An ethanolic extract of Lindera obtusiloba stems, YJP-14, improves endothelial dysfunction, metabolic parameters and physical performance in diabetic db/db mice

Abstract

Lindera obtusiloba is a medicinal herb traditionally used in Asia for improvement of blood circulation, treatment of inflammation, and prevention of liver damage. A previous study has shown that an ethanolic extract of Lindera obtusiloba stems (LOE) has vasoprotective and antihypertensive effects. The possibility that Lindera obtusiloba improves endothelial function and metabolic parameters in type 2 diabetes mellitus (T2DM) remains to be examined. Therefore, the aim of the present study was to determine the potential of LOE to prevent the development of an endothelial dysfunction, and improve metabolic parameters including hyperglycemia, albuminuria and physical exercise capacity in db/db mice, an experimental model of T2DM. The effect of LOE (100 mg/kg/day by gavage for 8 weeks) on these parameters was compared to that of an oral antidiabetic drug, pioglitazone (30 mg/kg/day by gavage). Reduced blood glucose level, body weight and albumin-creatinine ratio were observed in the group receiving LOE compared to the control db/db group. The LOE treatment improved endothelium-dependent relaxations, abolished endothelium-dependent contractions to acetylcholine in the aorta, and normalized the increased vascular oxidative stress and expression of NADPH oxidase, cyclooxygenases, angiotensin II, angiotensin type 1 receptors and peroxynitrite and the decreased expression of endothelial NO synthase in db/db mice. The angiotensin-converting enzyme (ACE) activity was reduced in the LOE group compared to that in the control db/db group. LOE also inhibited the activity of purified ACE, COX-1 and COX-2 in a dose-dependent manner. In addition, LOE improved physical exercise capacity. Thus, the present findings indicate that LOE has a beneficial effect on the vascular system in db/db mice by improving endothelium-dependent relaxations and vascular oxidative stress most likely by normalizing the angiotensin system, and also on metabolic parameters, and these effects are associated with an enhanced physical exercise capacity.

Conflict of interest statement

Competing Interests: Jung-Ok Lee, Moonkyu Kang and Kyoung Rak Kim are all employees of Hanwha Pharma. Co., Ltd, Chuncheon, Republic of Korea, whose company provided funding toward this study. Dong Hyun Park is an employee of YangJi Chemicals, Suwon, Republic of Korea, which is a daughter company of Hanwha. Hanwha has filed a European and US patent on YJP14 and endothelial dysfunction. The Korean FDA have approved further clinical studies regarding YJP-14 and endothelial dysfunction in diabetes. At present there are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. LOE and pioglitazone treatments improve blood glucose levels in db/db mice.

(A) Non-fasting and (B) fasting blood glucose levels from week 6 until week 14. All groups were fasted for 4 h before blood samples were taken. Values are shown as means ± S.E.M. (n = 13–14). *P<0.05 indicates a significant difference versus db/db group.

Figure 2. LOE and pioglitazone treatments improve endothelium-dependent relaxations and reduce endothelium-dependent contractile responses to acetylcholine in aortic rings of db/db mice.

(A) Concentration-relaxation curves to Ach in aortic rings with endothelium, (B) concentration-contraction curves to Ach in the presence of Nw-nitro L-arginine (an inhibitor of eNOS) in rings with endothelium, and (C) concentration-relaxation curves to sodium nitroprusside (a NO donor) in rings with endothelium in the presence of Nw-nitro L-arginine and indomethacin to avoid the formation of endothelium-derived vasoactive NO and prostanoids, respectively. Values are shown as means ± S.E.M. (n = 5–6). *P<0.05 indicates a significant difference between db/+ group versus db/db group, db/db+Pio group or db/db+LOE group, and † db/db+Pio group versus db/db+LOE group.pone.0065227.g003.tif

Figure 3. LOE inhibits the activity of cyclooxygenases.

(A) Cyclooxygenase-derived vasoactive prostanoids blunt endothelium-dependent relaxations to Ach in aortic rings of the db/+, db/db and db/db+Pio groups but not in the db/db+LOE group. Concentration-dependent relaxations induced by Ach in aortic rings with endothelium in the absence and presence of indomethacin (an inhibitor of cyclooxygenases). Values are shown as means ± S.E.M. (n = 5–7). *P<0.05 indicates a significant difference versus the respective control. (B) LOE inhibits COX-1 and COX-2 activity in vitro in a concentration-dependent manner. Values are shown as means ± S.D. (duplicate determinations).

Figure 4. Effect of LOE and pioglitazone treatments on vascular oxidative stress, and the expression of eNOS, NADPH oxidase subunits (NOX-1 and p47phox) and cyclooxygenases (COX-1 and COX-2) in aortic sections of db/db mice.

The formation of reactive oxygen species (ROS) was determined using the redox-sensitive probe dihydroethidine, and the expression of target proteins by immunofluorescent staining. Top: representative immunofluorescent staining; bottom: corresponding cumulative data. The lumen is on the right side of each image, and the scale bar corresponds to 50 µm. Values are shown as means ± S.E.M. (n = 6–7). *P<0.05 indicates a significant difference between db/+ group versus db/db group, db/db+Pio group or db/db+LOE group, †db/db group versus db/db+Pio group or db/db+LOE group, and #db/db+Pio group versus db/db+LOE group.

Figure 5. Effect of LOE and pioglitazone treatments on the expression of angiotensin II (Ang II) and angiotensin II type 1 receptors (AT1R) in aortic sections of db/db mice.

The expression of Ang II and AT1R was assessed by immunofluorescent staining. Top: representative immunofluorescent staining; bottom: corresponding cumulative data. The lumen is on the right side of each image, and the scale bar corresponds to 50 µm. Values are shown as means ± S.E.M. (n = 6–7). *P<0.05 indicates a significant difference between db/+ group versus db/db group, db/db+Pio group or db/db+LOE group, †db/db group versus db/db+Pio group or db/db+LOE group, and #db/db+Pio group versus db/db+LOE group.

Figure 6. LOE reduces plasma angiotensin converting enzyme (ACE) activity in db/db mice (A), and inhibits the activity of purified ACE (B).

Values are shown as means ± S.E.M. n = 6–7 (A) and 3 (B). *P<0.05 indicates a significant difference between db/+ group versus db/db group, db/db+Pio group or db/db+LOE group, †db/db group versus db/db+Pio group or db/db+LOE group, and #db/db+Pio group versus db/db+LOE group.

Figure 7. LOE but not pioglitazone improves slightly but significantly the physical exercise capacity of db/db mice.

The physical performance was assessed using a treadmill. (A) Time to exhaustion, (B) maximal distance run before exhaustion. Values are shown as means ± S.E.M. (n = 13–14). *P<0.05 indicates a significant difference versus db/db group.