The protocol in this study provides a combination strategy of two herbs for treating hypoxia-damaged PC12 cells, and a reference for optimizing the best combination mode of herbs. This technique can provide a simple, reliable, and efficient in vitro experimental method for screening the effective ingredient combination from herbs, against hypoxial cells. This strategy can also be applied to screen the component combinations against hypoxial cells from other herb preparation combinations, and illustrate their protective mechanism.
To begin, sub-culture the PC12 cells every two to three days at 37 degrees Celsius in an incubator supplemented with 5%carbon dioxide to get a passage of 4 to 8 for all subsequent experiments. Treat 70 to 80%confluent PC12 cells with one milliliter of 0.25%trypsin, and observe the cells under a microscope for cell detachment. Then stop the trypsin digestion by adding three milliliters of the complete medium.
Spin down the transferred cells in a 15-milliliter centrifuge tube at 840 x g for five minutes. After discarding the supernatant, resuspend the cell pellet with the complete medium and transfer the cell suspension to a 1.5-milliliter microcentrifuge tube. To count the cells, start the flow cytometry software and select the corresponding dilution multiple of the cell solution in the counting setting.
Click on Density Chart after loading the cell sample. Circle the cell population away from the x-axis and y-axis. Right-click the data table below the figure, and select X, Y, Count, and Abs.
Count to get the cell counting results under the data table of Abs.Count. After adjusting the cell concentration approximately to 1 times 10 to the 5th cells per milliliter with the complete medium, add 100 microliters of the cell suspension per well to a 96-well plate and incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, to the supernatant-discarded plate add 120 microliters of one milligram per milliliter MTT solution in each well, and incubate for four hours at 37 degrees Celsius.
After incubation, once the supernatant is discarded, add 150 microliters of DMSO to each well. Keep the plate under shaking in a vortex oscillator for 10 minutes at a speed of 240 times per minute. Then measure the absorbance at 490 nanometers in a microplate reader.
To screen the optimal combination of drugs by homogenous design method, culture the cells as demonstrated earlier and treat the injured PC12 cells with six different proportion concentrations of Astragalus injection and breviscapus capsule. To evaluate the protective effect of two optimal combinations, treat the injured PC12 cells with the two types of combination of drugs. Incubate the cells for 24 hours and calculate the cell viability as demonstrated earlier.
Seed the PC12 cells in a 12-well plate and incubate at 37 degree Celsius for 24 hours. Following drug treatment, treat the cells with 250 microliters of trypsin per well and centrifuge the cells at 840 x g for five minutes at room temperature. Then resuspend the cell pellet with 500 microliters of the binding buffer.
Add 5 microliters of Annexin V-FITC, 10 microliters of propidium iodide in triplicates for each group, and incubate the cells in the dark for 15 minutes at room temperature to check for apoptosis using flow cytometry. In the flow cytometer software, click Automatic Compensation in the Start menu, select FITC, PE, PerCP, and APC in the Select channel, and select Compensation at Height. Then click OK in the Statistical Item:Median.
Click Density Map and circle the effective cell population with the cell counting method demonstrated earlier. With FITC fluorescence as the x-axis parameter and other fluorescence as the y-axis parameter, create a density map. Click on Compensation Matrix in the Start menu and Coefficient in the overflow matrix.
Seed the PC12 cells to the coverslips placed on a 24-well plate well in triplicates, and incubate at 37 degrees Celsius for 24 hours. Following drug treatment, rinse the coverslips twice with PBS for five minutes each, fix them with 4%paraformaldehyde for 15 minutes, and permeabilize with 0.5%Triton X-100 for 20 minutes at room temperature. After rinsing the cells, block them with 10%goat serum for one hour at room temperature.
Add 200 microliters of diluted primary antibody caspase-3 to each coverslip and incubate overnight at 4 degrees Celsius. The following day, wash three times with TBST buffer for three minutes each, and incubate with 200 microliters of secondary antibody for one hour at room temperature in the dark. After incubation, incubate the coverslips with 300 microliters of 0.5 micrograms per milliliter of DAPI for 10 minutes, and wash the cells three times with TBST for five minutes each.
Capture the coverslip images under a fluorescence microscope and statistically analyze the fluorescence intensity with the imaging software. Wash the drug-treated PC12 cell coverslip three times with PBS for three minutes each and incubate with 400 microliters of 10 micromolar dichloro-dihydro-fluorescein diacetate at 37 degrees Celsius for 20 minutes. Wash the cells again with serum-free DMEM twice for three minutes each.
Add the fluorescence quenching agent and immediately capture the coverslip images under a fluorescence microscope to calculate the relative fluorescence intensity with the imaging software. Wash the drug-treated PC12 cells in a 6-well plate three times with PBS for five minutes each, and incubate on ice for 30 minutes in the prepared lysis buffer of 100 microliters per well. Following lysis, centrifuge the cells at 16, 000 x g for 20 minutes at 4 degrees Celsius to collect the supernatant.
After detecting the protein concentration by Bradford assay, run a 12%SDS-PAGE electrophoresis with 25 micrograms of protein in each lane and transfer the gel to the PVDF membrane. Block the membranes with 5%nonfat milk for 1.5 hours at room temperature, and incubate with five milliliters of the corresponding primary antibodies for 24 hours at 4 degrees Celsius. After incubation and washing the membrane with TBST three times for 10 minutes each, incubate with five milliliters of secondary HRP-conjugated antibodies at room temperature for two hours.
Finally, to the TBST-washed membrane, add chemiluminescent HRP substrate for protein band detection as per the manufacturer's instruction. Then capture the images using the chemiluminescence imaging system to quantify the gray values of the proteins, with beta-actin as an internal control. The cell viability on the normal PC12 cells was lower than 95%with Astragalus injection at concentrations greater than 12 micromolar, and greater than 5 micromolar for the breviscapus capsule.
Astragalus injection could improve the survival rate of the injured PC12 cells in the concentration range of 6 to 12 micromolar, and breviscapus capsule at 2 to 5 micromolar. Whereas the drug combination at a ratio of 6 to 1.8 micromolar exhibited the highest cell viability. The cell viability of the two combinations on the injured PC12 cells was significantly promoted, and the component combination was superior to the preparation combination.
The apoptosis rate showed that the percentages of early, late, and total apoptotic cells were significantly higher in the model group than in the normal group. Compared with the model group, the percentages of apoptotic cells at each stage were significantly lower in the treatment group. The fluorescence intensity of caspase-3 was significantly lower in each treatment group compared with the model group, and was relatively lower in the component combination group.
Relative expression of the genes Akt, Bcl-2, and Bax revealed that the component combination is superior to the preparation combination in promoting cell survival, which is related to the stronger anti-apoptosis effect. The component combination exhibited a better anti-oxidative damage effect than that of the preparation combination, and significantly higher expression of the Nrf2 protein. This method can be used to screen and evaluate the component combination with other potential pharmacological activities from the herb combination, such as anti-inflammatory, antibacterial, and more.
