The effects of ginseng (Panax quinquefolius) treatment on chronic diabetic complications Subhrojit Sen & Subrata Chakrabarti Dept. of Pathology University of Western Ontario

Introduction Diabetes mellitus is a heterogeneous clinical syndrome in which the central feature is a chronic elevation of the blood glucose concentration. It occurs due to deficiency of insulin (absolute) or resistance to insulin (relative). 40% diabetics develop long term complications affecting various organs including retina, heart and kidney.

Types of diabetes Type I diabetes Results from the body's failure to produce insulin. It is estimated that 5-10% of Americans who are diagnosed with diabetes have type 1 diabetes. Type II diabetes Results from insulin resistance (a condition in which the body fails to properly use insulin), combined with relative insulin deficiency. Most Americans who are diagnosed with diabetes have type 2 diabetes. Gestational diabetes Occurring during pregnancy. Gestational diabetes affects about 4% of all pregnant women - about 135,000 cases in the United States each year.

Chronic diabetic complications Diabetic Cardiomyopathy CHRONIC COMPLICATIONS OF DIABETES Retinopathy Nephropathy Chronic diabetic complications Diabetic Cardiomyopathy Diabetic Neuropathy Others

Progressive Background Retinopathy Progressive Background i) Neovascularization i) increased capillary permeability ii) closure of retinal capillaries iii) vascular leakage retinal edema iv) accumulation of lipids v) retinal ischemia

Nephropathy: Microalbuminuria elevated blood pressure progresses to macroproteinuria decline in renal function.

Diabetic Neuropathy: Peripheral Focal

Diabetic cardiomyopathy: microvascular narrowing micro aneurysm (increased risk of rupture) hypertrophy defects in collagen structure myocardial fibrosis perivascular fibrosis

Signalling pathways involved in diabetic complications Hyperglycemia Aldose reductase pathway Protein kinase C Oxidative stress AGE/RAGE Growth factors (TGF-β, VEGF etc.) ET vasoconstriction Permeability PKC, MAPK Blood flow Growth Factors Extracellular Matrix Protein Angiogenesis Khan et al, Current Vascular Pharmacology, 2006, Vol 4, No. 1

Ginseng has been shown to have antioxidant effect Ginseng has been shown to have antioxidant effect. Prevents oxidative lipid peroxidation and kidney damage in rats. Ginsenosides are the active components of ginseng found mainly in the roots. Structurally, ginsenosides are divided into two types: protopanaxadiol ginsenosides (PPDGs) and protopanaxatriol ginsenosides (PPTGS). North American Ginseng

Objective The present study will investigate the effects of ginseng on glucose-induced changes in human vascular endothelial cells (HUVECs) biochemical, functional and structural changes in heart, kidney and retina of insulin-dependent (IDDM) and noninsulin-dependent (NIDDM) mouse model.

Experiments on HUVECs HUVECs were incubated with 25 mM glucose for 24 hrs. Aqueous and ethanolic extracts of ginseng were given at conc. of 5 mg/ml. Glucose-induced i) fibronectin (FN) ii) FN protein level iii) EDB+FN iv) Vascular Endothelial Growth Factor (VEGF) v) endothelin (ET-1) mRNA levels were measured.

Animal models used in the present study Type 1 diabetes STZ mice Type 2 diabetes db/db mice

Experimental Design Mice Type 1 STZ induction (IP, 65 mg/kg BW) Type 2 db/db mice Control Diabetic DC Diabetic + Ginseng (DT) (200mg/Kg BW for 2 months after onset of diabetes by oral gavage) Control NC Diabetic db/db NC

Experimental parameters Body weight Heart weight Biochemical parameters from - blood : a) Glucose - urine a) Albumin by ELISA b) Creatinine by ALKALINE PICRATE METHOD

Preliminary results on HUVEC FN-mRNA levels in Endothelial cells 0.5 1 1.5 2 NG HG NGI HGI NGGE HGGE NGGA HGGA Groups mRNA/18s RNA * EDB+FN mRNA levels in endothelial cells 2.5 mRNA/18 sRNA NG: 5mM glucose; HG: 25mM High glucose; I = insulin; GA: aqueous extract of ginseng; GE = ethanolic extract of ginseng. * = significantly different from other groups ALCOHOLIC EXTRACTS APPEARED TO BE MORE EFFECTIVE

FN protein levels in endothelial cells * ** NG: 5mM glucose; HG: 25mM High glucose; I = insulin; GA: aqueous extract of ginseng; GE = ethanolic extract of ginseng. * = significantly different from other groups, * *= significantly different with HG and HGGA. ALCOHOLIC EXTRACTS APPEARED TO BE MORE EFFECTIVE

* * NG: 5mM glucose; HG: 25mM High glucose; I = insulin; GA: aqueous extract of ginseng; GE = ethanolic extract of ginseng. * = significantly different from other groups ALCOHOLIC EXTRACTS APPEARED TO BE MORE EFFECTIVE

Preliminary results on diabetic mouse model Groups Body wt. (gms) Heart (mgm) Fasting Blood Glucose mmol/L Urinary Albumin (μg/ml) Urinary Creatinine (mg/ml) NC 28±5.5 124.1±10.2 8.86±2.7 0.128±0.01 0.225±0.05 DC 24±1.6 121.12±2.5 27.4±5.5* 0.182±0.02* 0.350±0.025* DT (ginseng treated) 27.2±1.7 133.96±5.5 15.28±8.5 0.138±0.007 0.235±0.02 db/db 44.4±1.5* - 29.08±2.5* 0.184±0.02 0.312±0.01* THE ABOVE PARAMETERS WERE MEASURED AFTER 2 MONTHS OF ONSET OF DIABETES IN CASE OF IDDM MODEL. THE PARAMETERS IN db/db MICE WERE MEASURED AT 12 WEEKS OF AGE (Duration of diabetes 4 weeks).

Conclusion In HUVECs: Glucose caused upregulation of FN, EDB+FN, VEGF and ET-1. Treatment with both aqueous and ethanolic extract of ginseng (5mg/ml) caused diminution of the above parameters. In STZ mice: Treatment with ethanolic extract of ginseng (200mg/kg BW/day) for 2 months caused significant diminution of fasting blood glucose, urinary albumin and creatinine levels. In db/db mice: Enhancement of fasting blood glucose, urinary albumin and creatinine levels occurred after 12 weeks of age.

Acknowledgement I sincerely acknowledge the guidance of my supervisor, Dr. Subrata Chakrabarti and Dr. Ed Lui, Dept. of Physiology & Pharmacology. I also acknowledge the guidance and help provided by all members of my laboratory. Shali Chen 2) Biao Feng 3) Jane Chiu 4) Kara McArthur 5) Yuexiu Wu 6) Chunyan Wang 7) Biju George