Staphylococcus aureus, streptococcus hemolyticus, escherichia coli, pseudomonas aeruginosa, shigella flexneri, S. paratyphi B salmonella and bacterial culture medium were provided by Center for Disease Control and Prevention of Jiangsu Province and Microbiology Department of Southeast University. Methyl thiazolyl tetrazolium, dimethyl sulfoxide, and sodium dithionite were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Superior grade fetal bovine serum, endothelial cell growth additive, and heparin sodium were from Changzhou Xinhua Active Material Institute. Green streptomycin, penicillin and glutamine were received by Lianke Biotechnology Co., Ltd (Hangzhou, China). DMEM ordinary medium: 20% fetal bovine serum, 105 U/L penicillin, 100 mg/L streptomycin, 2 mmol/L L-glutamine, DMEM medium; DMEM complete medium: 20% fetal bovine serum, 105 U/L penicillin, 100 mg/L streptomycin, 2 mmol/L L-glutamine, 50 mg/L heparin, 7.5 µg/L endothelial cell growth additive, DMEM medium. Onion oil and garlic oil were prepared according to the method.
MCO-15AC CO2 incubator was purchased from Sanyo Trading Co., Ltd (Japan), H2S-H thermostatic water bath oscillator was from Harbin Donglian Electronic Technology Development Co., Ltd (Harbin, China), high-pressure steam sterilization pot and infrared sterilizer were purchased from Beijing Qincheng Scientific Instrument Co., Ltd, vortex mixer was from Gengchen Scientific Instrument Co., Ltd (Nanjing, China) and temperature and humidity regulating incubator was from Jinghong Experimental Equipment Co., Ltd (Shanghai, China).
Preparation of bacterial suspension
The freeze-dried bacteria tube was taken and open it under sterile conditions, appropriate amount of nutrient broth with capillary pipette was added, gently blew and sucked for several times to melt and dispersed the bacteria. The test tube containing 5.0~10.0 ml nutrient broth medium was taken, a little bacterial suspension was dropped and incubated at 37℃ for 18-24h.
The first generation of bacteria suspension was taken from the inoculation ring and inoculated on the nutrient agar medium plate, and cultured at 37℃ for 18-24h. The typical colonies of the second generation culture were selected and inoculated on the slant of nutrient agar and cultured at 37℃ for 18-24h, which was the third generation culture. The fresh slant culture (18-24h) of nutrient agar culture medium of the third to fourteenth generations of the strain and 3.0-5.0ml diluent with a 5.0ml pipette were taken, added it into the inclined tube, repeatedly blew and sucked, and the fungus coating was washed off. Then, the lotion was transferred to another sterile test tube with a 5.0ml pipette, mixed (oscillate) with an electric mixer for 20s, or vibrated 80 times on the palm of the hand to make the bacteria suspension evenly. The initial bacterial suspension was determined by turbidimetric method, and then diluted to 5×105 -5×106 cfu /ml.
Inhibition zone test
The qualitative filter paper was made into a round piece with a diameter of 6 mm by a punch. After high pressure sterilization, the qualitative filter paper was dried at 120℃ for standby. The treated filter paper was put into a clean plate, added onion/garlic oil diluent I 5μL, and should be used after natural drying at room temperature. At the same time, round filter paper containing n-hexane was made as control. The sterile cotton swab was dipped into the test bacterial suspension with the concentration of 5×105 -5×106 cfu /ml, and evenly applied on the surface of the culture medium plate for 3 times. The plate was rotated 60° for each application. Finally, the cotton swab was applied around the edge of the plate. The plate was covered and dried at room temperature for 5 min.
For each test, one bacterial plate was pasted, one test sample was pasted on each plate, and another negative control sample was made; or five test samples and one negative control sample were pasted on each plate, totally 6 pieces. The sample piece was pasted on the surface of the plate with sterile tweezers. The distance between the centre of each piece was more than 25mm, while the distance from the periphery of the plate was more than 15mm. After sticking, sterile tweezers were used to gently press the sample piece to make it close to the surface of the plate, then the plate was covered, and sample was incubated in 37℃ incubator for 24h to observe the bacteriostasis. When measuring the inhibition zone, the uniform and completely aseptic growth inhibition zone were chosen for measurement, calculated the diameter of the inhibition zone (including the patch) with digital vernier caliper, and recorded the measurement data. If the diameter of inhibition zone is more than 6 mm, it is judged that it has bacteriostatic effect. Otherwise it has no bacteriostatic effect.
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
One ml of onion/garlic oil diluent II was aspirated with sterile pipette gun and injected into a 90mm diameter aseptic culture dish, and then 15ml of dissolved medium (< 45℃) was added. After full mixing, the plate containing sample solution was prepared and numbered. Sterile water and 50% ethanol were used as control. 0.1ml of bacterial suspension or spore suspension on the surface of the plate was taken, evenly coated, incubated at 37℃ for 24h, and growth of the test bacteria was observed. The minimum inhibitory concentration of the tested sample was MIC. The plates of the above-mentioned non-growing bacteria were cultured for 24h. The minimum concentration of completely aseptic growth of onion/garlic oil was taken as MBC.
Culture of primary myocardial microvascular endothelial cells of rats
According to the method of Nishida, the method of in-vitro culture of intravascular endothelial cells in myocardial microvessels of neonatal rats was established. Four unborn 7~10d Wistar rats were killed by cervical dislocation, soaked in 75% alcohol for 2 minutes, and fixed with a pin. The chest was open, the heart was exposed, cut off and put into PBS solution, washed 3 times, the blood stains were washed, valves, large blood vessels and connective tissue with ophthalmology were cut off. The left ventricle was open and the heart was soaked in 75% ethanol for 30 seconds to inactivate the epicardial and endocardial endocardial epithelial cells. After rinsing with PBS solution, the myocardial tissue was cut up and then rinsed with PBS solution for 2 or 3 times. The washed tissue was precipitated with 2ml 0.2% type Ⅱ collagenase, blown, and incubated and digested 30min at 37℃. The same volume of pancreatic egg albumin enzyme with a final concentration of 0.02% was added, the tissue was gently blew for 10 times and digested 10min in a hot water bath at 37℃. 4ml containing serum culture medium was added to stop digestion, the cells were separated through 200 mesh sieve, the precipitation without sieve was discarded, filtrate was taken, separated from the heart, repeated twice. The obtained cells were re-suspended in 8ml DMEM ordinary medium, blown evenly, inoculated in 1% gelatin-treated culture flask, cultured at 37℃ for 4 hours, to remove unadhered cells, and then replaced with DMEM complete medium containing 50mg/L heparin and 7.5μg/L endothelial cell growth additive. The liquid was changed once every 2 or 3 days, and the monolayer of cells was grown until the monolayer was grown. After being identified as CMEC, the cells were subcultured and the third generation cells were used in the experiment.
Establishment of hypoxia/ reoxygenation injury model
The original culture medium was absorbed and added into the non-serum DMEM culture medium containing 0.5mol/L and sodium disulfite, and incubated in a CO2 incubator with a volume fraction of 5% at 37℃ for 60min ( anoxic treatment). The culture plate was taken out, non-serum DMEM complete medium without sodium disulfite was added and continued to be incubated for 60min in a CO2 incubator with a volume fraction of 5% at 37℃( reoxygenation treatment). The third generation neonatal rat myocardial microvascular endothelial cells were collected respectively, and the H/R model of isolated cells was established by hypoxia 1h/ reoxygenation 1h according to the above method.
Myocardial microvascular endothelial cells were randomly divided into 11 groups with 5 holes in each group: (1) blank control group: DMEM complete medium cultured in saturated humidity carbon dioxide incubator for 2h; (2) solvent control group: DMEM complete medium containing 1‰ DMSO, cultured in saturated humidity carbon dioxide incubator for 2h; (3) model control group A: hypoxia for 1h and reoxygenation for 1h. (4) Model control group B: hypoxia for 1h and reoxygenation for 1h, then cultured in DMEM complete medium containing 1‰ DMSO. (5) garlic oil group (group 7): garlic/onion oil was incubated with 200, 100, 50, 25, 12.5, 6.3 and 3.1mg/L at the same concentration (DMEM complete medium) for 24h, then hypoxia for 1h and reoxygenated lh. They were also randomly divided into 11 groups with 5 holes in each group, and the garlic oil group was replaced by onion oil group, and the other groups were the same.
Determination of cell survival rate and apoptosis rate of cardiomyocytes
The absorbance (A value) measured by MTT method is positively proportional to the number of living cells, so the cell survival rate can be expressed as A value, and the inhibition rate = experimental hole A value/control hole A value × 100%. The detection of MTT is completed on the enzyme-linked immunosorbent assay (Elisa).
Garlic oil and onion oil of 12.5mg/L were selected to detect the apoptosis rate of cells. After 6h of cell culture, the cells with precooled PBS were washed and resuscitated with diluted binding buffer and the cell density was adjusted to 5×105~1×106 per milliliter. 100μL cell suspension was taken into 5mL flow tube, AnnexinV- FITC 5μL and propidium iodide (PI, 20μg/mL) 5mL were added to cell suspension, gently shaken and incubated at room temperature and darked for 15 minutes, diluted binding buffer 400L was added into sample, and detected it on computer within 1h.
Results were expressed in the form of mean ± SD, statistical analysis was conducted by SPSS 19.0 software. The comparison between the two groups was conducted by t-test, and the comparison between multiple groups was conducted by analysis of variance (ANOVA). The comparison of inter group rates was performed by X2 test, with statistical significance set P < 0.05.