Background[1-3]
Human phosphorylated protein kinase C (P-PKC) ELISA kit uses a double-antibody sandwich enzyme-labeled immunoassay method to determine the level of p-PKC in the specimen. Coat the microwell plate with the purified antibody to make a solid-phase antibody. Add p-PKC, biotinylated anti-human p-PKC antibody, and HRP-labeled avidin to the microwells coated with the monoclonal antibody in sequence. After washing, the color was developed using TMB substrate. TMB is converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The depth of color is positively correlated with the p-PKC in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm and calculate the sample concentration.
Protein kinase C is an effector in the G protein-coupled receptor system. It is water-soluble in the inactive state and exists freely in the cytosol. It becomes a membrane-bound enzyme after activation. Activation of protein kinase C is lipid-dependent, requiring the presence of the membrane lipid DAG, and is Ca2+-dependent, requiring an increase in the Ca2+ concentration in the cytosol. When DAG appears in the plasma membrane, protein kinase C in the cytosol is bound to the plasma membrane and then activated under the action of Ca2+. Like protein kinase A, protein kinase C is a multifunctional serine and threonine kinase. Protein kinase C is a cytoplasmic enzyme. In unstimulated cells, PKC is mainly distributed in the cytoplasm in an inactive conformation. Once the second messenger exists, PKC will become a membrane-bound enzyme. It can activate enzymes in the cytoplasm and participate in the regulation of biochemical reactions. It can also act on transcription factors in the nucleus and participate in the regulation of gene expression. It is a Multifunctional enzyme. Protein kinase C is involved in the control of gene expression in at least two ways. One way is that protein kinase activates a phosphorylation cascade system to phosphorylate MAP protein kinase. The phosphorylated MAP protein kinase phosphorylates the gene regulatory protein Elk-1, causing it to be activated. Activated Elk-1 binds to a short DNA sequence (called a serum response element, SRE), which then works with another factor (a serum response factor, SRF) to regulate gene expression. Another way is that protein kinase phosphorylates and activates the inhibitory protein Iκ-B, releasing the gene regulatory protein NF-κ-B, allowing it to enter the nucleus and activate the transcription of specific genes.
Application[4][5]
Study on the protective effect and mechanism of beraprost sodium on rat mesangial cells injured by high glucose
Diabetic nephropathy (DN) is one of the unique microvascular complications of diabetes mellitus (DM). Its characteristic pathological changes include the accumulation of extracellular matrix (ECM) in the mesangial area, renal glomerulonephritis, Sclerosis and tubulointerstitial fibrosis ultimately lead to progressive increase in urinary protein/albumin excretion and decrease in renal function. The pathogenesis of DN is complex. Currently, it is believed that hyperglycemia, abnormal renal hemodynamics, increase in advanced glycation end products (AGEs), polyols and protein kinase C (PKC) Various factors such as pathway activation and oxidative stress generation jointly participate in the occurrence and development of DN. How to effectively prevent and treat diabetic nephropathy has always been a hot and difficult issue in clinical practice.
Monocyte chemoattractant protein-1 (MCP-1) plays a key role in macrophage infiltration and inflammation in diabetic tissues. MCP-1 is a secreted protein that can be expressed by a variety of cells and has chemotactic effects on monocytes, T lymphocytes, etc. After MCP-1 binds to its receptor, it induces the expression of downstream adhesion molecules, causing monocytes/macrophages to gather to the lesion site to exert its pathological effects. Mesangial cells cultured with high glucose can promote the expression of MCP-1 through the activation of PKC and oxidative stress. Blocking the binding of MCP-1 to its receptor can reduce the infiltration of macrophages and the expression of TGFβ1 and IV collagen in the renal tissue of diabetic mice, effectively preventing the progression of DN glomerulosclerosis.
