The Effect Of Shenshuai Yangzhen Capsule Containing Sera On Leptin And C/EBPα Expression In Insulin-induced Rat Adipocytes

BACKROUND:Malnutrition is common complications in patients with CRF. Its prevalence both in the no-dialysis period and during dialysis is high. More than 65% ESRD have varing degrees of malnutrition reported in the domestic literature. Malnutrition is known to induce a state of immunodeficiency and aggravate anemia, or lead to multiple system organ failure. This is a problem of substantial importance because many measures of nutritional status correlate with the risk of morbidity and mortality. The researches of malnutrition in CRF patients become a key task. Leptin is a 16 kDa hormonal protein synthesized mainly by adipose tissue. Leptin ,a weight homeostasis regulating hormone, reduces appetite and increases energy consumption. Recent epidemiologic studies have suggested that increased serum levels of leptin may reduce nutrient intake and contribute to the development of malnutrition. Leptin has long been associated with malnutrition in CRF patients. It is believed that high leptin levels is common in CRF patients. Serum leptin levels are high in CRF patients, mainly because leptin is degraded in the kidney, but serum leptin levels are also closely associated with inflammation, hyperinsulinemia and hypercholesterolaemia that can upgrade ob-RmRNA expression. High leptin levels can lead to malnutrition. Medical nutrition therapy is important in malnutrition of CRF patients. In western medicine, Ketosteril is main medicine treatment on malnutrition of CRF patients, but its curative effete is limited and its price is exorbitant. TCM has better effects on treatment of malnutrition of CRF patients, but mechanisms of TCM has not been explored. Capusle of Shenshuai Yangzhen is experiential compound that is placed by our supervisor Wei Lian-bo. Capusle of Shenshuai Yangzhen that has owned national invent patent is imbursed by Guangdong Scientific and Technological center tackling key problems and Guangdong TCM management department. It has been tested that Shenshuai Yangzhen can impove obviously malnutrition of CRF rats and can decline leptin contents in clinical and animal researches.OBJECTIVE:Observe the effects of shenshuai yangzhen capsule on Leptin secretion,OB mRNA and C/EBPαmRNA expression in insulin-induced rat adipocytes. Explore the possible mechanisms of Shenshuai Yangzhen Capusle on improving malnutrition in CRF rats.METHODS:1 .We collected rat sera containing herbs after rats were intragastricly administered with the shenshuai yangzhen capsule,Ketosteril medicine and normal sodium.2. we established rat adipocytes successful which were differentiated from primary culture of preadipocytes and identified by oil red O stained. The Adipocytes were randomly dibided into 5 groups:insulin group,shenshuai yangzhen pharmacologic serum group,Ketosteril pharmacologic serum group,normal rat blood serum group,as well as normal adipocytes group in . Adipocytes induced by insulin were incubated with heat-inactivated pharmacologic serum for 48 hours .3. The Leptin secreted in culture supernatant were measured by radio immunoassay.4. The expression of OB mRNA and C/EBPαmRNA in adipocytes was detected by RT-PCR: extracted the total mRNA by Trizol and the OB mRNA and C/EBPα mRNA expression was measured by semi-quantitative RT-PCR: using the One-Step RT-PCR Kit:5μl of total RNA and 0.3μl (20mmol/L) anti-sense Leptin specific primers. 5 minutes later at the temperature of 70℃, added the sense Leptin specific primers and 0.1 % DEPC pretreated water to 20μl.The same method with the internal control GAPDH was carried out. Synthesized the cDNA template at 42℃for 1 hour, and inactivated the reverse transcriptase and predenaturalized the cDNA at 94℃for 5 minutes. The final product was electrophoresed in the 1.5 % agarose gel, visualized by EB, analyzed by Gel Doc1000,the content of OB and C/EBPαfragment and GAPDH fragment were compared.RESULTS:1. We confirmed that primary culture of rat preadipocytes can be derivated into adipocytes by incubated with IBMX,insulin and dexameth. successful Red-stained particles were observed in the oil red O-stained cells under a light microscope, indicating that the cells had differentiated into adipocytes.2. We observed that the leptin contents of adipocytes were significantly elevated which were induced by insulin (P<0.001) .Shenshuai yangzhen pharmacologic serum could downgrade the exorbitant leptin contents. Comparing with insulin group, the leptin contents of Shenshuai yangzhen pharmacologic serum group were significant decreased (P=0.027) , and no significant differences between Ketosteril pharmacologic serum group and insulin group(P = 0.512). The Leptin contents of Shenshuai yangzhen pharmacologic serum group were lower than Ketosteril 's,but there was not significant differences between them(P>0.05).3. There was significant differences on the gene expression of OB among the five groups(F= 10.323, P < 0.001). Insulin can increase the gene expression of OB significantly (P < 0.001) . And Shenshuai yangzhen pharmacologic serum significantly inhibit the excessive gene expression of OB induced by insulin (p = 0. 005). Ketosteril pharmacologic serum also reduced the excessive gene expression of OB induced by insulin, but there was not significant difference between them(P = 0.433). The gene expression of OB in Shenshuai yangzhen pharmacologic serum group was markedly lower than that in Ketosteril (P<0. 05) .4. There were also significant differences on the C/EBPαgene expression among the five groups ( F=5.421, P=0.003) . Insulin can increase the gene expression of C/EBPαsignificantly (P=0.001) . And Shenshuai yangzhen pharmacologic serum significantly inhibit the excessive gene expression induced by insulin (P=0. 002). Ketosteril pharmacologic serum also reduced the excessive C/EBPαgene expression induced by insulin, but there were no significant differences between them(P=0.260).Conclusion:Shenshuai Yangzhen Capsule Containing Sera can inhibit the excessive expression of Leptin and C/EBPαgene in adipocytes induced by insulin and reduce leptin concentrations significantly. These effects maybe one of the mechanisms in accordance with which Shenshuai Yangzhen Capsule adjusts malnutrition of CRF.