The Protective Effect Of Sesquiterpene Lactones Derivative On Islet Cell Of Db/db Mice

Background and objectivesIt is generally known, we have to face more and more threat of chronic non-communicable diseases (CNCDs) as the rising standards of living and the rapid evolution of aging population. The incidence of diabetes mellitus (DM) is on the rise, and it has been one of diseases which seriously jeopardized the health of human beings on global. There are more than 100 diabetes complications in the world,such as diabetic nephropathy, diabeticretinitis and diabetic foot. In our country, the morbidity rate of type 2 diabetes mellitus more than 90%, so it is very imperative to prevent and cure diabetes mellitus. However the etiology and mechanism of type 2 diabetes mellitus is complicated and yet not well clarified. Age, obesity, oxidative stress, β cell dysfunction, lipid accumulation, hyperglycaemia, tissue inflammation and endoplasmic reticulum stress are all related to the development of DM. DM is also a chronic inflammatory disease. Insulin resistance and increase rate of islet β cell dysfunction were considered as the main pathogenesis of type 2 diabetes mellitus for now. In the pre-diabetes, there is islet cell hyperplasia and the high secretion of insulin. Then the incidence of DM would be accelerate if islet beta cell dysfunction and the reduction of inslin secretion. Islet P cell dysfunction is indispensable factor of the development of DM and P cell apoptosis may be the main pattern of cell dysfunction. It is know that glucose toxicity, fatty toxicity, free fatty acids and so on could enhance the apoptosis. So our main research direction is how to prevent islet beta cell apoptosis and decrease beta cell dysfunction.Nuclear factor-kappa B (NF-κB) has been considered as an important transcription factor which is found widely in cells and tissues of mammals. NF-κB plays an important role in inflammatory response, oxidative stress, immune responses and tumorigenesis and cancer progression. It also participates in cellular proliferation, apoptosis, invasion, differentiation, metabolism and angiogenesis. Studies have shown that the NF-κB expression was up-regulated in the pancreatic tissue of diabetic animal models and diabetic patients. Many factors could stimulate the activation of NF-κB, then regulat a large of inflammatory cytokines, such as IL-1β, TNF-α, inducible nitric oxide synthase (iNOS), which could damage the function of islet cell, however, these factors also can increase the activation of NF-κB. The activation of NF-κB may also regulate the expression of Bax and Bcl-2, which way is related to apoptosis. Plenty of experiments implicated that the specific inhibitor of NF-κB could decrease the active of NF-κB then down-regulator the expression of inflammatory agents and decrease the generation of oxidative stress. This pathway plays a key role in improving islet cell dysfunction, decreasing the apoptosis of beta cell, then retarding the progression to type 2 diabetes mellitus.Parthenolide (PTL), a naturally occurring sesquiterpene lactones (SLs) is originate in feverfew (Tanacetum parthenium). This plant extract exert was being used to skin infections, rheumatic fever,migraines, joint pain, periodic fever, asthma and so on. However SLs possess not just these activity but also exert a strong anti-inflammatory, anti-oxidant activity, anti-cancer and antibacterial activities. PTL been known as an inhibitor of NF-κB, it could inhibit the activation of NF-κB by suppressing the IKK complex and decreasing the phosphorylation of IκB, and eventually inhibiting the avtivation of NF-κB. This research we used a new derivative chemical compounds (named ACT001), which synthesized from PTL. We have proved that ACT001 could inhibit the activation of NF-κB in podocyte and rat mesangial cells, and it have a marked advantage on preparation technology, stability, bioavilability and fewer side effects compares to PTL. This derivative shows the wide prospect of development and clinical application.In our experiment, we use db/db mice as an object of experimental research. In order to elucidate whether ACT001 possess the function of preventing islet beta cell and its related mechanism of action. The impact of fasting plasma glucose, plasma iusulin, blood lipids, inflammatory factors as well as the expression of NF-κB, iNOS, Bcl-2 and Bax in pancreatic tissues were observed.Contents and methodsTwelve db/m mice and twenty-four db/db mice,15weeks of age, male and female mice,were purchased from Model Animal Research Center of Nanjing University. After acclimating for one week, db/m mice were set as normal control group (n=12), twenty-four db/db mice were randomly divided into two groups, respectively provided by stroke-physiological saline solution (diabetes mellitus model group, n=12) and ACT001 (ACT001 treat group, n=12,25mg/kg/d) for 12 weeks, every mice took food and water freely in experimental period, body weigh and fasting blood glucose were monitored. After 12 weeks treatment, the mice were fasted for 12 hours before each experiment, every mice was tranquilized by pentobarbital, fast blood glucose, fasting insulin concentrations, liqid level, IL-1(3, TNF-a were observed. Every mice’s pancrease were taken for hematoxylin-eosin staining and immunohistochemical of NF-κB、iNOS、Bcl-2、Bax, the protein expression of NF-κB、iNOS was measured by western blotting assay.1. The concentration of blood glucose, insulin and blood lipids:fasting blood glucose were measured by ACCU-CHEK glucose meter, fasting insulin concentrations were measured by ELISA kit; blood lipids were measured by autoanalyzer.2. The concentration of IL-1β and TNF-α:the level of serum IL-1β and TNF-α were measured by ELISA kit.3. Histological morphological examination:Pancrease were fixed in 4% paraformaldehyde, then dehydrate using graded ethanol, then embedded in paraffin and paraffin section. HE staining were used to abserve the morphology of mice islet and immunohistochemical staining of NF-κB, iNOS, Bcl-2, Bax also been observe.4. The protein expressin of NF-κB, iNOS in pancrease of mice were determined with western blotting.5. Statistics:The statistical analysis of all data were mean±SEM. Statistical soft ware SPSS 19.0 was performed to statistical comparison of all groups. Comparison between two groups was done with two-sample T-test. Difference of measurement data was compared with single factor analysis of variance. Welch test and Dunnett’s T3 test were used for the unequal variances. Data was evaluates at the significance level P<0.05, test criteriona=0.05.Result1. The general condition of mice in every group:The general condition of normal control group was good, have a great mental state, move easily, shiny hair, normal diet, excretory functions were normal and no death; model control group and ACT001 group have a poor mental status, sluggishness and inactivity, polyphagia, polydispia, polyuria; compared with model group, the symptoms of diabetes in ACT001 group were improved.2. Body weight changes of mice in every group:At the end of this experiment, the level of body weight of model control group and ACT001 group were higher compared to normal control group (P<0.05); there are no difference between model control group and ACT001 group.3. The change of secrum biochemical parameters in every group:the level of secrum biochemical parameters of normal control group were lower than the other tow groups (P<0.05); comared with model control group, the expression of secrum biochemical parameters of ACT001 group were dcreased (P<0.05)4. The expression of serum IL-1β, TNF-α in every group:the expression level of secrum IL-1β and TNF-a in model control group and ACT001 group are higher than normal control group(P<0.05); however, the expression level of secrum IL-1β,TNF-α in ACT001 group are lower than model control group after experiment(P<0.05)5. HE staining on pancreatic tissues:Under light microscope, islet in pancrease tissues of the normal control group were morphologically intact with clear membrane and plenty of secreting granules; Instead, the number of islet was decreased with irregular morphology and edgies in model control group and ACT001 group; The lesions of the pancreatic islets were significantly improved in ACT001 group compared to model group after experiment.immunohistochemical staining on pancreatic tissues of every group:Compared with normal control group, the expression of NF-κB, iNOS, Bax in other two groups were markedly increased (P<0.05) and the expression of Bcl-2 was decreased; compared to model control group, the expression of NF-κB, iNOS, Bax were decreased and the expression of Bcl-2 was increased in ACT001 group (P<0.05)6.The change of protein expression of NF-κB, iNOS in every group:Compared with normal control group, the protein expression of NF-κB and iNOS was increased in model control group and ACT001 group (P<0.05); the expression of NF-κB and iNOS in ACT001 group were reduced compared to model control group (P<0.05)Conclusion1. Db/db mice had a higher body weight, food and water intake than db/m mice; compared with db/m mice, the fasting blood glucose, serum insulin and serum lipids of db/db mice were markly increased.2. Db/db mice expressed a higher level of serum IL-1β、TNF-α than db/m mice.3. The up-regulated expression of NF-κB, iNOS, Bax and down-regulated expression of Bcl-2 could be observed in the tissues of the pancreas of db/db mice, which were related to pancreatic islet beta cell dysfuntion.4. Sesquiterpene Lactones derivative could reduced the level of fasting blood glucose, serum insulin and lipids as well as the expression of serum IL-1β、TNF-α in db/db mice.5. Sesquiterpene Lactones derivative could inhibite the activation of NF-κB, then down-regulator the expression of iNOS, which was helpful for protecting islet beta cell.6. Sesquiterpene Lactones derivative down-regulated the expression of Bax and up-regulated the expression of Bcl-2, then improve the apoptosis of islet beta cell.7.In this research, we found that our new kind of Sesquiterpene Lactones derivative (named ACT001) may have an effect to inhibit the apoptosis of cell and protect pancreatic islet β cells.