Effect Of Hypoglycemic Components Of Hypoglycemic And Xiaoke Granules On Mouse Pancreatic Islet Cells Based On Apoptosis And Anti - Apoptotic Mechanism

BackgroundDiabetes mellitus (DM) is a metabolic disorder syndrome characterized by relative and/or absolute shortage of insulin. In recent years, prevalence of DM is rising from less thanl% in 1980 to 9.7% in 2007 in China. The function and form of pancrease βcell is essential to pathogenesis of DM. Regenerating gene protein plays an important role in the process of Pcell regeneration; Traditional Chinese herbs also have a definite effect in the treatment of diabetes.Jiang Tang Xiao Ke Granule (JTXK) was a series of formula constructed above the theory of treatment of DM based on regulating the function of liver, Spleen and kidney and the guide of the ample clinical experience and comprehensive understanding of TCM and western medicine theories.Berberine is extracted from Rhizoma Coptidis or Cortex Phellodendri, which is one of the most important herbs in JTXK going towards spleen and stomach meridians with the function of dispelling dampness and clearing heat producd by dysfunction of liver, spleen and kidney. AndBase on the regenerationg gene and component of Chinese herbs in JTXK, we explore the apoptosis and anti-apoptosis mechanism to help the treatment of DM.ObjectivesThere are two parts in the present stydy.Part 1:we now demonstrate that mReg2 induces glucose-regulated peptide 78(GRP78) expression via the Akt-mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity.Part2:Berberine induced apoptosis via changing the expression level of pro-/anti-apoptotic proteins of mitochondrial sigal passway in mouse insulinoma cell. It will provide experimental data for the using of Chinese herbs with function of dispelling dampness and clearing heat in the treatment of diabetes.MethodsParti:FCM assay measure the apoptosis ratio of Min6-VC and Min6-mReg2 cell; Measure the difference of ATF6 and GRP78 level via the phosphorylation of IREla and eIF2ato estimate the UPR and ER stress induced by thapsigargin and glucolipotoxicity. Measure the level of Akt、pSer473Ak、mTOR、pSer2448mTOR, p70 S6K、Raptor、GβL protein to estimate the effect of mReg2 toAkt-mTOR signal passway after inhibitor of Akt and mTORC1.Part2:Treat the Min6 cell with different concentration of berberine for different time and observe the cell viability by MTTassay and flow cytometry(FCM).Compare the effect after treatment of berberine and/or Streptozotocin (STZ) in Min6 cell. We detect DNA fragmentation in cells using ELISA assay and the Bax, Bcl-2, Caspase3, PARP1, AIF, cytochrome C protein expression.ResultsPart1:1.Min6-VC cell displayed robust UPR in response to ERstress induced by Tg (1uM) for 4h as evidenced by 3-fold increase in the phosphorylation of IRE1α and eIF2α.2.Cleavage of ATF6 is less in Min6-mReg2 than in Min6-VC.3.Apoptosis ratio of Min6-VC increased 4-fold after treatment of Tg (1uM) for 4h; And there is no difference in Min6-mReg2 between before and after treatment.4.Overexpression of mReg2 decreased the phosphorylation of IRE1α.5.The basal expression of GRP78 in Min6-mReg2 is more than 5-fold than that in Min6-VC.6.mReg2 promoted the phosphorylation of Akt and mTOR and active mTORC1. The level of pSer473Akt and pSer2448mTORis higher than in Min6-VC, and the ratio is 1.7 and 1.5-fold.P<0.01.7. Phosphorylation of IRE1αrised after the treatment of Akt and mTORC1 inhibitior.Part2:1. Berberine decrease the Min6 cell viability in a dose-dependent way. The cell viability decreased 44% after exposure to berberine 5uM for 16h, and 83% after 20uM.2. We observed a time-dependent decrease in Min6 cell viability after berberine treatment. The high dose group(10uM) showed 25%,53% and 81% decrease after treatment for 4h, 8h, and 16h. And low dose group(5uM) showed 28% and 65% decrease at 8h and 16h. P<0.01.3.The viability of murine primary islet cell showed the similar dose-dependent effect. P<0.05.4.Min6 cell showed DNA fragmentation after exposure of berberine for 16h from 1.25uM.5.FCM assay showed the different apoptosis ratio in high (10uM) and low dose (5uM) group for 8h and 16h. P<0.01.6.STZ (5mM) has the synergistic effect when added to cell with berberine (10uM). The viability decreased to 5.5%.7.The level of Bcl-2, an anti-apoptosis protein, decreased 50%, compared a pro-apoptosis protein, Bax, raised 12-fold; And cytochrome C, AIF and Apaf-1 increased 3.9,2.3, and 2-fold respectively. Caspase-e and parp-1 increased 3.7 and 2.5-fold respectively. P<0.01.ConclusionPart1:1. mReg2 protect the insulin producing cells.2. mReg2 decreased UPR in Min6 via increasing the Raptor and GβL expression level and promoting the phosphorylation of Akt and mTOR,activing Akt-mTOR signaling axis and increasing the basal expression of GRP78.Part2:1. Berberine induced Min6 cell apoptosis in dose-/time-dependent way.2. Berberine induced Min6 cell apoptosis via increasing proapoptosis protein level, decreasing anti-apoptosis level, increasing cytochrome C-Apaf-caspase3 and AIF in mitochondria.In the present study, we have studied the protective effect of murine regenerating protein 2, a current hot factor, on the insulin-producing cells. The underlying mechanism was revealed from the perspective of Akt-mTORC1 signaling pathways and GRP78, which provides a new thought and method for the researches on the pro-and anti-apoptosis process of islets cells, as well as a new target for screening the herbal drugs which protect the function of islet cells.The relationship between the pancreas and spleen of TCM, and the function of berberine in onset and progression of diabetes was explored from the perpective of zang-fu visceral theory in TCM. On the basis of the previous clinical and experimental researches, we confirmed the rationality and safety of theory that treating diabtes by regulating function of liver, spleen and kidney together from the toxicology point of view. In addition, the adverse reaction and the mechanism of the drugs which could removing heat and dampness were also studid through pro-and anti-apoptotic process of islet cells.