Berberine protects rat heart from ischemia/reperfusion injury via activating JAK2/STAT3 signaling and attenuating endoplasmic reticulum stress

Abstract

Aim: Berberine (BBR), an isoquinoline-derived alkaloid isolated from Rhizoma coptidis, exerts cardioprotective effects. Because endoplasmic reticulum (ER) stress plays a pivotal role in myocardial ischemia/reperfusion (MI/R)-induced apoptosis, it was interesting to examine whether the protective effects of BBR resulted from modulating ER stress levels during MI/R injury, and to define the signaling mechanisms in this process.

Methods: Male rats were treated with BBR (200 mg · kg(-1) · d(-1), ig) for 2 weeks, and then subjected to MI/R surgery. Cardiac dimensions and function were assessed using echocardiography. Myocardial infarct size and apoptosis was examined. Total serum LDH levels and CK activities, superoxide production, MDA levels and the antioxidant SOD activities in heart tissue were determined. An in vitro study was performed on cultured rat embryonic myocardium-derived cells H9C2 exposed to simulated ischemia/reperfusion (SIR). The expression of apoptotic, ER stress-related and signaling proteins were assessed using Western blot analyses.

Results: Pretreatment with BBR significantly reduced MI/R-induced myocardial infarct size, improved cardiac function, and suppressed myocardial apoptosis and oxidative damage. Furthermore, pretreatment with BBR suppressed MI/R-induced ER stress, evidenced by down-regulating the phosphorylation levels of myocardial PERK and eIF2α and the expression of ATF4 and CHOP in heart tissues. Pretreatment with BBR also activated the JAK2/STAT3 signaling pathway in heart tissues, and co-treatment with AG490, a specific JAK2/STAT3 inhibitor, blocked not only the protective effects of BBR, but also the inhibition of BBR on MI/R-induced ER stress. In H9C2 cells, treatment with BBR (50 μmol/L) markedly reduced SIR-induced cell apoptosis, oxidative stress and ER stress, which were abolished by transfection with JAK2 siRNA.

Conclusion: BBR ameliorates MI/R injury in rats by activating the AK2/STAT3 signaling pathway and attenuating ER stress-induced apoptosis.

Figures

Figure 1

Chemical structure of berberine (BBR). Molecular weight: 336. Molecular formula: C20H18NO4+.

Figure 2

BBR or AG490 treatment had no significant toxic effect on sham-operated rat hearts. Normal rats pretreated with BBR or AG490 were subjected to the sham operation. After 24 h of reperfusion, echocardiography was performed. After 6 h of reperfusion, the myocardial infarct size and apoptosis index were evaluated. (A) Representative M-mode echocardiographic images. (B) Left ventricular ejection fraction (LVEF). (C) Left ventricular fractional shortening (LVFS). (D) Representative heart section images. The Evans blue-stained areas (blue) indicate the non-ischemic/reperfused area; the TTC-stained areas (red) indicate ischemic but viable tissue; and the Evans blue/TTC-unstained (negative) areas (white) indicate infarcted myocardium. (E) Representative images of apoptotic cardiomyocytes by TUNEL staining. The green fluorescence shows the TUNEL-positive nuclei; the blue fluorescence shows the nuclei of all cardiomyocytes; original magnification, ×400. (F) The myocardial infarct size expressed as the percentage of area-at-risk. (G) Percentage of TUNEL-positive nuclei. (H) Representative blots. (I) p-JAK2/JAK2 ratio. (J) p-STAT3/STAT3 ratio. BBR, berberine; AG, AG490. The results are expressed as the mean±SEM. n=8/group. cP<0.01 vs the sham group. fP<0.01 vs the sham+BBR group.

Figure 3

BBR effectively reduced the MI/R injury, whereas AG490 treatment blocked this effect. Normal rats pretreated with BBR or AG490 were subjected to the MI/R operation. After 24 h of reperfusion, echocardiography was performed. After 6 h of reperfusion, the myocardial infarct size and apoptotic index were evaluated. (A) Representative M-mode echocardiographic images. (B) Left ventricular ejection fraction (LVEF). (C) Left ventricular fractional shortening (LVFS). (D) Representative images of apoptotic cardiomyocytes by TUNEL staining. The green fluorescence shows the TUNEL-positive nuclei; the blue fluorescence shows the nuclei of all cardiomyocytes; original magnification, ×400. (E) Percentage of TUNEL-positive nuclei. (F) Representative heart section images. The Evans blue-stained areas (blue) indicate the non-ischemic/reperfused area; the TTC-stained areas (red) indicate ischemic but viable tissue; and the Evans blue/TTC-unstained (negative) areas (white) indicate infarcted myocardium. (G) The myocardial infarct size expressed as the percentage of area-at-risk. BBR, berberine; AG, AG490. The results are expressed as the mean±SEM. n=8/group. cP<0.01 vs the MI/R+V group; fP<0.01 vs the MI/R+BBR group.

Figure 4

BBR effectively ameliorated cardiac necrosis and oxidative stress, whereas AG490 treatment blocked these effects. Normal rats treated with BBR or AG490 were subjected to the MI/R operation. All measurements were performed after 6 h of reperfusion. (A) Serum LDH levels. (B) Serum CK levels. (C) Cardiac superoxide generation. (D) gp91phox expression. Top images: representative blots. (E) Myocardial MDA contents. (F) Myocardial SOD contents. BBR, berberine; AG, AG490. The results are expressed as the mean±SEM. n=8/group. cP<0.01 vs the MI/R+V group; fP<0.01 vs the MI/R+BBR group; iP<0.01 vs the MI/R+BBR+AG group.

Figure 5

BBR up-regulated cardiac JAK2/STAT3 signaling and down-regulated myocardial apoptosis, whereas AG490 treatment blocked these effects. Normal rats pretreated with BBR or AG490 were subjected to the MI/R operation. All measurements were performed after 4 h of reperfusion. (A) Representative blots. (B) p-JAK2/JAK2 ratio. (C) p-STAT3/STAT3 ratio. (D) Caspase-3 expression. (E) Bcl-2 expression. (F) Bax expression. BBR, berberine; AG, AG490. The results are expressed as the mean±SEM. n= 8/group. cP<0.01 vs the MI/R+V group; fP<0.01 vs the MI/R+BBR group; iP<0.01 vs the MI/R+BBR+AG group.

Figure 6

BBR significantly attenuated the PERK/eIF2α/ATF4-mediated myocardial ER stress, whereas AG490 treatment blocked this effect. Normal rats pretreated with BBR or AG490 were subjected to the MI/R operation. Western blotting analysis was performed after 4 h of reperfusion. (A) Representative blots; (B) p-PERK/PERK ratio; (C) p-eIF2α/eIF2α ratio; (D) ATF4 expression; (E) CHOP expression. BBR, berberine; AG, AG490. The results are expressed as the mean±SEM. n=8/group. cP<0.01 vs the MI/R+V group; eP<0.05, fP<0.01 vs the MI/R+BBR group; iP<0.01 vs the MI/R+BBR+AG group.

Figure 7

In the cultured H9C2 cells subjected to SIR treatment, BBR significantly reduced the cellular apoptosis, whereas JAK2 siRNA markedly blocked these effects. H9C2 cells were exposed to BBR treatment for 4 h at the concentrations of 0.5, 5, 50, or 500 μmol/L. Then, cell viability was measured using MTT. Then, the cells were exposed to the SIR treatment with or without BBR or JAK2 siRNA treatment. (A) The cardiomyocyte viability was calculated by dividing the optical density of samples by the optical density of sham control. (B) The cardiomyocyte viability was evaluated using MTT. (C) Representative photomicrographs of TUNEL staining. The green fluorescence shows the TUNEL-positive nuclei; the blue fluorescence shows the nuclei of all cardiomyocytes; original magnification, ×200. (D) Percentage of TUNEL-positive nuclei. The results are expressed as the mean±SEM of 8 separate experiments with triplicate samples for each experimental condition within each experiment. cP<0.01 vs the control group; fP<0.01 vs the SIR group; iP<0.01 vs the SIR+BBR group.

Figure 8

In the cultured H9C2 cells subjected to the SIR treatment, BBR effectively reduced the oxidative stress, whereas the JAK2 siRNA blocked this effect. The cells were exposed to the SIR treatment with or without BBR or JAK2 siRNA treatment. Then, the cardiomyocyte oxidative stress markers were measured. (A) Cardiomyocyte superoxide generation. (B) gp91phox expression; top images: representative blots. The results are expressed as the mean±SEM of 8 separate experiments with triplicate samples for each experimental condition within each experiment. cP<0.01 vs the SIR group; fP<0.01 vs the SIR+BBR group; iP<0.01 vs the SIR+BBR+JAK2 siRNA group.

Figure 9

In the cultured H9C2 cells subjected to the SIR treatment, BBR effectively up-regulated the cardiac JAK2/STAT3 signaling and down-regulated the myocardial apoptosis, whereas JAK2 siRNA blocked these effects. The cells were exposed to the SIR treatment with or without BBR or JAK2 siRNA treatment. Then, cardiomyocyte protein expression was measured. (A) Representative blots. (B) p-JAK2/JAK2 ratio. (C) p-STAT3/STAT3 ratio. (D) Caspase-3 expression. (E) Bcl-2 expression. (F) Bax expression. The results are expressed as the mean±SEM. n=8/group. cP<0.01 vs the SIR group; fP< 0.01 vs the SIR+BBR group; iP<0.01 vs the SIR+BBR+JAK2 siRNA group.

Figure 10

In the cultured H9C2 cells subjected to the SIR treatment, BBR significantly attenuated the PERK/eIF2α/ATF4-mediated myocardial ER stress, whereas JAK2 siRNA abolished this effect. (A) Representative blots. (B) p-PERK/PERK ratio. (C) p-eIF2α/eIF2α ratio. (D) ATF4 expression. (E) CHOP expression. The results are expressed as the mean±SEM. n=8/group. cP<0.01 vs the SIR group; fP<0.01 vs the SIR+BBR group; iP<0.01 vs the SIR+BBR+JAK2 siRNA group.