plays a key role in the radiotherapy response of HCC.
Apoptosis/*radiation effects ; Blotting, Western ; Carcinoma, Hepatocellular/metabolism/pathology/radiotherapy ; Cell Line, Tumor ; Cell Survival/drug effects ; G1 Phase Cell Cycle Checkpoints/radiation effects ; *Gamma Rays ; Glutathione/metabolism ; Hep G2 Cells ; Humans ; Iodine Radioisotopes/chemistry/pharmacology/therapeutic use ; Liver Neoplasms/metabolism/pathology/radiotherapy ; Phosphorylation ; Reactive Oxygen Species/metabolism ; Tumor Suppressor Protein p53/*metabolism