| Colistin, also known as polymyxin E, is an aerobic spore-forming polypeptide antibioticobtained from the culture of an rod Bacillus Polymyxa Var. colistinus, and its sulfate has becameavailable for clinical use. But early colistin was restricted because of its nephrotoxicity andneurotoxicity. In recent years, with the ravages of multi-resistant bacteria, the research anddevelopment of new drugs against multidrug resistant limited and colistin sulfate has significantefficacy against multi-drug-resistant Gram-negative bacilli, colistin sulfate is received attention inclinical applications again. Although neurotoxicity and nephrotoxicity problem of colistin stillpersists, it’s important significantly that we are looking for a new attenuated measures and clinicaltreatment by clearing the mechanism of toxicity. Using an in vitro system to detect neurotoxicity isa trend and many in vitro models were used to detect potential neurotoxicity and mechanisms oftoxicity. Especially, rat adrenal medulla pheochromocytoma （PC12cells） has been widely used inneurobiology and neurotoxicology of research. In this paper, the mechanism of neurotoxicityinduced by colistin was incestigated in PC12cells combined with the acute toxicity test doseselection and discussed the possible mechanism of colistin sulfate-induced neuronal apoptosis.The acute toxicity test of the colistin sulfate was carried out by K rber method in18～22gKunming mice by tail vein injection administration and the effect of colistin sulfate acute toxicitywas detected on routine blood, blood biochemistry, liver, kidney and other organs. Based on theacute toxicity test results,1mg·mL-1colistin sulfate was selected as an in vitro cytotoxicityexperiments the highest dose because it’s not significant difference between this dose treatmentand the control group in the symptoms of poisoning, routine blood and blood biochemicalparameters, liver and kidney tissue structure. Combined three doses of250,125,62.5μg·mL-1colistin sulfate were screened and used to follow-up test by determination of cell viability test（MTT）, AO/EB double staining, Hoechst33258staining, ultrastructure observation bytransmission electron microscopy, determination of LDH levels and ROS concentration. In order toobserve sulfate colistin-induced apoptosis in PC12cells circumstances, mitochondrial membranepotential and apoptosis cycle was detected using Rhodamine-123staining by the flow cytometryand PI single staining, respectively. Caspase-3, caspase-9activity was determined by the kit, DNAfragments was detected by DNA Ladder method. In order to discuss mechanism of sulfatecolistin-induced apoptosis in PC12cells, free Ca2+concentration of intracellular andextracellular was detected, expression of apoptosis-related protein: p53, Bax, Bcl-2, cytochrome c, Fas, FasL, GnRP78, caspase-3, caspase-8, caspase-9and caspase-12was detected by thewestern-blot assay, expression of apoptosis-related gene: caspase-3, caspase-8, caspase-9,caspase-12mechanism was detected by the real-time quantitative PCR dye assay. Mechanism ofsulfate colistin-induced apoptosis in PC12cells derived from three classic signaling pathwayinduced apoptosis. The test results showed as follows:（1） The result of MTT test showed that cell viability reduced. The result of LDH test showedthat LDH release increased with increasing of time and dose. These proved colistin sulfate wasstrong toxicity on PC12cells.（2） By observations of the inverted microscope, fluorescent inverted microscope andtransmission electron microscopy, the dose of125and250μg·mL-1colistin sulfate could causePC12cell typical characteristics of apoptosis and dose-dependent relationship, i.e protrusionsshorter, intracytoplasmic vacuolesformation, nuclear condensation, nuclear fragmentation,increased mitochondrial swelling, and accompanied by vacuolization and the endoplasmicreticulum ribosome release.（3） Colistin sulfate could elevate ROS activity, damage the body’s antioxidant system, lead tooxidative stress, resulting in PC12cells oxidative damage, and a dose-response relationship andtime-effect relationship. This indicated that oxidative stress might be one of the mechanisms ofcolistin sulfate induced neurotoxicity.（4） The dose of125,250μg·mL-1colistin sulfate could reduce the mitochondrial membranepotential, interfere with DNA normal synthesis process, cause the S phase arrest in the cell cycle,and cause DNA strand breaks by the flow cytometry. The result showed that colistin sulfate couldactivate apoptosis in PC12cells with time and dose-dependent relationship.（5） The dose of125,250μg·mL-1colistin sulfate can cause DNA strand breaks and leading toDNA damage in the DNA Ladder detection and caused activation of caspase-3, caspase-9.Thedegree of was significantly enhancing, and the dose-time dependent relationship.（6） Western-blot analysis results showed that125,250μg·mL-1colistin sulfate couldsignificantly increase the expression of p53, cytochrome c, Bax, of caspase-9and caspase-3andsignificantly reduce the expression of Bcl-2. This suggested that and colistin sulfate could inducethe occurrence of apoptosis through the mitochondrial pathway by damaging cellularDNA-induced activation of p53, and increasing the expression of Bax while decreasing expressionof Bcl-2, and increase Bax/Bcl-2ratio, and promoting cytochrome c to release apoptotic materialinto the cytoplasm, and then causing caspase-9release, the last activation of caspase-3.（7） The dose of125,250μg·mL-1colistin sulfate could significantly increase expression ofFas, FasL and caspase-8in PC12cells by Western-blot analysis. These indicated that colistinsulfate could induce occurrence of apoptosis induced by the death receptor pathway by increasingexpression of Fas, FasL receptor and release of caspase-8, and then activation of caspase-3. （8） The dose of125,250μg·mL-1colistin sulfate could increase Ca2+concentration in theintracellular of PC12cells and reduce Ca2+concentration in the cell culture medium, and lead todamage cell calcium homeostasis. The results showed that the endoplasmic reticulum chaperoneGnRP78increased expression, and caspase-12increased release and then caspase-3was activatedby western-blot method. These indicated that colistin sulfate could induce the occurrence ofapoptosis by endoplasmic reticulum pathway.（9） By detecting expression levels of caspase-8, caspase-9, caspase-12gene in PC12cells byreal-time quantitative PCR, sulfate colistin-induced apoptosis in PC12cells is mainly caused bythe death receptor pathway, next mitochondrial pathway, and finally endoplasmic reticulumpathway.In summary, high concentrations of colistin sulfate in vitro can cause the morphologicalchanges of PC12cells and lead to neuronal intracellular oxidative stress and induce apoptosis bythe joint action of death receptor pathway, mitochondria pathway and endoplasmic reticulumpathway. In this paper, it’s suggested that the colistin sulfate-caused neurotoxicity may be relatedto nervous cells apoptosis, which leaded to damage of the central nervous system. |
