Novel Dose Dependent Effects of Andrographolide on the Enhancement of Chondrogenesis and Osteogenesis in HumanSuprapatellar Fat Pad Derived Mesenchymal Stem Cells
Thitianan Kulsirirat1, Sittisak Honsawek2, Mariko Takeda-Morishita3, Nuttanan Sinchaipanid4, Jiraporn Leanpolchareanchai1, Korbtham Sathirakul1*
1Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand 10210
2Osteoarthritis and Musculoskeleton Research Unit, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand 10330
3Laboratory of Drug Delivery Systems, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Hyogo, Japan 650-8586
4Department of Manufacturing Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand 10400
*Corresponding author. Korbtham Sathirakul, Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand
E-mail address: pyksk2001@yahoo.com.sg
Keywords: Mesenchymal stem cells, Suprapatellar fat pad, Andrographolide, Osteoarthritis, Regenerative medicine
ABSTRACT:
Background and Purpose: Andrographolide (AG) is a labdane diterpenoid herb, which is isolated from the leaves ofAndrographis paniculate, and widely used for its potential medical properties. However, there are no reports on the effects of AG on the human suprapatellar fat pad of osteoarthritis patients. In the present study, our goal was to evaluate the innovative effects of AG on viability and Tri-lineage differentiation of human mesenchymal stem cells from suprapatellar fat pad tissues.
Experimental approach: The effects of Andrographolide on viability and differentiation of human primary mesenchymal stem cells obtained from human suprapatellar fat pad tissues were evaluated by staining assay. Moreover, the effects on molecular expression were quantitatively measured by mRNA expression in real time PCR.
Key Results: The results revealed that AG had no cytotoxic effects when the concentration was less than 12.5 µg/mL. Interestingly, AG had significantly enhanced, dose dependent, osteogenesis and chondrogenesis as evidenced by a significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, AG can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runx2 , OPN ,Sox9 , and Aggrecan in mesenchymal stem cells from human suprapatellar fat pad tissues. In contrast, AG suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and expression levels for adipogenic-specific genes forPPAR-γ2 and LPL .
Conclusions and Implications: These findings confirm that AG can specifically enhance osteogenesis and chondrogenesis of mesenchymal stem cells from human suprapatellar fat pad tissues. It has potential as a therapeutic agent derived from natural sources for regenerative medicine.
Abbreviations: AG, andrographolide; OA, osteoarthritis; IL-1β, interleukin-1β; MMPs, matrix metalloproteinases; BMPs, bone morphogenetic proteins; IGF1, insulin growth factor 1; TKA, total knee arthroplasty; SPFP, Suprapatellar fat pad; MSCs, mesenchymal stem cells; PBS, phosphate buffer solution; DMEM-HG, Dulbecco’s Modified Eagle Medium-high Glucose; FBS, fetal bovine serum; SPFP-MSCs, suprapatellar fat pad derived mesenchymal stem cells; ISCT, International Society for Cellular Therapy; DMSO, dimethyl sulfoxide; OD, optical density; IBMX, 3-isobutyl-methyl-xantine; TGF-β3, transforming growth factor-β3; ITS, insulin-transferrin-selenium; H&E, Haematoxylin & Eosin; CT, cycle threshold; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OM, osteogenic induction medium; Runx2 , Runt-related transcription factor 2; OPN , Osteopontin; AM, adipogenic induction medium; PPAR-γ2 , Peroxisome proliferator-activated receptor gamma 2; LPL , lipoprotein lipase; CM, chondrogenic medium; Sox9 ,SRY -related transcription factor 9; NF-κB, nuclear factor-kappaB; ERK, extracellular-signal-regulated kinase