Mechanism of Kemeng Fang’s Inhibition of Podocyte Apoptosis in Rats with Membranous Nephropathy through the PI3K/AKT Signaling Pathway

The scope of this study is the of the mechanism of traditional Chinese medicine in the treatment of membranous nephropathy. Experimental methods are used to prove that traditional Chinese medicine plays a role that cannot be ignored in the treatment of MN. In order to clarify the mechanism of trauma in the treatment of MN.

There are many steps to pay attention to in , such as the voltage and time during electrophoresis, and the transfer, the concentration, and the reaction time of primary and secondary antibodies and so on, which will affect your final result.

Through experimental research, it was confirmed that prescription can slow down podocyte apoptosis and improve MN by activating the PI3K AKT signaling pathway. Modern research methods were used to confirm the effectiveness of the traditional Chinese medicine compound, and the feasible direction for the treatment of MN.

[Narrator] To begin, obtain the cationized bovine serum albumin, or C-BSA. For pre-immunization of the rat, grasp its back skin with the left hand, and turn the abdomen upward, with the abdominal skin tightened. Using a 2.5-milliliter syringe, inject the C-BSA emulsifier subcutaneously into the axilla and groin of the rat. For formal immunization, remove the rats from their cages and place them on the wire bar lid, with their tail facing the experimenter. Wipe the rat's tail with an alcohol cotton ball and pinch both sides of the rat's tail with the thumb and forefinger of the left hand to fill the vein and keep it facing up. Hold a one-milliliter syringe in the right hand so that the needle is at 30 degrees to the tail vein. After gently pricking into the skin, keep the tip of the needle beveled upward and the needle parallel to the vessel. Then, use a dry cotton ball to press the injection point for about a minute to stop any bleeding. After the animal recovers, grasp the rat's skin on its back with the left hand. Turn its abdomen upward and tighten the skin. Holding the needle of a 10-milliliter syringe in the right hand, insert it into the rat's mouth from one side, sliding along the palate, the posterior pharynx wall, and further to the stomach. Using the index finger of the right hand, slowly administer the appropriate drugs to each animal group for a total duration of four weeks. Pull the gastric needle out after the administration is complete. To begin, obtain the renal tissue for biochemical assays from the membranous nephropathic rat treated with KMF. Add 500 microliters of the prepared lysis solution into a microcentrifuge tube. Add 100 milligrams of frozen kidney tissue to the tube with lysis solution, and grind thoroughly with a high-speed tissue grinder until there is no visible tissue mass. Place the microcentrifuge tube on ice for 30 minutes, and then centrifuge at 15,984 G for 10 minutes. Aspirate the supernatant and store it at minus 80 degrees Celsius. Detect the protein concentration according to the instructions of the bicinchoninic acid protein concentration assay kit and add PBS solution drop-wise to ensure consistent protein concentration in each group. Then, incubate the sample with 5x protein upsampling buffer at 100 degrees Celsius for 15 minutes to fully denature proteins. Separate the proteins by 12.5% SDS-PAGE gel electrophoresis, using 80 volts first, and then shifting to 130 volts, until the bromophenol blue runs out from the bottom of the gel plate. After removing the gel, wet the filter paper with membrane transfer buffer. Activate the polyvinylidene fluoride, or PVDF, membrane in methanol for 30 seconds. Then, arrange the sponge mesh, filter paper, gel, and the PVDF membrane in the clip for the transfer. Place the gel in the negative pole, and the PVDF membrane in the positive pole, to transfer the membrane with a current of 200 milliamperes for 90 minutes. Then, remove the PVDF membrane and wash with TBST once for five minutes. Add the closure solution at room temperature while shaking for 90 minutes. Then, wash with TBST three times for five minutes each. Incubate the membrane with the primary antibodies in a wet box overnight at four degrees Celsius. After washing three times with TBST, add the secondary antibody in one to 10,000 dilution drop-wise, and incubate at 37 degrees Celsius for 90 minutes. Then, wash with TBST three times for three minutes each. Finally, using the developing solution, develop the membrane on the machine as per the instructions. Compared with the control group, the model group showed a significant increase in the expression levels of PI3K, PIK3CA, AKT, phospho-AKT, BAD, BAX, and C-caspase-3, while the expression levels of phospho-BAD and BCL2 decreased significantly. KMF treatment reduced the expression levels of PI3K, PIK3CA, AKT, phospho-AKT, BAD, BAX, and C-caspase-3, and increased the expression levels of phospho-BAD and BCL2.