| Backgrounds and aimsWith the improvement of economic conditions in our country, diabetes mellitus presents the trend of the outbreak. According to the epidemiological survey counducted by professor NingGuang, Shanghai Ruijin Hospital, the Chinese adult over the age of 18 diabetes mellitus morbidity reached to 11.6%. Data released by the International Diabetes Federation, China currently has the world's maximal diabetes population.Increased incidence of diabetes and poor control levels cause high incidence of the chronic complications of diabetes and bring us a serious social and economic problems.The international diabetes federation and the Chinese medical association of common complications study also pointed out that as high as 173.4 billion yuan each year is directly related to diabetes in China, accounting for almost 13% of China's medical expenditure. Chronic complications of diabetes are mainly neuropathy, retinopathy,kidney disease, vascular lesions. Those complcations bring economic burden to patients and at the same time cause a drop in the quality of life, also may cause disability, as a result of a series of social problems. Vascular complications in diabetes complications affect the most importantly. The Chinese Center for Disease Control and Prevention diabetic chronic complications investigation group report that in the type 2 diabetes inpatients of the tertiary referral center complications prevalence is respectively:hypertension 34.2%, cerebrovascular disease 12.6%, lower extremity vascular disease17.1%, cardio-vascular disease 5.2%.Now we think that the vascular endothelial dysfunction is the initiating factor in and major pathophysiological basis of diabetes vascular lesions, even in patients without chronic vascular complications of diabetes the endothelial function was obviously dmaged. Hyperglycemia can damage vascular endothelial cell function through various mechanisms, inflammation, oxidative stress, protein non enzymatic glycosylation,secondary lipid metabolic disorder and so on. The above ways can partly explain the mechanism of diabetes vascular lesions and present the important prevention and control targets. It is very important for the prevention of vascular complications of diabetes.H2S is considered as the third gas signal molecule. The first and second gas signal molecules are the nitric oxide (NO) and carbon monoxide (CO). H2S plays an important role in human body. Its molecular weight is 34.076. It is a flammable acidic gas under the standard condition, colorless, smells like the rotten egg in low concentration,relative density 1.19 (air = 1). Exogenous H2S with relatively high concentration is a kind of poisonous gas to the human body. So in the past the researches of H2S are all about the mechanism of toxicology. Since the 1990's, it is found that the human body can produce H2S gas itself and is effected in a lot of tissues.The metabolism of sulfur amino acids is involved in the generation of H2S. Three enzymes, cystathionine- y -lyase, cystathionine- ? -synthase, 3-mercaptopyruvate sulfur transferase, are the main producer. In the body H2S mainly exist in two ways: physical dissolved H2S gas accounted for one third of the total; about two-thirds in the form of NaHS.Under the physiological condition, H2S has many roles. It facilitates the induction of hippocampal long-termpotentiation by enhancing the activity of NMDA receptors. The astrocytes are the type of glial cells, which surround and regulate synapses. H2S can induce Ca2+ influx in them. H2S can modify synaptic activity by regulating the activity of both neurons and astro cytes.In unnormal cases, such as hyperglycemia, the changed role of H2S in cardiovascular tissue has become one of the mechanisms of vascular lesions. Our previous study showed that hyperglycemia increased the levels of ROS while reducing superoxide dismutase activity and upregulated the ratio of Bax/Bcl-2 in HUVECs. It also activated caspase-3. Pretreatment of sodium hydrosulfide could prevent all the above responses.But our previous studies also found that the synthesis of H2S is reduced when treated with high concentration of glucose in the endothelial cells. So the vascular protective effect of H2S will be weakened. Then why in hyperglycemia environment the synthesis of H2S has been reduced, in other words, what is the mechanism of reduction? I will try to answer the question by my research.Materials and methods1.cell culture: I collected the umbilical cords from healthy full-term pregnancy mothers during delivery in the fifth people's hospital of Jinan or Shandong provincial hospital affiliated to Shandong university. I separate its vein endothelial cells as the experimental subject. Our experiment plan was approved by Shandong University Research Ethics Committee and all donors gave the informed consents. We lavaged the umbilical vein with 0.125% trypsin+0.1%EDTA solution. And the endothelial cells drifted down about ten minutes later. The medium which was used for culturing human umbilical vein endothelial cells was M199 medium supplemented with fetal bovine serum, penicillin-streptomycin and recombinant bovine basic fibroblast growth factor.2. cell treatment: We use the 50% D-glucose solution as the hyperglycemia provider and the proper amount was calculated by the formula designed by me. The inhibitor of phorsphated p38MAPK we choosed was SB203580.3. Real-time fluorescence quantitative PCR?Take the cells used for the test out of the incubator,and wash the dish 2-3 times with cold PBS. Add 1ml of Trizol, disintegrate the cells, and put lysate into the EP tube.?Add 200?l of chloroform to each EP tube, shake for 30 seconds, and keep it at room temperature for 15 minutes; shake again for 30 seconds, let it stand at room temperature for 15 minutes.?Pre-cool the low-temperature centrifuge, so that the centrifugal temperature can be maintained at 4?. After centrifugation, the liquid in the EP tube is divided into three layers. The upper aqueous phase contains RNA; the middle layer contains a white precipitate containing protein and DNA; in addition to the protein and RNA, the lower layer also contains red phenol and chloroform.?Carefully absorb the upper aqueous phase to another new EP tube, and add the equal volume of isopropyl alcohol and mix upside down, letting it stand at room temperature for 5-10 minutes.?After standing, put it in 4? centrifuge to centrifuge for 10 minutes, with white precipitate seen at the bottom, which is RNA precipitate.?Discard the supernatant. Wash the remaining RNA precipitate with 75% ethanol formulated prepared with DEPC water, and while washing, pay special attention to avoiding the removal of RNA from the tube.?Centrifuge and wash 2 times again, suck most of the supernatant, and at room temperature, place it in the fume hood for 5-10 minutes. Complete drying is not allowed until the precipitate becomes moist, translucent, white, and excessive drying will reduce the solubility, and decrease the OD260 / 280 ratio.?Gradually dilute with DEPC water until the precipitate is completely dissolved.Take 2 ?ll of it for the measurement of RNA concentration, and at OD260/280 ratio,determine the purity of RNA, with the ratio of the samples required being 1.8-2.2,otherwise they are abandoned.?Add the extracted mRNA to the gDNA Eraser for the genomic removing reaction,and then prepare a reverse transcription system to for reverse transcription of mRNA into cDNA.?Prepare cDNA and SybrGreen in each group into mixture, prepare the designed primer and deionized water into mixture, and then carry out the fluorescence real-time quantitative PCR reaction. After the end of the reaction, record the cp values of the samples, and compare the differences of 2-??cp in the groups.4.Detection on Protein Expression by Western Blot?Prepare an ice box, and the treated cells are placed and operated on the ice. Absorb the medium, and add cold PBS, washing 3 times. 80-100 ?l of protein extract is added to the culture dish in 60 mm diameter.?With a cell scratcher, scrape the cells into the protein extract, and then transfer them into the EP tube ice for lysis for 30 minutes.?After lysis on ice, ultrasound is used for lysis of cells. The ultrasonic probe is not allowed to touch the wall and the bottom of the EP tube, and the probe, before entering into the EP tube, shall be washed with alcohol and pure water, to avoid other protein contamination.?After ultrasonic lysis, place them into the low-temperature centrifuge at 4?, and take the supernatant after centrifugation.?Dilute the concentration of extracted protein,prepare AB liquid,and by BCA method, measure the protein concentration. Calculate the loading quantity of sample.Open the water bath, and perform albuminous degeneration of samples to be added.?Prepare the electrophoresis buffer, gel, and put gel band filled with gel into the electrophoresis tank for pre-electrophoresis. The buffer in the electrophoresis tank must submerge the thin plate, otherwise there will be poor conductivity. Then the comb is pulled up vertically to avoid damage to the sample adding holes. Sample adding: Inject protein marker2-5?l in a comb hole, and inject the protein sample into each comb hole according to the calculated adding quantity of sample.?Electrophoresis: The first stage is operated,with the conditions of 30 volts,for 60 minutes. In this way, the protein sample is pressed into a line in the upper layer of gel.At the second stage, voltage is 100 volts, and a different time is selected depending on the different molecular weight.?Transfer to membrane: Pour appropriate amount of transferring buffer in the tray,so that the liquid can submerge the clip and fiber cloth. My reference is GAPDH(glyceroldehyde-3-phosphate dehydrogenase), and the gel is cut according to the molecular size of the target protein and reference protein. For internal reference GAPDH and CSE, constant voltage 100 volt is selected for 40 min. MAPK-14 and phosphorylated MAPK-14 have the same transferring conditions. The transferring conditions for Spl with a larger molecular weight and phosphorylated Spl are 100 volts,for 70 min.?Incubation of primary and secondary antibodies? Development5. Chromatin immunoprecipitation?Cross-linking and lysis of living cells: Take out the culture dish (the culture dish is a large dish with the diameter of 15cm, and 20 ml of culture solution is added in it) and add 550 ?l of formaldehyde (37%) solution, shake and mix for 10 minutes at room temperature. After washing, add 2 ml of PBS containing Cocktail ?, scrape the cells into 1 Ep tube with a cell scraper, add the lysate, and then cut the DNA by ultrasound.?Protein co-immunoprecipitation: Prepare three Ep tubes, add 100 ?l of the cut DNA-protein suspension to each tube, add 900 ?l of dilution buffer, add the antibody of Spl, and after centrifuging and washing several times, reserve the precipitate, which is protein-DNA coprecipitate.?Elution of protein / DNA complex: 200?l of prepared elution buffer to the input tube, and stand at room temperature for use in subsequent steps. Add 100 ?l to the remaining 3 tubes respectively, incubate for 15 minutes at room temperature, and centrifuge it again to remove the centrifugal precipitate; then, take the supernatant, add 100 ?l of buffer to each tube again, repeat the previous step for washing,and finally, get 200 ?l of liquid.?Free DNA: For all samples,add 5mol / L NaCl 8?l,1?lRNase,and 4?l 0.5mol / L EDTA solution, and 8?l lmol/L Tris-HCl and 1?l proteinase K, and incubate appropriately.?Purification of DNA with an adsorption column: Take out 4 adsorption columns and 4 collection tubes. Binding agent is added to 4 Ep tubes incubated in the previous step, and the filtrate obtained by multiple centrifugations is the required DNA.?PCR: Obtained DNA is subjected to PCR amplification. The amplified bands are analyzed.Results1. For CSE protein expression of endothelial cells in hyperglycemic environment,regardless of 11.1, 16.7 or 25 mmol / L glucose concentration, after 48 hours, the protein synthesis of CSE decreases, and as compared with the normal glucose group, the expression of CSE in each high glucose group has decreased (P <0.05).2. The results of RT-PCR show that the mRNA levels of CSE are different at different glucose concentrations. 48 hours after treatment in high glucose concentration,the mRNA mRNArna level of CSE in 11.1, 16.7 or 25 mmol / L glucose concentration groups decreases significantly, and the difference is significant (P <0.05) compared with normal glucose concentration group. This suggests that the decrease in CSE synthesis begins at the transcriptional level.3.The results of CHIP test show that the input group is positive,the experimental group, to which Spl antibody is added, is also positive, and the results of the AC group and NC group are positive and negative, respectively. These results show that Spl is indeed bound near starting transcription point of the CSE gene.4. After blot analysis of human umbilical vein endothelial cells in normal blood glucose and hyperglycemia, it is found that the total amount of Spl does not change but the phosphorylation level increases and the phosphorylation level of p38MAPK also increases in the condition of hyperglycemia (P <0.05). After SB203580 is given to block the function of phosphorylated p38MAPK, changes to phosphorylation of Spl and CSE disappear.Conclusions1. Under the environment of elevated glucose concentration, the expression of CSE is declining, and the decline occurred at the transcriptional level.2.In human umbilical vein endothelial cells, the Spl is the transcripition factor binding to CSE genes upstream.3. It is increased phosphorylation of Spl itself rather than quantity of it regulating CSE transcription. And p38 MAPK activity is involved in the process of regulation. |
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