Molecular Signal Mechanism For Respiratory Burst Dysfunction Induced By High Glucose In Phagocytic Cells

BACKGROUNDThe diabetes incidence rate roars up globally with improvement of people’s living standards, change in lifestyle and global aging. Diabetes has become another severe chronic non-communicable disease following the cardiovascular diseases and tumor which damage people’s health seriously. Nowadays there are more than one hundred and fifty million diabetics around the world and the number is supposed to hit three hundred million in2025.Diabetes are predisposed to infections because of various immune deficiencies. Infection is the principal causes of diabetic death before insulin treatment. The death rate of diabetics was still high in the1950"s. In the past few years, As a consequence, the death rate of diabetes complicated infection was decreased by using insulin and antibiotic drugs. However, infection remains is the second reason of diabetic death. An increase of36.8%in risk of infection following diabetes has reported. It is very important to enhance diabetics’ resistance of infection with the result that a decreasing death rate.Nowadays, people believe that various complicating infections of diabetics are primarily related to hyperglycemia, impairment of resistance capability, complications of diabetes, dystrophy and other factors in which impairment of resistance of organisms is even of more concern. Diabetics with poor control or ketoacidosis are extremely susceptible to infection to a great extent as they are often accompanied by different deficiency of resistance as well as functional inhabitation in all stages of response to invading microbes. Leukocytes play a critical role in anti-infection by involving in nonspecific and specific immunologic processes of engulfing and killing pathogens as the first anti-infection line of body defense. Many researchers have studied the bacteria engulfing and killing function of the leukocytes of diabetics and found the chemotaxis. adhesion and bacteria engulfing and killing functions of neutrophilic granulocytes of those with poor blood glucose control are lower than normal individuals and have some connection with the degree of disease control and metabolic disturbance. In addition, the leucocytic ultra-structures of most diabetics are disordered. In recent years, people attach increasing importance to the research of the mechanism impairing the bacteria engulfing and killing functions of leukocytes of diabetics and many domestic and foreign scholars have worked over the membrane fluidity, activity of alkaline phosphatase. lysosomal enzyme, opsonin, Ca2+concentration, metabolic sorbitol and inositol and other aspects of the leukocytes of diabetics. However, they are still open questions.The bactericidal functions of neutrophils were mediated by respiratory Burst. Following activation, leukocytes primarily perform the function of sterilization mainly by respiratory burst. After engulfing pathogens, leukocytes effectively kill them by the joint functioning of the large quantity of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) during the respiratory burst. NOX is the key enzyme for respiratory burst which is substantially the process of NOX activation.At present, it is thought that NOX is mainly composed of2membrane protein subunits(gp91phox and p22phox),3cytoplasmic subunits (p47phox, p67phox and p40phox), a small-molecular-mass protein Rac and newly-identified p29peroxiredoxin. P47phox is essential for the maintenance of in vivo neutrophilic granulocytes’normal bacteria-killing function[9]. which is the initial point of NOX activation. When neutrophilic granulocytes are activated through surrounding stimulation, firstly p47phox in cytoplasm is phosphorylated in large amount which changes its conformation, the compact combination between molecules being opened up. P67phox then combines SH3and PX structure domain exposed after phosphorylation of p47phox and SH3domain located in C terminal, and phosphorylated p47phox takes p67phox, p40phox to move to cell membrane, combines p22phox’s end part protruding from cytoplasm with SH3domain exposed in N-terminal, thus realizes the aggregation and assembling of subunits of cell membrane and subunits of cytoplasm in cell membrane, finally forms active NOX in cell membrane and makes its biological effect[10-12]. So the phosphorylation level of p47phox can directly reflect the activation level of NOX.Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme for the pentose phosphate pathway in cells. Its activity relates closely to the formation of NADPH which is the necessary substance of respiratory burst. Since the1980’s, the research of G6PD had been started in DNA level and focused on gene mutation mainly. In the past few years, Joslin Diabetes Center of Harvard medicine discovered the close relationship between G6PD and diabetic nephropathy and endothelial cell’s function. G6PD not only relating with neutrophil’s respiratory burst, but also relating with diabetes and its complications.The present study will make in vitro human monocyte strain THP-1cultured under different concentration of glucose and peripheral white blood cells(PWBC) of diabetic patients who meets the diagnostic standard of ADA2011as subjects, observing effect of glucose concentration in surrounding environment of cells on their function of respiratory burst, and explore the possible mechanism of signal transduction causing the functional change of respiratory burst mediated by high-level glucose.OBJECTIVEThrough detection of the effect of the change in G6PD activity, NOX activity and ROS level on respiratory burst of THP-1cells in different groups when they are activated with fMLP, we made an approach to the possible pathway of the suppression of respiratory burst in THP-1cells under high-glucose circumstance.METHOD1.Human monocyte THP-1cells were distributed into3groups:high-glucose group(33.3mmol/LD-GLU), control group(11.1mmol/L D-GLU), G6PD repressing group (33.3mmol/LD-GLU+100umol/L DHEA),6cases for each group.2. After culture for48h, the subjects were treated with10umol/L FMLP for30min. using spectrophotometric method to determine G6PD and NOX activity of THP-1cells; determining ROS content by fluorescent probe DCFH-DA.3. The experimental data and measurement data were indicated by means±standard deviation (x±s). Statistical analysis was taken using SPSS16.0software. The data comparison among several groups was made by means of one-way ANOVA. The test standard a was set to0.05.RESULT1.In comparison with control group, the G6PD activity, NOX activity and ROS level decreased in high glucose group and G6PD repressing group, and there were significant differences in both group (P<0.01)2. In contrast to G6PD repressing group, the NOX activity and ROS level descending amplitude being greater in high glucose group (P<0.01). However, the descending amplitude of G6PD activity were similar in high glucose group and G6PD repressing group (P>0.05)CONCLUSIONS1. There is a functional disorder of respiratory burst in THP-1cells by repressing G6PD activity only.2. High glucose circumstance influence on the respiratory burst of THP-1cells by repressed G6PD activity and NOX activity directly. Chapter2Effect of cAMP-PKA signal pathway on the dysfunction of respiratory burst in THP-1cells mediated by high glucoseOBJECTIVETo probe into the effect of cAMP-PKA pathway on the change in the G6PD activityand respiratory burst dysfunction of the THP-1cells mediated by high glucoseconcentration by pre-treating THP-1cells cultured with culture solution of differentglucose concentrations using PKA stimulator Forskokin and specific PKA inhibitorPKI.METHOD1.Subject: Human monocyte cell line THP-1，divided into4groups: high glucosegroup(33.3mmol/LD-GLU), control group(l1.lmmol/L D-GLU)，PKA inhibitinggroup (33.3mmol/LD-GLU+10umol/L PKI) and PKA stimulating group(11.1mmol/LD-GLU+20umol/L Forskolin)，6cases in each group.2.After culture for48h，the subjects were treated with lOumol/L FMLP for30min.Then the G6PD and NOX activity in THP-1cells were tested by spectrophotometry，and the ROS content in the cells and cAMP content determined by means offluorescence probe and ELISA respectively.3.The experimental data and measurement data were indicated by means土standarddeviation (x±s). Statistical analysis was taken using SPSS16.0software. The datacomparison among several groups was made by means of one-way ANOVA. The teststandard a was set to0.05.RESULTS1.Compared with control group, G6PD activity，NOX activiyt and ROS productionwere all apparently decreased in high glucose group and PKA stimulating group, andthere were significant differences in both groups (P<0.01). There were no obviouschange in NOX activity and ROS output (P>0.05). Compared with PKAstimulating group, there was no significant difference in G6PD activiyt, NOX activityand ROS production in high glucose group (P>0.05).2.Compared with control group，cAMP content in high glucose group and PKAstimulating group enhanced greatly and both showed significant difference (P<0.01). There was no significant difference in cAMP content in PKA inhibiting group compared with control group (P>0.05). And there is also no significant difference in cAMP content in high glucose group compared with PKA stimulating group (P>0.05)CONCLUSIONS1.With continuous high-concentration glucose intervention, the G6PD activity, NOX activity and ROS production in the activated THP-1cells are obviously decreased and the p47phox phosphorylation is inhibited, which causes its respiratory burst dysfunction.2.PKA-inhibitor may continuously enhance the G6PD activity, NOX activity and ROS production and increase the47phox phosphorylation level of the THP-1cells cultured in solution of the high glucose concentration, thereby partially recovering the respiration burst dysfunction of THP-1cells caused by high glucose concentration. This indicates that the respiration burst dysfunction of THP-1cells mediated by high glucose concentration had some connection with the activation of the PKA pathway.3.The NOX activity and ROS production can also be decreased to inhibit p47phox phosphorylation by suppressing the G6PD activity, thereby producing an effect on respiratory burst function of the THP-1cells. However, it has weaker inhibiting effect on respiration burst function than high glucose concentration, suggesting that high glucose inhibits the G6PD and NOX activity by activating PKA pathway and thus influences the respiratory burst function of the THP-1cells. OBJECTIVE Through detection of the phosphorylation level of G6PD,Raf-1,ERK and p47phox in different groups, we made an approach to the possible mechanism of the suppression of respiratory burst in THP-1cells under high-glucose circumstance by cAMP-PKA pathway.METHOD1.Human monocyte THP-1cells were distributed into4groups:high-glucose group(33.3mmol/LD-GLU), control group(11.1mmol/L D-GLU), PKA repressing group (33.3mmol/LD-GLU+10umol/L PKI) and PKA agitating group (11.1mmol/LD-GLU+20umol/L Forskolin),6cases for each group.2. After culture for48h, the subjects were treated with10umol/L FMLP for30min. The expression and phosphorylation level of G6PD, Raf-1, ERK, JNK, p38MAPKand p47phox in different treatment groups were determined by Western-blot method.3. The experimental data and measurement data were indicated by means±standard deviation (x±s). Statistical analysis was taken using SPSS16.0software. The data comparison among several groups was made by means of one-way ANOVA. The test standard a was set to0.05.RESULT1.In comparison with control group, the phosphorylated expression of G6PD raised in high glucose group and PKA agitating group, and there were significant differences in both group (P<0.01); Meanwhile, the phosphorylated expression of G6PD in PKA repressing group didn’t change significantly (P>0.05).2. In contrast to control group, the phosphorylated expression of Raf-1in high glucose group and PKA agitating group augmented, while that of ERK, JNK and p38MAPK reduced in both groups, all showing significant differences (P<0.01); the phosphorylated expression of Raf-1and ERK, JNK and p38MAPK did not change significantly in PKA repressing group (P>0.05)3. In contrast to control group, the phosphorylated expression of p47phox in high glucose group and PKA agitating group lessened, there being significant difference in both group (P<0.01); There was no significant change in p47phox phosphorylated expression in PKA repressing group (P>0.05)CONCLUSIONS1.Continuous high-density glucose intervention can apparently scale up phosphorylated expression of Raf-1in THP-1cells under the state of activation, at the same time step down the phosphorylated expression of ERK, JNK and p38MAPK, therefore p47phox phosphorylated expression was lessened and G6PD phosphorylation augmented, which can be supposed that cAMP-PKA pathway blocked ERK. JNK and p38MAPK shunt in MAPK pathway, further inhibiting the activation of p47phox and G6PD activity, which resulted in the respiratory burst dysfunction.2. PKA depressor can reduce Raf-1phosphorylation level and elevating ERK. JNK and p38MAPK phosphorylation level, therefore raising p47phox phosphorylation level, which illuminates that repression of cAMP-PKA pathway can reverse the inhibition of respiratory burst caused by high glucose, and this further proves that ERK, JNK and p38MAPK shunt participates in the activation of cAMP-PKA pathway mediated by high glucose which causes respiratory burst dysfunction.3.After treating with PKA depressor, phosphorylation level of p47phox was enhanced apparently but was still lower than that in normal glucose level, which suggests that high glucose may suppress NOX activity through other signal pathway and affects the normal function of respiratory burst.