| Diabetic mellitus is a metabolic disorder syndrome with character of chronic hyperglycemia; its incidence is increasing every year. Improper treatment can lead to chronic complications and endanger the heart, brain, kidney, peripheral nerves, eyes, feet, seriously affecting the life quality of patients. Central nervous system damage caused by diabetes, leading to cognitive impairment and changes in brain physiology and structure, known as diabetic encephalopathy clinical manifestations of understanding decrease, memory loss, and indifferent expression, slow and so on. The pathogenesis of central nervous system damage induced by diabetic mellitus is complicated. In recent years, studies have shown that neuronal apoptosis induced by high glucose is one of the important causes of central nervous system injury induced by diabetic mellitus. Autophagy plays an important role in maintenance of intracellular homeostasis in cells. m TOR is a key factor of regulating autophagy. AMPK is a negative regulator of m TOR by inducing phosphorylation of TSCl/2. TSC2 could negative regulation of Rheb. AMPK promote the formation of autophagy by inhibiting the activity of m TOR.Berberine(BBR), an isoquinoline alkaloid isolated from Rhizoma Coptidis and Cortex Phellodendri, is extensively used in Asia for the treatment of diarrhea. It has been reported that BBR has multiple pharmacological activities, including hypoglycemia, hypolipidemia, anti-inflammatory effects and anti-oxidative effects. However, the poor oral bioavailability of BBR limits its clinical application as an anti-diabetic drug. Our laboratory developed the Huang-Gui solid dispersion(HGSD), which is an amorphous solid dispersion of berberine with the absorption enhancer sodium caprate(SC). We have demonstrated that HGSD could significantly increase the bioavailability of berberine without any serious mucosal damage. Our preliminary work also indicated that HGSD exhibited a good hypoglycemic activity by promoting AMPK activation. But, the effect and mechanism of HGSD on neuronal apoptosis induced by diabetic mellitus is not clearly.Method: First, we have discussed the Huang-gui solid dispersion(HGSD) is able to against neuronal injury in vivo. High fat diet and intraperitoneal injection of STZ were used to establish the type 2 diabetic model in C57BL/6J mice. Mice were randomly divided into four groups(n=15): control group, mice were given equivalent amount of saline; Model group, T2 DM mice were given equivalent amount of saline; BBR-40 group, T2 DM mice were given BBR(40 mg/kg, b.w.); HGSD-40 group, T2 DM mice were given HGSD(40 mg/kg, b.w.). After 4 weeks of admininstration, HE staining was used to detect pathological changes in hippocampus CA1 area tissue; TUNEL staining was used to detect apoptosis in hippocampus CA1 area tissue; Western blotting was used to detect the expressions of apoptosis related proteins, autophagy related proteins, p-AMPK/AMPK and p-m TOR/m TOR. To further explore the mechanism of the neuroprotective effect of HGSD, we determined the influence of BBR on SH-SY5 Y cells under a high glucose condition. Using high glucose established apoptosis model of SH-SY5 Y cells, dividing into control group, BBR group, HG group, BBR+HG group. The changed of cell morphology were observed by inverted phase contrast; the level of autophagy was assayed by MDC; the changed of nucleus were assayed by Hoechst33258. Western blotting was used to detect the expressions of apoptosis related proteins, autophagy related proteins, p-AMPK/AMPK and p-m TOR/m TOR. To further reveal the relationship between autophagy and apoptosis as well as the function of BBR, SH-SY5 Y cells were pretreated with 3-MA, an inhibitor of autophagy. the changed of cell morphology were observed by inverted phase contrast; the level of autophagy was assayed by MDC; the changed of nucleus were assayed by Hoechst33258. SH-SY5 Y cells were treated with compound C and AICAR, the level of autophagy was assayed by MDC; the changed of nucleus were assayed by Hoechst33258. Western blotting was used to detect the expressions of apoptosis related proteins, autophagy related proteins, p-AMPK/AMPK and p-m TOR/m TOR.Results: In vivo, compared with the control group, the hippocampus CA1 area in the model group appeared to have neuronal loss and a loose arrangement of neurons as shown by HE staining; significant numbers of TUNEL-positive cells appeared in the hippocampus CA1 area; the expression of Caspase-3 clearly increased and Bcl-2 clearly decreased; the expressions of p-ULK1/ULK1, LC3-II/LC3-I and Beclin-1 clearly decreased. Compared with model group, HGSD-40 significantly decreased neuronal loss and improved neuronal arrangement; there were significantly fewer TUNEL-positive cells; HGSD-40 significantly decreased the activity of Caspase-3 and increased the activity of Bcl-2. The expression levels of p-ULK1/ULK1, LC3-II/ LC3-I and Beclin-1 were significantly increased. Compared with control group, the indicators have improved in BBR-40 group, but the difference was not statistically significant. In vitro, compared with the control group, Hoechst 33258 staining was used to detect DNA condensation and nuclear fragmentation in model group; the fluorescence intensity of the SH-SY5 Y cells decreased detected by MDC; the expression of Caspase-3 clearly increased, and the expression of Bcl-2 clearly decreased; the expressions of p-ULK1/ULK1, LC3-II/LC3-I and Beclin-1 clearly decreased. SH-SY5 Y cells were pretreated with 3-MA, compared with BBR group, 3-MA abolished the BBR-induced cell morphology renewed; anti-apoptotic effect of BBR was diminished by 3-MA. SH-SY5 Y cells were treated with compound C and AICAR, that anti-apoptotic, promote autophagy and activing AMPK effects of BBR was diminished by Compound C, while enhanced by AICAR significantly.Conclusion : Results showed that the AMPK/m TOR signaling pathway exerted a protective role in the high glucose-induced cellular apoptotic death via the induction of autophagy. HGSD enhanced autophagy by increasing the expression of p-AMPK, thereby inhibiting apoptosis. |
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