Andrographolide and 14-deoxy-11,12-didehydroandrographolide from Andrographis paniculata attenuate high glucose-induced fibrosis and apoptosis in murine renal mesangeal cell lines
Graphical abstract
This study showed that andrographolide (AP1) and 14-deoxy-11,12- didehydroandrographolide (AP2), which were isolated from Andrographis paniculata reduced the induced ECM fibronectin, cytokine TGF-β, plasminogen activator inhibitor-1, and apoptosis marker caspase-3 in hyperglycemic MES-13 cells.
Introduction
Andrographis paniculata Nees (Acanthaceae) is a medicinal plant widely cultivated in tropical regions in Asia. Traditionally it is used for the treatment of cold, fever, laryngitis and infection in many Asian countries. Extract of the plant is reported to possess immunological, antibacterial, anti-inflammatory, antithrombotic, hepatoprotective, anti-hypertensive, and anti-diabetic activities (Mishra et al., 2007). The extract of the plant is a rich source for flavonoids and labdane diterpenoids (Rao et al., 2004, Geethangili et al., 2008). Of the diterpenoids, andrographolide (AP1) and 14-deoxy-11,12-didehydroandrographolide (AP2) were isolated from the 95% alcoholic extract obtained from the aerial parts of Andrographis paniculata, following the extraction and isolation procedures we described previously (Rao et al., 2004, Geethangili et al., 2008). AP1 is a diterpene containing a β-lactone ring connected to a decalin ring system via an unsaturated C-2 moiety. AP2 is different from AP1 in dehydrogenation at 11, 12 positions in the diterpene, and deoxygenation in the 14 position of the β-lactone ring (Fig. 1).
AP1 regulates several factors of the innate as well as the adaptive immune response (Chiou et al., 1998, Burgos et al., 2005, Iruretagoyena et al., 2005, Qin et al., 2006), protects the cardiovascular system (Thisoda et al., 2006, Woo et al., 2008), possesses anti-hepatotoxic activities (Kapil et al., 1993, Akowuah et al., 2008), and shows significant anti-viral and anti-cancer activities (Kumar et al., 2004, Kim et al., 2005, Wiart et al., 2005, Ji et al., 2007, Sheeja and Kuttan, 2007, Geethangili et al., 2008, Shi et al., 2008, Zhao et al., 2008). AP2 shows a similar spectrum of activities as found in AP1 including antioxidant (Zhang et al., 1998, Kumar et al., 2004), cardioprotective (Zhang and Tan, 1999, Yoopan et al., 2007, Woo et al., 2008), anti-viral (Wiart et al., 2005), and antitumor abilities (Kumar et al., 2004, Geethangili et al., 2008). Effect of anti-diabetes was reported for AP1. It enhances the uptake of glucose and level of GLUT4 (Yu et al., 2003), and causes reduction in peak blood glucose in diabetic rats (Subramanian et al., 2008). Its action mechanism is suggested via activating α1-angiotensin II receptor to enhance the secretion of β-endorphin which can stimulate the opioid micro-receptors to reduce hepatic gluconeogenesis and to enhance the glucose uptake in soleus muscle (Yu et al., 2008). It is suggested that AP2 works via adrenoceptors, autonomic ganglia receptor and angiotensin-converting enzyme (Zhang et al., 1998).
Diabetic nephropathy (DN) is characterized by renal hypertrophy, accumulation of extracellular matrix components, glomerulosclerosis, tubular atrophy and interstitial fibrosis. DN is due to longstanding diabetes mellitus, and is a prime cause for dialysis in many Western countries. Glomerulosclerosis leads to fluid filtration deficits, hypertension, arteriosclerosis of the renal artery, and proteinuria. For the existing therapy, the first line of treatment is controlling blood-glucose level, however anti-hypertension drugs delay the progression of end-stage nephropathy (Salvetti et al., 1999). At the moment angiotensin-converting enzyme (ACE) and angiotensin II receptor inhibitor drugs (ARB), both treating the vascular system by its renal vasodilatation activities resulting in increased renal blood flow and dilatation of the efferent arterioles, are suggested drugs. However, renal protection of drug faces the challenge of side effect (dry cough), as well as patient resistance. Renal protection for DN benefits from combination of drugs (Codreanu et al., 2005) and multifactoral treatment (Fioretto and Solini, 2005), therefore development of a new drug or food supplement that acts on multiple factor of DN, is very much needed.
Herbal extracts used in the ancient culture often act on multiple aspects of the disease and were passed on because of their excellent activities. It is possible that extract of Andrographis paniculata Nees was similarly versatile. While both hyperglycemia and hypertension contribute to the diabetic nephropathy, effects of AP1 and AP2 for ameliorating both were reported (Yu et al., 2003, Hsu et al., 2004, Reyes et al., 2006, Yoopan et al., 2007, Yu et al., 2008). Although the constituents of Andrographis paniculata are reported to have anti-diabetic potency, the precise active compounds responsible for diabetic nephropathic activity of this plant have not been clearly identified. In the continuation of our search for the bioactive natural products (Rao et al., 2006, Rao et al., 2009, Lee et al., 2009, Tzeng et al., 2009), we are intended to test how AP1 and its related compound AP2 from Andrographis paniculata act on high glucose afflicted renal cells. High blood glucose-induced advanced glycation end product and transforming growth factor (TGF)-β secretion of tubular cells, and subsequent fibronectin/extracellular matrix (ECM) deposition and cell death are proposed for the etiopathology of DN (Ayo et al., 1990, Ziyadeh et al., 1994, Oh et al., 1998, Ha and Lee, 2000). Plasminogen activator inhibitor type 1 (PAI-1) inhibits fibrinolysis and ECM degradation contributes to TGF-β mediated ECM deposition (Rerolle et al., 2000). Part of the DN pathology could be reproduced in vitro by culturing tubular cell lines or primary mesangial cells in a high glucose concentration. Induction of increased TGF-β, PAI 1, and fibronectin secretion could be measured in the culture medium, and cell apoptosis measured by level of cleaved caspase-3 (MacKay et al., 1988, Wolf et al., 1992, Kang et al., 2003). We took advantage of this system to test the single compounds AP1 and AP2 from Andrographis paniculata for their possible activities on DN. The results are reported herein.
Section snippets
Extraction and isolation of AP1 and AP2
The compounds andrographolide (AP1) and 14-deoxy-11,12-didehydroandrographolide (AP2) (Fig. 1) were obtained from the 95% alcoholic extract obtained from the air-dried and powdered aerial parts of Andrographis paniculata. The extraction and isolation procedures were described in our previous reports (Rao et al., 2004, Geethangili et al., 2008).
Murine mesangial cell (MMC) culture
SV40-transformed MMCs (MES-13) were obtained from the Bioresource Collection and Research Center, Food Industry Research and Development Institute,
AP1 and AP2 protected cells from high glucose-induced apoptosis
The cells were treated with 25 mM glucose (‘high glucose’group) in a DMEM/Ham's F-12 based serum free medium, or high glucose with the addition of 1–20 μM AP1 or AP2, for 1 day, before caspase-3 levels were examined. With high glucose treatment, the cleaved caspase-3 level was up-regulated to 2.5 times of the ‘normal glucose’ treatment (100 mg/dl). AP1 at 10 μM down-regulates the level of activated caspase-3 to be lower than the level with high glucose treatment (p < 0.05), and at 20 μM the inhibition
Discussion
High glucose increase the level of reactive oxygen species in mesangial cells (Lee et al., 2003, Yamagishi et al., 2005), which in turn stimulate downstream signaling pathways to TGF-β secretion and extracellular matrix (ECM) deposition (Yamagishi et al., 2003), and/or to nuclear factor (NF)-κB mediated apoptosis (Kang et al., 2003). Thus control of blood-glucose level prevents the progression to diabetic nephropathy. In glomerusclerosis, reduction of intraglomerular capillary pressure reduces
Conclusion
In conclusion, for the first time our data demonstrated that pure compounds andrographolide and 14-deoxy-11,12-didehydroandrographolide from Andrographis paniculata are capable of reducing the production of extracellular molecule fibronectin, cytokine TGF-β, and plasminogen activator inhibitor-1, as well as apoptosis marker caspase-3 in a diabetic nephropathy cell line MES-13 cells. This indicates that apart from anti-hyperglycemia and anti-hypertension, compounds of Andrographis paniculata are
Acknowledgment
This study was supported by National Science Council of Taiwan, ROC (NSC 99-2811-M-324-003).
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