Interaction Of Nanobacteria With Cultured Human Normal Hepatocytes

Partâ… Detection,Identification and Culture of Nanobacteria Isolated from Cirrhotic liver TissuesObjective:To investigate possible infection by nanobacteria in the liver tissues of patients with cirrhotic liver disease.Nanobacterial isolates were cultured after identification by several methods.Methods:Twelve cases of cirrhotic liver tissues and 12 cases of normal liver tissues were achieved during operation.Parts of every specimen were prepared for detection of nanobacteria with the method of immunohistochemistry(IHC),the rest were cooled down rapidly in liquid nitrogen followed by storage in—70℃refrigerates for isolation of nanobacteria.Identification of cultured isolates was accomplished with immunofluorescent double staining of hoechst33258 and monoclonal antibody against nanobacteria,also observed under transmission electron microscope(TEM).Confirmed nanobacterial isolates were recultured and consumption of electrolytes and glucose from culture media was dynamically measured during the progress.Results:Nanobacteria were detected in ten cases of cirrhotic liver tissues and one case of normal liver tissues,the difference was statistically significant(P＜0.05).In indirect immunostaining, nanobacteria gave out green fluorescence.Size of nanobacteria was measured ranging from 100nm to 300nm with electron microscope scanning.During nanobacterial culture,no remarkable changes were observed on the concentration of K⁺,Na⁺,Cl^-,Mg²⁺and glucose,while that of Ca²⁺and P^3-gradually decreased weekly(P＜0.05).Conclusions:Nanobacteria were found in cirrhotic liver tissues. Indirect immunofluorescence and electron microscope were perfectly helpful methods in identification of nanobacteria.Nanobacteria consumed Ca²⁺and P^3-instead of K⁺,Na⁺,Cl^-,Mg²⁺and glucose during culture. Partâ…¡Cytotoxicity of Nanobacteria Exerted on Cultured Human Normal HepatocytesObjective:To study cytotoxic effects of nanobacteria exerted on cultured human normal hepatocytes.Methods:Cytotoxic effects of nanobacteria exerted on human cultured normal hepatocytes were verified by MTT[3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]viability assay,LDH(lactate dehygrogenase)release test and direct optical microscopy.NO(nitric oxide)assay was carried out during cultured hepatocytes exposed to nanobacteria.Results:Hepatotoxicity was greatly dependent on the concentrations of nanobacteria and time of exposure.Under direct optical microscopy,various cellular morphological changes could be observed, such as oncosis,ambiguous appearance,rupture of the plasma membrane, cell lysis,even unorganized dismantling of swollen organelles.With higher concentrations and longer time of exposure,the damage got worse. After incubation with nanobacteria for 48 hours,MTT viability-measurement test results showed that difference was significant between control and treatment group except OD=0.001 group,as well as between different concentration groups(P＜0.05).After 72 hours,MTT results showed no difference between OD=0.137 and OD=0.246,while difference reached significance between other treatment groups(P＜0.05). LDH values increased gradually with longer exposure time(P＜0.05).NO values rose up with exposure time prolongation until reached peak value followed by obvious decrease,while difference arrived at statistical significance when compared each treatment concentration with the control group(P＜0.05).Conclusions:Nanobacteria demonstrated concentration-and-time dependently cytotoxic effects on cultured human normal hepatocytes. During exposure to nanobacteria,peak values of NO production went up with increased concentrations of nanobacteria and longer exposure time, while followed by obvious decrease. Partâ…¢Mechanism of Cytotoxicity of Nanobacteria on Cultured Human Normal HepatocytesObjective:To explore mechanism responsible for nanobacteria -induced cytotoxicity in cultured human normal hepatocytes.Methods:Status of mitochondrial permeability transition pore at different time points was observed under microscopy using mitochondrial probe when cultured hepatocytes incubated with different concentrations of nanobacteria.Mitochondrial ultra-structural changes were examined under electron microscopy.Cellular adenosine trisphosphate concentration was measured and apoptosis was monitored by flow cytometer.Results:After exposure to nanobacteria at concentration of OD=0.001 for 6 hours,PTP in cultured hepatocytes was observed opening to some extent,as well as mitochondrial potential slight decrease. Exposure for 36 hours caused a large decrease in calcein fluorescent intensity with many fewer mitochondria being detected,also Mitotracker Red dye diffusely distributed in the cells as a result of mitochondrial rupture.Transmission electron microscopy at the same time point revealed obviously mitochondrial dilation,cristae disappearance and vacuolization.At exposure time point of 72 hours,little calcein fluorescence was observed,mitochondrial potential completely lost as large decrease of Mitotracker Red dye ring fluorescence,although cellular nucleate appearance remained morphologically intact during the whole exposure period.Nanobacteria,at concentrations of OD=0.246,rapidly induced opening of permeability transition pores after exposure for 3 hours.At exposure time point of 6 hour,calcein fluorescence intensity greatly diminished,also mitochondrial potential lost apparently.After exposure for 14 hours,we observed MitoTracker CMXRos distributed in the plasma resulted from rupture of most mitochondria.Transmission electron microscopy at the same time point revealed obviously mitochondrial dilation,chaotic cristae,and decreased matrix density.At 16 hour point,little calcein fluorescence disappeared and transmission electron microscopy at the same time point revealed obviously mitochondrial dilation,cristae disappearance and vacuolization,although nuclear remains morphologically intact.22 hour exposure time point saw vestige of calcein and Mitotracker CMXRos dye,at the same time cellular nucleus ruptured and morphological appearance disappeared.Cell apoptosis was induced by nanobacteria at concentrations of OD=0.001 when compared with a little apoptosis rate by nanobacteria at concentrations of OD=0.018 after exposure for 24 hours.While cell apoptosis was induced by nanobacteria at concentrations of OD=0.246 after exposure for 6 hours.ATP measurement reached statistic significance when compared different concentrations of nanobacterial intervention(P=0.013),while differences between OD=0.001 or OD=0.018 and control group were not statistically significant(P=0.614, 0.213),also existed between OD=0.137 and OD=0.246 group.When compared with different time point of exposure,apart form difference between 0.5 hour and 0 hour,the rest inner-time point was statistically significant(P＜0.05).Nanobacteria produced concentration-dependent decreases in cellular ATP levels.Conclusions:Cytotoxicity of nanobacteria exerted on cultured hepatocytes was a consequence of mitochondrial permeability transition pores opening followed by disruption of mitochondrial function and structure,which preceded cell death.Apoptosis could be induced by exposure to nanobacteria in cultured hepatocytes,while apoptotic cell death would shift to necrotic cell death under the circumstances of higher concentrations of nanobacteria and longer exposure time.First of all, opening of permeability transition pores and subsequent ATP depletion contributed to fate of cultured hepatocytes exposed to nanobacteria.