Dissertation
Dissertation > Medicine, health > Clinical > Therapy > Emergency, first aid

Experimental Research of Renal Dysfunction and Mitochondrial Biogenesis in Septic Rats

Author LiMuSheng
Tutor ZengQiYi
School Guangzhou Medical College
Course Pediatrics
Keywords Sepsis Mitochondria Mitochondrial bioremediation Mitochondrial respiratory chain complex Reactive oxygen species Reactive nitrogen family
CLC R459.7
Type Master's thesis
Year 2011
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Purpose Sepsis is a systemic inflammatory response caused by various microorganisms and immunogenic substances and host immunologic injury, by its very nature, a large number of inflammatory cytokines and inflammatory mediators uncontrolled release due to a host of autoimmune damage (host autoimmunity injury). Sepsis is one of the major challenges facing medicine today, one of the causes of death in the pediatric diseases, further development can cause severe sepsis (severe sepsis) and septic shock (septic shock) and multiple organ dysfunction consolidated levy (Multiple Organ Dysfunction Syndrome, MODS) or even death. Germany annually sepsis incidence and 79000 (11.6%), the incidence of severe sepsis / septic shock 75,000 (11%), 454 ICU ward infections 30.8% of patients with severe sepsis / septic shock. There are 751,000 cases of severe sepsis / septic shock patients in the United States each year, the case fatality rate was 28.6%. Not very clear in the pathogenesis of sepsis pathogenesis may include: inflammatory cytokines storm, coagulation system disorders and microcirculation, mitochondrial dysfunction, etc.. Mitochondrial dysfunction in sepsis, in particular, played an important role in the progression of the disease in patients with septic shock. Sepsis recovery process, the mitochondrial mechanism is not clear. The mitochondrial biogenesis repair (Mitochondrial biogenesis) is to maintain and restore mitochondrial structure and function of a programmed cell plays an important role in sepsis recovery. The start of some of the factors (not yet clear), sepsis, organ mitochondrial regeneration and repair of related gene expression and increase mitochondrial DNA replication and transcription, mitochondria-related functions and structural protein expression increased to restore the function of mitochondria. The kidneys are vital organs of the body, is vulnerable organ when sepsis. Sepsis rat kidney mitochondria whether bioremediation occurred mitochondrial, and mitochondrial bioremediation of sepsis impact of this study. The subject of the functional status of the kidneys of rats with sepsis, kidney mitochondrial function status, ultrastructure and mitochondrial biogenesis repair gene expression studies to explore the relationship between sepsis kidney function damage and mitochondrial damage and repair, and learn more about pus the pathophysiology of thyrotoxicosis. . Establish animal models and packet specific pathogen free (SPF) 50-class male SD rats were randomly divided into: control group, endotoxin 4 hours group (LPS 4h group), 24 hours endotoxin group (LPS 24h group) 48 hours of endotoxin (LPS 48h group) and endotoxin 72 hours group (LPS 72h group), 10 in each group. The endotoxin model group were treated with intraperitoneal injection of endotoxin (10 mg / kg) to establish the volume of normal saline sepsis rat model, intraperitoneal injection of the control group. After testing, the model rats with varying degrees of heart rate, respiration, body temperature, increased white blood cells, the performance of the systemic inflammatory response, and the emergence of endotoxemia. Method and significance of the major indexes 2.1 uses automatic biochemical analyzer serum Cr, BUN levels reflect the functional state of the rat kidney. 2.2 by the observation of mitochondrial morphology and function testing of mitochondrial damage state. Electron microscopy mitochondrial morphological changes Flameng score of mitochondrial ultrastructure semiquantitative score; use flow cytometry mitochondrial membrane potential and the analysis of the degree of swelling, to reflect mitochondrial function and structure of the state. 2.3 by mitochondrial oxidative stress factor changes of mitochondrial damage mechanisms. Oxidative stress factors including manganese superoxide dismutase (Mn-Superoxidedismutasc, Mn-SOD) activity, malondialdehyde (Malondialdehyde, MDA) concentration, reduced glutathione glycoside peptide (Reduced glutathione hormone GSH) activity, nitric oxide (Nitric oxide, NO) concentration and nitric oxide synthase (Nitric oxide synthase, NOS) activity. 2.4 by real-time quantitative PCR detection of kidney tissue mitochondrial DNA copy number level. Mitochondrial DNA controls the basic nature of the mitochondria, mitochondrial DNA copy number and mitochondrial oxidative phosphorylation function is closely related to, by detecting the level of the functional status of the reflection of the mitochondria, is also one of the reflect the indicators repair of mitochondrial biogenesis. 2.5 by real-time quantitative PCR detection of kidney tissue mitochondrial biogenesis repair regulatory factor mRNA levels. The detection factors include peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 2 (NRF2), mitochondrial transcription factor A (Tfam). Tfam can be controlled by regulating the transcription of the the mtDNA light chain and the heavy chain of mtDNA transcription and replication, Tfam may be both mtDNA transcription regulation, the mtDNA stability requires a simple protein, Tfam content associated with the mtDNA content; NRF2 either directly regulating nuclear genome encoded respiratory chain subunit of expression and regulation Tfam and regulating mitochondrial genome transcription, translation, etc., which directly or indirectly from the regulating mitochondrial genome encoding the respiratory chain subunit expression role also may maintain mtDNA of the stability; PGC-1α is a major regulator of the matter (masterregulator) of the mitochondrial populations. Its expression can enhance mitochondrial biogenesis and increase in the number of mitochondria. PGC-1α can also be assisted activation of nuclear respiratory factor, PGC-1α activation through the auxiliary control cell metabolism in a group of specific nuclear receptors and non-nuclear receptor transcription factor to improve mitochondrial generation cellular respiration rate and energy substrate uptake and utilization, changes in order to meet the energy demand of the cells in the environment changes. 3. Statistical methods of experimental data using the statistical software SPSS 13.0 for analysis, using Excel 2003 mapping; your data first test of normality (Kolmogorov-Smirnov One-Sample Test) indicators meet the normality test, measurement data description using (x | -) ± s said, were analyzed using one-way ANOVA, between groups using Tukey's method, correlation analysis using Pearson correlation is 0.05, α defined. Sepsis, renal injury experimental results of this study: sepsis model group, serum Cr, BUN levels in the early (LPS 4h group) had significantly higher recovery trend, but the serum Cr level in LPS 72h group to normal. Compared with the control group, LPS 24h group, LPS 48h group, LPS 72h BUN levels was significantly higher (P lt; 0.01); compared with LPS 4h group, LPS 48h group, LPS 72h group was significantly decreased (P lt; 0.05 , P lt; 0.01). Serum Cr levels compared with the control group, LPS 4h, 24h group of LPS, LPS 48h group Cr levels were significantly higher (P lt; 0 01, P lt; 0.05, P lt; 0.05), LPS the 72h group of Cr levels not with a statistically significant difference (P gt; 0.05). Sepsis, kidney tissue mitochondria injury 2.1 sepsis kidney tissue mitochondrial morphological changes in the electron microscopy the mitochondrial ultrastructure results show: sepsis each group have different levels of damage, mainly to mitochondrial swelling and transparent matrix, Mitochondrial cristae, matrix solidification, the complete disappearance of inner and outer membranes, vacuolization, but LPS 72h basically normal. Flameng scoring showed the group LPS 24h injury is the most obvious, but a reversal of trend. Compared with the control group, LPS 4h group, LPS 24h group, LPS the 48h group of mitochondrial electron microscopy semiquantitative score was significantly higher (P lt; 0.01), LPS 72h group the mitochondrial electron microscopy semi-quantitative score showed no statistical significance (P gt; 0.05 ). At the same time, the flow cytometric analysis results show that the degree of swelling of mitochondria: sepsis mitochondrial swelling degree of change and ultrastructure the Flameng rated basic. Compared with the control group, LPS 4h group, LPS 24 h group kidney mitochondrial swelling degree was significantly higher (P lt; 0.05, P lt; 0.05), 48h group of LPS, LPS 72h group kidney mitochondrial swelling degree of the difference was not statistically difference (P gt; 0.05, P gt; 0.05). 2.2 sepsis kidney tissue altered levels of mitochondrial membrane potential by flow cytometry analysis of mitochondrial membrane potential results: sepsis, renal tissue mitochondrial membrane potential at an early decline to LPS 24h group was significantly, but LPS 48h group returned to normal . Compared with the control group, LPS 4h group, LPS 24h group kidney mitochondrial membrane potential was significantly lower (P lt; 0.05, P lt; 0.05) 48h group, LPS, LPS 72h group kidney mitochondrial membrane potential does not have a statistically significant difference (P gt ; 0.05, P gt; 0.05). . The mitochondrial endogenous oxidative stress related factor 3.1. Kidney mitochondrial total SOD of MnSOD activity compared with the control group, LPS 72h group kidney mitochondrial SOD activity was significantly lower (P lt; 0.01) 4h group, LPS, LPS 24h LPS 48h group kidney mitochondrial SOD activity does not have a significant difference (P gt; 0.05, P gt; 0.05, P gt; 0.05). 48h group of LPS and LPS 72h group kidney mitochondrial MnSOD activity was significantly lower (P lt; 0.01, P lt; 0.01) compared with the control group, LPS 4h group and LPS 24h group kidney mitochondrial MnSOD activity does not have a statistically significant difference (P gt ; 0.05, P gt; 0.05). Compared to 3.2 kidney mitochondrial GSH activity was detected with the control group, LPS 4h group, LPS 48h group of kidney mitochondrial GSH activity was no statistical difference (P gt; 0.05). LPS 24h-kidney mitochondrial GSH activity increased significantly (P lt; 0.01), the kidney mitochondrial GSH LPS 72h group activity was significantly decreased (P lt; 0.01). The 3.3 kidneys mitochondrial levels of TNOS, of iNOS activity was detected with the control group compared to LPS 4h group, LPS 24h group, LPS 48h group, LPS 72h group the kidney mitochondria TNOS activity was significantly higher (P lt; 0.01, P lt; 0.01, P lt; 0.01, P lt; 0.01). Compared with the control group, LPS 4h group, LPS 24h group, LPS 48h group, LPS 72h group myocardial mitochondrial iNOS activity was significantly higher (P lt; 0.01, P lt; 0.01, P lt; 0.01, P lt; 0.05). Of 3.4 kidney tissue the mitochondrial MDA in LPS group rat kidney mitochondrial malondialdehyde (MDA), the difference between the groups was not statistically significant (P gt; 0.05). 3.5 kidney tissue mitochondrial NO levels detected with the control group compared to, LPS 4h group, LPS 48h group, LPS 72h group kidney mitochondrial NO levels decreased significantly (P lt; 0.05, P lt; 0.01, P lt; 0.05), LPS 24h group kidney the mitochondrial NO level change is not obvious (P gt; 0.05). Compared with LPS 24h group, LPS 48h group kidney mitochondrial NO activity showed no statistical significance (P gt; 0.05), LPS 72h group kidney mitochondrial NO activity was significantly decreased (P lt; 0.01). Kidney tissue mitochondrial DNA relative copy number of real-time fluorescence quantitative PCR results show the relative copy number analysis of mitochondrial DNA has occurred: sepsis (LPS 4h group) in the early mitochondrial DNA relative copy number decreased, but significantly decreased LPS 24h group . Compared with the control group, LPS 24h renal mitochondria copy number decreased significantly (P lt; 0.05) 4h group, LPS, LPS 48h group, 48h group of LPS relative copy number of the difference was not statistically significant (P gt; 0.05, P gt; 0.05, P gt; 0.05). 5. Kidney mitochondria the bioremediation PGC-1α Ffam, NRF-2 regulatory factor mRNA levels detect real-time fluorescence quantitative PCR analysis of mRNA levels of each factor results show: each cytokine mRNA expression levels in LPS 4h significantly increased. Reached a peak the Tfam level in the LPS 24h group, after back to normal levels; NRF2 PGC-1α peak in LPS 4h group began to decrease, LPS 48h and 72h group back to restore to normal levels. Compared with the control group, LPS 4h group, LPS 24h group kidney Tfam expression was significantly higher (P lt; 0.01, P lt; 0.01), 48h group of LPS, LPS 72h group renal Tfam mRNA expression was no significant difference (P gt ; 0.05). Compared with the control group, LPS 4h group, LPS 24h group kidney NRF2 expression was significantly higher (P lt; 0.05, P lt; 0.05), 48h group of LPS, LPS 72h group renal NRF2 mRNA expression was no significant difference (P gt ; 0.05, P gt; 0.05). Compared with the control group, LPS 4h group, LPS the 24h group kidney PGC-1αmRNA expression was significantly higher (P lt; 0.05, P lt; 0.05), LPS 48h group,, LPS 72h group kidney express PGC-1αmRNA, was not statistically significant ( P gt; 0.05). Correlation analysis of of 6.1 rat renal mitochondrial membrane potential correlation analysis correlation analysis results: changes in septic rats BUN levels and kidney mitochondrial membrane potential level change has a significant negative correlation, the correlation coefficient was r = -0.124 (P lt; 0.05). The analysis showed correlation analysis of 6.2 rats changes in the level of mitochondrial membrane potential and mitochondrial DNA relative copy number changes: changes in the level of mitochondrial membrane potential of rat kidney mitochondrial DNA level changes in the relative copy number of a statistically significant positive correlation, the correlation coefficient r = 2.483 (P lt; 0.01) 6.3 rat kidney mitochondrial DNA copy number and mitochondrial biogenesis repair gene expression levels of the correlation analysis of the correlation analysis results show: the rat kidney mitochondrial DNA copy number and Tfam, NRF2, PGC-1α expression levels statistically significant negative correlation, the correlation coefficient (r = -0.913 P lt; 0.05; r = -0.919 P lt; 0.05; r = -0.894 P lt; 0.05), with the decline in mitochondrial DNA copy number, gene expression increased . Conclusion 1. Early stage of sepsis that exist kidney mitochondrial damage associated with renal dysfunction induced renal dysfunction induced by one of the reasons, the kidney mitochondrial damage was reversible. Sepsis kidney mitochondrial DNA copy number dropped one of the reasons for the decline of mitochondrial membrane potential, mitochondrial function recovery and the recovery of DNA copy number. Sepsis mitochondrial biogenesis repair gene expression began to increase in the early stage of sepsis associated with mitochondrial DNA copy number and mitochondrial membrane potential. 4. Sepsis mitochondrial oxidative stress-induced mitochondrial damage, oxidative stress may be the initiating factor of mitochondria bioremediation.

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