Discussion

Xiaokeping showed significant effects on reducing blood glucose in both animal model experiments［15］and clinical investigations［16］；however，the underlying molecular mechanism causing the reduction has not been identified.In this investigation，a potential mechanism was illustrated by measuring autophagy formation，apoptotic rate and autophagy/apoptosis-related protein expression.We found that XKPinduced autophagy in pancreaticβ-cells via the mTOR pathway.Inβ-cells under a high glucose stress，XKP decreased their apoptotic rate and increased autophagy.To investigate whether autophagy contributes to the anti-apoptotic effect of XKP，3-MA was applied to inhibit autophagy activity，and consequently the anti-apoptotic effect of XKP was attenuated.Therefore，the protective effect of XKPagainst apoptosis might be a result of XKPinducing autophagy in cells under high glucose stress.Autophagy plays an important role in endocellular processes ofβ-cells，such as cell cycle，signal transduction，transcriptional regulation，DNA repair，and cell apoptosis［10］.Loss of autophagy diminishesβ-cell mass and function with the consequence of hyperglycemia［17］.Autophagy is either positively regulated by the AMPK pathway or negatively regulated by the mTOR pathway［18］.Our present investigation indicates that XKPinduced autophagy inβ-cells via the mTOR pathway.However，autophagy can also protectβ-cells against apoptosis induced by cholesterol or free fatty acid(FFA)via the AMPK pathway［18］.Thus the function of autophagy may depend on different autophagy inducers.AMPKis a cellular energy sensor that is sensitive to theintracellular ratio of adenosine triphosphate(ATP)to AMP.The activity of AMPK is allosterically enhanced by AMP binding to theγsubunit［19］.Moreover，a variety of cellular stresses that decrease ATP generation can activate AMPK，including metabolic toxins as well as pathologic cues such as nutrient starvation，ischemia，and hypoxia.On the other hand，mTORis regulated by cellular energy and nutrient status.mTOR interacts with raptor to form a complex which is the central component of signaling pathways that are nutrientand hormone-sensitive and regulate cell growth.Raptor regulates the activity of S6K and mTOR.The inhibition of that pathway can stabilize the mTOR-raptor association and inhibit mTOR kinase activity［20］.In our experiment，we found that XKPinhibited the mTORsignaling pathway by inducing autophagy in the response ofβ-cells to high glucose concentrations.Future studies should focus on identifying the specific components in XKP to further elucidate the molecular mechanism of XKPeffects onβ-cells.

A common symptom of diabetes patients is the loss of pancreaticβ-cell mass.The pancreaticβ-cell is a highly differentiated cell，and slow to proliferate in adults.Protectingβ-cells from apoptosis due to a high glucose stress may ameliorate symptoms of diabetes.Our data suggest that XKP has a protective effect against high glucose-induced apoptosis in pancreaticβcells.The protective effect is similar to other medicines that protectβ-cells from apoptosis under high glucose stress.For example，metformin prevents glucotoxicity by alleviating oxidative and endoplasmic reticulum(ER)stress in pancreaticβ-cells［21］；Liraglutide increasesβcell mass to amelioration of glucolipotoxicity［22］；Thorn grape seed oil protectsβ-cells from high glucose-induced apoptosis［23］.(https://www.daowen.com)

The purpose of modern pharmacology is to explore the molecular mechanisms that can be targeted by therapeutic compounds.Xiaokeping is a typical TCM，which has shown therapeutic effects on T2DM.Though we have demonstrated a potential mechanism of antidiabetic effect of XKP related to autophagy/apoptosis pathways，the specific effective compounds of XKPare unknown.Coincidentally，berberine，one of the components of XKP，has been reported to activate autophagy and mitigate high glucose-induced apoptosis of mouse podocytes［24］.Thus the anti-diabetic effects of XKPmay be due to berberine as well as other potentially effective components that remain to be further explored.Traditional Chinese medicine is based on hundreds of years of clinical application in China.Most TCMs are extracted from one or more medicinal herbs which make their effective components difficult to identify.However，the existence of multiple bioactive ingredients makes many TCMs potential novel resources for the discovery of new drugs including anti-diabetic drugs.