| ?Background?Insulin resistance(IR) refers to the pathological condition when the sensitivity of the response of insulin-target tissues(liver, skeletal muscle and fat tissue) to physiological level of insulin is decreased, leading to the abnormal metabolism of glucose, lipid and protein. Now the well accepted theory of "common soil" consider that IR is the commo n pathogenesis of a series of diseases, including type 2 diabetes mellitus(T2DM), metabolic syndrome(Met S), hypertension, hyperuricemia, non-alcoholic fat liver disease(NAFLD), etc. The individuals with IR are more liable to develop cardiovascular and cerebrovascular accidents.As one of the main target organs, the liver can synthesize and store glycogen and release glucose, which is closely associated with fat metabolism. High energy diet can reduce glycogen synthesis and increase the decomposition of glycogen, which is one of the main reasons for the increase of blood glucose level. At present, the IR cell model was usually established in 3T3-L1(fat cell), Hep G2(human hepatocellular carcinoma cell) cell lines, and so on. The establishment of IR model in normal liver cells is helpful to study the molecular mechanism of IR and to develop potential treatment.Various types of drugs have been developed to treat T2 DM. However, most of these drugs has limited effects on IR and exhibits certain side-effects. In the last few years, considerable progress has achieved in studies of traditional Chinese medicine to treat T2 DM. Researchers have a better understanding of the effective parts and mechanism of TCM and have made progress in develop novel options for the treatment of T2 DM. Jiangzhitong(JZT) is a total flavones extracted from 6 kinds of the traditional Chinese medicine. Oleanolic acid(OA) is a kind of pentacyclic triterpene compound. Both of the m have protection effect on liver. It is very important to investigate the effect of Jiangzhitong and nano OA on IR hepatic cell and to study them as candidate medicine for T2 DM. ?Objective?Investigating possibility of insulin resistance in BRL-3A cells incubating with palmitic acid(PA) and detecting its stability. Detecting oxidative stress levels in IR process induced by PA to explore the role of oxidative stress during IR. The n the effect of JZT and nano OA on glucose and lipid metabolism and oxidative stress levels in IR cell models were studied. Based on these studies, we expected to evaluate the potential of JZT and nano OA as therapeutic agents for T2 DM.?Methods? 1. Cell viability:MTT method was used to detect the relative survival rate of cells in control, PA and solvent group after incubation. 2. Effect of PA on basic glucose consumption in cells:After incubation, cell culture fluid in hole was abandoned. The RPMI-1640 culture solution was added to the culture medium for 24 h, and the glucose consumption was detected. 3. Effects of PA on insulin-stimulated glucose consumption in cells: After incubation, cell culture fluid in hole was abandoned. Glucose consumption was detected in the control group and the insulin-stimulated group. 4. Effect of PA on biochemical parameters in cells: After incubation, triglycerides, total cholesterol, high density lipoprotein cholesterol, low-density lipoprotein cholesterol levels were detected. 5. Evaluation of Stability in IR cells:BRL-3A cells were incubated with appropriate PA concentration and for certain time periods, the glucose consumption of glucose and insulin-stimulated glucose consumption were detected. 6. Effect of JZT on oxidative stress level in IR cells: BRL-3A cells were incubated with different concentrations of JZT before PA exposure, and then reactive oxygen species levels in cells were detected by flow cytometry, and catalase, total antioxidant capacity, malondialdehyde and glutathione levels were detected using commercial assay kits by microplate reader. 7. Effect of JZT on biochemical parameters in IR cells: BRL-3A cells were incubated with different concentrations of JZT before PA exposure, then levels of basal glucose consumption, triglycerides, total cholesterol, high density lipoprotein cholesterol, low-density lipoprotein cholesterol were detected. 8. Effect of nano OA on BRL-3A cells: Relative survival rate, glucose consumption and reactive oxygen levels of cells in control, OA and nano OA group were detected after incubation. 9. Effect of nano OA on biochemical parameters in IR cells: triglycerides, total cholesterol, high density lipoprotein cholesterol, low-density lipoprotein cholesterol were detected after incubation. 10. Effect of nano OA on oxidative stress state in IR cells: reactive oxygen levels and malondialdehyde levels were detected after incubation.?Results? 1. Incubation of cells with low concentrations of PA(50,100?mol/L) within 12 h had no significant effect on cell viability(P>0.05). Along with the increase of PA concentrations and incubation time, cells survival could be significantly decreased(P<0.05), indicating a dose and time-dependent manner. 2. The ability of basic glucose consumption in cells was not altered by 50?mol/L PA incubation for different time periods(P>0.05). Along with the increase of PA concentrations and incubation time, glucose consumption in cells was decreased gradually. Incubati ng of cells with 50?mol/L PA could significantly decrease basic glucose consumption(P<0.05). 3. Along with the increase of PA concentrations and incubation time, difference between insulin-stimulated and basic glucose consumption in cells decreased gradually. After incubating over 12 h, the ability basic glucose consumption in cells was not altered by insulin(P>0.05). 4. PA could significantly increase levels of triglycerides, total cholesterol, low-density lipoprotein cholesterol, while decrease high density lipoprotein cholesterol in cells. 5. The ability of basic and insulin-stimulated of glucose consumption remained unchanged after 72 h continuing culture. 6. JZT could significantly decrease level of reactive oxygen, malondialdehyde, while increase total antioxidant capacity and catalase activity in IR cells. 7. JZT could significantly increase level of basic glucose consumption, high density lipoprotein cholesterol, while decrease triglycerides, total cholesterol, low-density lipoprotein cholesterol in IR cells. 8. Nanocrystallization could reduce OA-induced effect relative survival rate, reactive oxygen and glucose consumption in cells. 9. Compared with OA, nano OA could significantly increase glucose consumption, high density lipoprotein cholesterol, while decrease triglycerides, total cholesterol, low-density lipoprotein cholesterol in cells. 10. Compared with OA, nano OA could significantly decrease level of reactive oxyge n and malondialdehyde in cells.?Conclusion? 1. Incubation of BRL-3A cells with 100?mol/L of PA for 12 h could decrease glucose consumption ability, weaken insulin sensitivity, induce disorder of lipid metabolism, resulting in IR. 2. IR remains unchanged after 72 h continuing culture.3. JZT can reduce the level of oxidative stress, and improve the level of glucose and lipid metabolism in IR cells. 4. Nanocrystallization could reduce OA-induced effect on relative survival rate, reactive oxygen and glucose consumption in cells. 5. Nano OA can reduce the level of oxidative stress, and improve the level of lipid metabolism in IR cells. 6. JZT and nano OA can be used as candidate drugs to treat IR and T2 DM and further studies are needed. |
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