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