Discussion

TGSO showed remarkable anti-diabetic effects in animal models，however，functional components in TGSO and its anti-diabetic mechanism have not been identified.In this investigation，the compositions of TGSO were analyzed by GC-MS and its mechanism was illustrated by measuring apoptotic rate，insulin release and related gene expression.

Glucose with supraphysiological level has deleterious effect on pancreaticβ-cell function and consequently results in dysfunction and apoptosis［4］.As in our previous study，25 mM glucose was used in this investigation to construct apoptotic cell models with high apoptotic rate and low insulin secretion［38］.Mechanisms of high glucose inducedβ-cell apoptosis include increasing iNOSexpression together with regulation of apoptosis-related gene expressions involved in three pathways.In consistent with our previous observation，high glucose induced apoptosis via Fas signal pathway and imbalance expression of Bcl-2 versus Bax.The anti-apoptotic gene Bcl-2 expression did not response to high glucose stimulation，whereas pro-apoptotic Bax gene was increased［38］，leading to the alteration of the mitochondrial homeostasis，and subsequent activation of apoptosis.In addition，up-regulated expressions of ATF-3，JNK，p38 and down-regulated expressions of Akt revealed the high glucose induced apoptosis is closely related with ER stress pathway too.This is agreed with the review of Bensellam et al.that ER stress pathway plays an important role inβ-cell glucotoxicity［4］.Among these tested genes in ER stress pathway，the modification of transcription factor ATF-3 expression in model group is the highest(9.17 folds).This might due to its role as a downstream target of both NF-kBand JNK/SAPK signaling pathways and the induction of NO which is synthesized by iNOS［21］.(https://www.daowen.com)

After co-culture with TGSO，the cells were protected against high glucose-induced apoptosis and insulin secretion was reserved［31］.This may due to the UFA it contained with the ratio of UFA/SFA up to 670%.Though SFA was reported to induce apoptosis by mitochondrial dysfunction and production of reactive oxygen species［40，41］，UFA could protect RINm5F cells against SFA-induced cell death，not only induction of apoptosis prevented［32］，but alsoβ-cell proliferation promoted［42］.It is reported that the antiapoptotic UFA displays considerable structural specificity and it appears to regulate a signaling pathway that controls the activity of effector caspase enzymes in the cells［43］.UFA protection from apoptosis mediated through a PI3-kinase-dependent signaling pathway，since PI3-K inhibitor abolished the UFA protection［32］.These were supported by our observation that treatment ofβ-cell with TGSO causes attenuated expression of caspase-3 and increased expression of Akt，the downstream targets of PI3K.

The regulation of apoptosis-related gene expressions suggested that the protect activity of TGSO was via all of the three pathways，namely ER stress pathway，mitochondrial pathway and Fas signal pathway.In ER stress pathway，ATF3，as a member of the ATF/c AMP response element binding protein(CREB)family of transcription factors，its induction was reported to play an important role inβ-cell destruction［44，45］.Li et al.［20］found that ATF-3 down-regulate the expression of insulin receptor substrate 2(IRS2)while Allen-Jennings et al.［46］demonstrated its inhibition ofβcell proliferation.Hartman et al.speculated that ATF3 may in turn affect the ability of NO to modulate the cell death machinery directly or indirectly［21，47］.Burak Kutlu et al.observed that iNOS inhibitor reduced cytokine-induced ATF-3 expression in INS-1βcell［48］.In this investigation，the extreme high expression of ATF-3 gene induced by high glucose was reduced to the comparable level of control which may be a result of declined NO formation.Besides，the p38 expression might illustrate that the anti-apoptotic action played in ER stress pathway was mainly through the p38 branch rather than JNK.In view of the cytochrome c release block and Bcl-2 expression decrease，mitochondrial pathway was reported to contribute to the protection of MUFA against saturated fatty acid-induced apoptosis in humanβ-cells［31］.In this research，the expression of Bcl-2/Bax was declined which implied that the protection of TGSO，containing 87.02% UFA，against high glucose was via mitochondrial pathway.In addition to these two pathways，the down-regulation of Fas gene revealed that Fas signal pathway was involved in this anti-apoptotic activity as well.