Breviscapine ameliorates hypertrophy of cardiomyocytes induced by high glucose in diabetic rats via the PKC signaling pathway

Abstract

Aim: To investigate the influence of breviscapine on high glucose-induced hypertrophy of cardiomyocytes and the relevant mechanism in vitro and in vivo.

Methods: Cultured neonatal cardiomyocytes were divided into i) control; ii) high glucose concentrations; iii) high glucose+PKC inhibitor Ro-31-8220; iv) high glucose+breviscapine; or v) high glucose+NF-kappaB inhibitor BAY11-7082. Cellular contraction frequency and volumes were measured; the expression of protein kinase C (PKC), NF-kappaB, TNF-alpha, and c-fos were assessed by Western blot or reverse transcription-polymerase chain reaction (RT-PCR). Diabetic rats were induced by a single intraperitoneal injection of streptozotocin, and randomly divided into i) control rats; ii) diabetic rats; or iii) diabetic rats administered with breviscapine (10 or 25 mg x kg(-1) x d(-1)). After treatment with breviscapine for six weeks, the echocardiographic parameters were measured. All rats were then sacrificed and heart tissue was obtained for microscopy. The expression patterns of PKC, NF-kappaB, TNF-alpha, and c-fos were measured by Western blot or RT-PCR.

Results: Cardiomyocytes cultured in a high concentration of glucose showed an increased pulsatile frequency and cellular volume, as well as a higher expression of PKC, NF-kappaB, TNF-alpha, and c-fos compared with the control group. Breviscapine could partly prevent these changes. Diabetic rats showed relative cardiac hypertrophy and a higher expression of PKC, NF-kappaB, TNF-alpha, and c-fos; treatment with breviscapine could ameliorate these changes in diabetic cardiomyopathy.

Conclusion: Breviscapine prevented cardiac hypertrophy in diabetic rats by inhibiting the expression of PKC, which may have a protective effect in the pathogenesis of diabetic cardiomyopathy via the PKC/NF-kappaB/c-fos signal transduction pathway.

Figure 1

Breviscapine decreased the expression of PKC-α in high glucose cultured cardiomyocytes. Cardiomyocytes cultured in high glucose levels showed higher expression and activity of PKC-α compared with control group. After adding PKC inhibitor Ro-31-8220 (50 nmol/L) and breviscapine (10, 20, and 60 mmol/L), the expression and activity of PKC-α decreased. n=4 or 5. Mean±SEM. ^bP<0.05 vs control group. ^eP<0.05 vs high glucose group. (L: control glucose group; H: high glucose group; Ro: high glucose+PKC inhibitor Ro-31-8220 group; B-10: high glucose+10 mmol/L breviscapine; B-20: high glucose+20 mmol/L breviscapine; B-60: high glucose+60 mmol/L breviscapine group).

Figure 2

Breviscapine decreased the expression of PKC-β2 in high glucose cultured cardiomyocytes. Cardiomyocytes cultured in high glucose levels showed higher expression and activity of PKC-β2 compared with control group. After adding PKC inhibitor Ro-31-8220 (50 nmol/L) and breviscapine (10, 20, and 60 mmol/L), the expression and activity of PKC-β2 decreased. The results were shown in Figure 2 expressed as means±SEM (n=4 or 5). ^bP<0.05 vs control group. ^eP<0.05 vs high glucose group. (L: control glucose group; H: high glucose group; Ro: high glucose+PKC inhibitor Ro-31-8220 group; B-10: high glucose+10 mmol/L breviscapine; B-20: high glucose+20 mmol/L breviscapine; B-60: high glucose+60 mmol/L breviscapine group).

Figure 3

Breviscapine decreased the expression of NF-κB in high glucose cultured cardiomyocytes. Cardiomyocytes cultured in high glucose levels showed higher expression and increased activity of NF-κB compared with control group. After adding PKC inhibitor Ro-31-8220 (50 nmol/L), NF-κB inhibitor BAY11-7082 (5 μmol/L) and breviscapine (10, 20, and 60 mmol/L), the expression and activity of NF-κB decreased as shown in Figure 3. The results were expressed as means±SEM. n=4 or 5. ^bP<0.05 vs control group. ^eP<0.05 vs high glucose group. (L: control glucose group; H: high glucose group; Ro: high glucose+PKC inhibitor Ro-31-8220 group; B-10: high glucose+10 mmol/L breviscapine; B-20: high glucose+20 mmol/L breviscapine; B-60: high glucose+60 mmol/L breviscapine group; BAY: high glucose+NF-κB inhibitor BAY11-7082 group).

Figure 4

Breviscapine decreased the expression of TNF-α in high glucose cultured cardiomyocytes. Cardiomyocytes cultured in high glucose levels showed higher expression of TNF-α compared with control group. After adding PKC inhibitor Ro-31-8220 (50 nmol/L), NF-κB inhibitor BAY11-7082 (5 μmol/L) and breviscapine (10, 20, and 60 mmol/L), the expression of TNF-α decreased compared with the cardiomyocytes cultured in high glucose levels as shown in Figure 4. The results were expressed as means±SEM. n=4 or 5. ^bP<0.05 vs control group. ^eP<0.05 vs high glucose group. (L: control glucose group; H: high glucose group; Ro: high glucose+PKC inhibitor Ro-31-8220 group; B-10: high glucose+10 mmol/L breviscapine; B-20: high glucose+20 mmol/L breviscapine; B-60: high glucose+60 mmol/L breviscapine group; BAY: high glucose+NF-κB inhibitor BAY11-7082 group).

Figure 5

Breviscapine decreased the expression of c-fos in high glucose cultured cardiomyocytes. Cardiomyocytes cultured in high glucose levels showed higher expression of c-fos compared with control group. After adding PKC inhibitor Ro-31-8220 (50 nmol/L), NF-κB inhibitor BAY11-7082 (5 μmol/L) and breviscapine (10, 20, and 60 mmol/L), the expression of c-fos decreased as shown in Figure 5. The results were expressed as means±SEM. n=4 or 5. ^bP<0.05 vs control group. ^eP<0.05 vs high glucose group. (L: control glucose group; H: high glucose group; Ro: high glucose+PKC inhibitor Ro-31-8220 group; B-10: high glucose+10 mmol/L breviscapine; B-20: high glucose+20 mmol/L breviscapine; B-60: high glucose+60 mmol/L breviscapine group; BAY: high glucose+NF-κB inhibitor BAY11-7082 group).

Figure 6

Ultrastructure of myocardium in rats. Control rat (×20 000); diabetic rat (DM, ×20 000); diabetic rat treated with low dose of breviscapine (DM+LDB, ×20 000); diabetic rat treated with high dose of breviscapine (DM+HDB, ×20 000); 1: the loss and derangement of myofibrils; 2: swollen and distended of mitochondria; 3: dilated and fragmented sarcoplasmic reticulum.

Figure 7

Ultrastructure of myocardium in rats. Control rat (×50 000); diabetic rat (DM, ×50 000); diabetic rat treated with low dose of breviscapine (DM+LDB, ×50 000); diabetic rat treated with high dose of breviscapine (DM+HDB, ×50 000). 1: the loss and derangement of myofibrils; 2: swollen and distended of mitochondria; 3: dilated and fragmented sarcoplasmic reticulum.

Figure 8

Breviscapine decreased the expression of PKC-α in diabetic rats hearts. Diabetic rats (DM) showed higher expression of PKC-α in heart tissues compared with normal control ones (Control) analyzed by Western blot. Treatment with breviscapine reduced the expression of PKC-α in diabetic cardiomyopathy, the reduced expression of PKC-α was more obvious in diabetic rats treated with breviscapine 25 mg·kg⁻¹·d⁻¹ (DM+HDB) than those received 10 mg·kg⁻¹·d⁻¹ breviscapine (DM+LDB), β-actin was an endogenous control. Each bar represents the mean±SEM. n=8−10 rats for each group. ^bP<0.05 vs control group. ^eP<0.05 vs high glucose group.

Figure 9

Breviscapine decreased the expression of PKC-β2 in diabetic rats hearts. Diabetic rats (DM) showed higher expression of PKC-β2 in heart tissues compared with normal control rats (Control). Treatment with breviscapine could reduced the expression of PKC-β2 in diabetic cardiomyopathy, the reduced expression of PKC-β2 was more obvious in diabetic rats treated with 25 mg·kg⁻¹·d⁻¹ (DM+HDB) breviscapine than those received 10 mg·kg⁻¹·d⁻¹ breviscapine (DM+LDB), β-actin was an endogenous control. Each bar represents the mean±SEM. n=8−10 rats for each group. ^bP<0.05 vs control. ^eP<0.05 vs DM.

Figure 10

Breviscapine decreased the expression of NF-κB in diabetic rats hearts. Diabetic rats (DM) showed higher expression of NF-κB in heart tissues compared with normal control rats (Control). Treatment with breviscapine reduced the expression of NF-κB in diabetic cardiomyopathy, the reduced expression of NF-κB was more obvious in diabetic rats treated with breviscapine 25 mg·kg⁻¹·d⁻¹ (DM+HDB) than those received 10 mg·kg⁻¹·d⁻¹ breviscapine (DM+LDB), β-actin was an endogenous control. Each bar represents the mean±SEM. n=8−10 rats for each group. ^bP<0.05 vs control. ^eP<0.05 vs DM.

Figure 11

Breviscapine decreased the expression of TNF-α in diabetic rats hearts. Diabetic rats (DM) showed higher expression of TNF-α in heart tissues compared with normal control rats (Control) analyzed by RT-PCR. Treatment with breviscapine reduced the expression of TNF-α in diabetic cardiomyopathy, the reduced expression of TNF-α was more obvious in diabetic rats treated with breviscapine 25 mg·kg⁻¹·d⁻¹ (DM+HDB) than those received 10 mg·kg⁻¹·d⁻¹ breviscapine (DM+LDB), GAPDH was an endogenous control. Each bar represents the mean±SEM. n=8−10 rats for each group. ^bP<0.05 vs control. ^eP<0.05 vs DM.

Figure 12

Breviscapine decreased the expression of c-fos in diabetic rats hearts. Diabetic rats (DM) showed higher expression of c-fos in heart tissues compared with normal control rats (Control). Treatment with breviscapine reduced the expression of c-fos in diabetic cardiomyopathy, the reduced expression of c-fos was more obvious in diabetic rats treated with breviscapine 25 mg·kg⁻¹·d⁻¹ (DM+HDB) than those received 10 mg·kg⁻¹·d⁻¹ breviscapine (DM+LDB), β-actin was an endogenous control. Each bar represents the mean±SEM. n=8−10 rats for each group. ^bP<0.05 vs control. ^eP<0.05 vs DM.