Amorphous solid dispersion of Berberine mitigates apoptosis via iPLA2β/Cardiolipin/Opa1 pathway in db/db mice and in Palmitate-treated MIN6 β-cells

Abstract

Aims: Berberine (BBR) improves beta-cell function in Type 2 diabetes (T2D) because of its anti-apoptotic activity, and our laboratory developed a new preparation named Huang-Gui Solid Dispersion (HGSD) to improve the oral bioavailability of BBR. However, the mechanism by which BBR inhibits beta-cell apoptosis is unclear. We hypothesized that the Group VIA Ca²⁺-Independent Phospholipase A₂ (iPLA₂β)/Cardiolipin(CL)/Opa1 signaling pathway could exert a protective role in T2D by regulating beta-cell apoptosis and that HGSD could inhibit β-cell apoptosis through iPLA₂β/CL/Opa1 upregulation. Methods: We examined how iPLA₂β and BBR regulated apoptosis and insulin secretion through CL/Opa1 in vivo and in vitro. In in vitro studies, we developed Palmitate(PA)-induced apoptotic cell death model in mouse insulinoma cells (MIN6). iPLA₂β overexpression and silencing technology were used to examine how the iPLA₂β/CL/Opa1 interaction may play an important role in BBR treatment. In in vivo studies, db/db mice were used as a diabetic animal model. The pancreatic islet function and morphology, beta-cell apoptosis and mitochondrial injury were examined to explore the effects of HGSD. The expression of iPLA₂β/CL/Opa1 was measured to explore whether the signaling pathway was damaged in T2D and was involved in HGSD treatment. Results: The overexpression of iPLA₂β and BBR treatment significantly attenuated Palmitate- induced mitochondrial injury and apoptotic death compared with Palmitate-treated MIN6 cell. In addition, iPLA₂β silencing could simultaneously partly abolish the anti-apoptotic effect of BBR and decrease CL/Opa1 signaling in MIN6 cells. Moreover, HGSD treatment significantly decreased beta-cell apoptosis and resulted in the upregulation of iPLA₂β/CL/Opa1 compared to those of the db/db mice. Conclusion: The results indicated that the regulation of iPLA₂β/CL/Opa1 by HGSD may prevent beta-cell apoptosis and may improve islet beta-cell function in Type 2 diabetic mice and in palmitate-treated MIN6 cells.

Keywords: Apoptosis; Berberine.; Beta-cell dysfunction; Cardiolipin; Dynamin-related protein(Opa1); Group VIA Ca2+-Independent Phospholipase A2 (iPLA2β); Type 2 diabetes.

Figure 1

Palmitate(PA) induce the apoptosis in MIN6 cells and over-expression iPLA₂β can mitigate this injury. The influence of PA-induced cytotoxicity and the mediation of over-expression iPLA₂β were assessed by MTT assay(A). Data was expressed as means ± SEM (n=6). Compared with control group, *P<0.05, **P<0.01; compared with PA group, ^#P<0.05, ^##P<0.01. Basal insulin release and content were measured in MIN6(B). Data was expressed as means ± SEM (n=6). Compared with control group, **P<0.01; compared with PA group, ^#P<0.05. After treated with PA for 24h in normal cells and iPLA₂β over-expressed cells, the apoptosis were observed by Hoechst staining, bright blue means the apoptotic cells and marked with arrows(C). The protein was extracted from cells and cytosolic cytc and caspase3 were measured and analyzed by Western Blot(D). Data was expressed as means ± SEM (n=4). Compared with control group, *P<0.05, **P<0.01; compared with PA group, ^#P<0.05, ^##P<0.01. Mitochondrial membrane potential in MIN6 were observed by JC-1 fluorescent dying and the intensity has been analyzed, ratio of green fluorescence to red fluorescence can represent the loss of mitochondrial membrane potential(E) Data was expressed as means ± SEM (n=4).Compared with control group,**P<0.01; compared with PA group, ^##P<0.01.

Figure 2

iPLA₂β/CL/Opa1 play an important role in protecting cells from injury induced by PA. After treated with PA in normal cells and iPLA₂β over-expressed cells, mitochondria were isolated from cells and the protein was extracted from mitochondria. iPLA₂β was analyzed by Western blot and analyzed(A). Distribution of iPLA₂β was detected by fluorescence microscopy. Mitochondria was marked by Mito-Tracker Red and iPLA₂β was marked by Green fluorescence respectively, then the two colors were merged to observe the iPLA₂β's distribution and the intensity of yellow area has been analyzed via IOD/AREA (B-C) Data was expressed as means ± SEM (n=4). Compared with control group,**P<0.01; compared with PA group, ^##P<0.01. Lower limit of quantitation(LLOQ) and standard sample were detected to ensure the accuracy of measurement (D,E). The Cardiolipin content of mitochondria isolated from cells was observed by HPLC/MS(F). The content of CL in samples were detected by high resolution HPLC/MS and analyzed later(G). Data was expressed as means ± SEM (n=4). Compared with control group, **P<0.01; Compared with PA group, ^##P<0.01. Opa1 was detected by Western blot and analyzed(H). Data was expressed as means ± SEM (n=4). Compared with control group, **P<0.01; Compared with PA group, ^#P<0.05.

Figure 3

Anti-apoptotic activity, MMP maintenance and insulinotropic effect of BBR partly diminished by iPLA₂β silence. The influence of BBR's anti-apoptosis function and the block of silence iPLA₂β were assessed by MTT assay(A). Data was expressed as means ± SEM (n=6). Compared with control group, **P<0.01; compared with PA group, ^##P<0.01; compared with BBR treated group in vector cells, ^xP<0.05. Basal insulin release and content were measured in MIN6(B). Compared with control group, **P<0.01; compared with PA group, ^#P<0.05; compared with BBR treated group in vector cells, ^xxP<0.01. After using BBR to treat PA-induced apoptosis for 24h in normal cells and iPLA₂β silenced cells, the apoptosis were observed by Hoechst staining, bright blue means the apoptotic cells and marked with arrows(C). The protein was extracted from cells and cytosolic cytc and caspase3 were measured and analyzed by Western Blot(D). Data was expressed as means ± SEM (n=4). Compared with control group, *P<0.05; compared with PA group, ^#P<0.05, ^##P<0.01; compared with BBR treated group in vector cells, ^xP<0.05, ^xxP<0.01. Mitochondrial membrane potential in MIN6 were observed by JC-1 fluorescent dying and the intensity has been analyzed, ratio of green fluorescence to red fluorescence can represent the loss of mitochondrial membrane potential(E,F) Data was expressed as means ± SEM (n=4). Compared with control group,**P<0.01; compared with PA group, ^#P<0.01, compared with BBR treated group in vector cells, ^xP<0.05.

Figure 4

BBR may relieve the PA impairment via iPLA₂β/CL/Opa1 pathway. After using BBR to treat PA-induced apoptosis in vector cells and iPLA₂β silenced cells, mitochondria were isolated from cells and the protein was extracted from mitochondria. iPLA₂β was analyzed by Western blot and analyzed(A). Compared with control group, Data was expressed as means ± SEM (n=6), *P<0.05; compared with PA group, ^#P<0.05; compared with BBR treated group in vector cells, ^xxP<0.01. Distribution of iPLA₂β was detected by fluorescence microscopy. Mitochondria was marked by Mito-Tracker Red and iPLA₂β was marked by Green fluorescence respectively, then the two colors were merged to observe the iPLA₂β's distribution and the intensity of yellow area has been analyzed via IOD/AREA (B,C) Data was expressed as means ± SEM (n=4). Compared with control group,**P<0.01; compared with PA group, ^##P<0.01. The Cardiolipin content of mitochondria isolated from cells was observed by HPLC/MS analyzed(D). Data was expressed as means ± SEM (n=4). Opa1 was extracted from mitochondria detected by Western blot and analyzed(E). Data was expressed as means ± SEM (n=4). Compared with control group, *P<0.05; **P<0.05; compared with PA group, ^#P<0.05, ^##P<0.01; compared with BBR treated group in vector cells, ^xxP<0.01.

Figure 5

The effect of BBR and HGSD on higher weight, blood glucose, blood lipid and insulin resistance in db/db mice. Body weight was tested from 10-week to 13-week (A). At the end of 12 week, fasting blood glucose and random blood glucose were measured(B). Blood lipid included triglyceride(TG), total cholesterol(T-CHO) were measured by biochemical kits at 12-week old (C, D). Plasma glucose concentrations in different phases were measured in Oral Glucose Tolerance Test(OGTT) and Insulin tolerance test (ITT) at 12-week old(E-H). Data was expressed as means ± SEM (n=6). Compared with CON group, **P<0.01. Compared with DB/DB group, ^#P<0.05, ^##P<0.01.

Figure 6

The effect of BBR and HGSD on insulin secretion and islet morphology in db/db mice at the end of 12 week. Fasting blood insulin was measured in mice serum(A). After stimulation of glucose, plasma insulin concentrations were measured in different phases during glucose stimulated insulin secretion (GSIS) (B, C). Histology were used to observe islet morphology. Islet was severely damaged in T2D mice as the images marked with arrow. (D) (200×). Pancreatic insulin level in db/db mice are measured using Immunohistochemistry (E) (200×) and insulin was analyzed. IOD/AREA means the average insulin of islet cells (F). Data was expressed as means ± SEM (n=3). Compared with CON group, **P<0.01. Compared with DB/DB group, ^#P<0.05, ^##P<0.01.

Figure 7

The effect of BBR and HGSD on apoptosis of beta cells and the injury of mitochondria in db/db mice at the end of 12 week. Apoptotic cells were detected by TUNEL using brown staining(200×) and IOD/AREA has been calculated (A,B). Injury of mitochondria and insulin vesicle was showed by electron microscope images and marked with arrows.(C) Data was expressed as means ± SEM (n=3). Compared with CON group, **P<0.01. Compared with DB/DB group, ^#P<0.05, ^##P<0.01.

Figure 8

HGSD improved the deterioration of iPLA₂β/CL/Opa1 and mitigate apoptosis via regulating cytc and caspase3 in db/db mice islets. Mitochondria was isolated from mice pancreas and the protein was extracted from mitochondria. iPLA₂β was analyzed by Western blot and analyzed (A). The Cardiolipin content of mitochondria isolated from mice pancreas was observed by HPLC/MS and the content of CL in con and db/db mice were analyzed (B). Opa1 was analyzed by Western blot and analyzed(C). Data was expressed as means ± SEM (n=4). Compared with CON group, *P<0.05, **P<0.01. Compared with DB/DB group, ^#P<0.05. Protein was exacted from islets. Cytosolic cytc and caspase-3 were measured by Western blot and analyzed. (D-E). Data was expressed as means ± SEM (n=4). Compared with CON group, *P<0.05, **P<0.01. Compared with DB/DB group, ^#P<0.05.