Journal: Frontiers in Pharmacology

Article Title: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

doi: 10.3389/fphar.2018.01016

Figure Lengend Snippet: Ulinastatin inhibits the formation of osteoclasts and bone loss in OVX mice, meanwhile decreases the serum level of uPAR. (A) Distal femurs were embedded with paraffin and sliced up, then H&E and TRAP staining were performed. (B,C) Distal femurs were embedded with paraffin and sliced up, then TRAP staining were performed. The osteoclast number/bone surface (N.Oc/BS, N/mm) was quantified. Data are represented as mean ± SD. n = 10 and ∗ P < 0.05. (D) The serum level of uPAR was detected by ELISA. Data are presented as mean ± SD. n = 10 and ∗ P < 0.05.

Article Snippet: The serum level of uPAR was evaluated by mouse uPAR ELISA kit (Boster, Wuhan, China).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

doi: 10.3389/fphar.2018.01016

Figure Lengend Snippet: Ulinastatin reduces the expression of uPAR, NFATc1 and osteoclast marker genes induced by RANKL. C represents control group, R represents RANKL group, R + ulinastatin represents RANKL + 800 units/mL ulinastatin group. (A) Ulinastatin reduces RANKL-induced mRNA expression of cathepsin K, Trap, Rank, NFATc1, and uPAR. BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL), and treated with or without ulinastatin (800 units/mL) for 4 days. mRNA expression was detected by qRT-PCR. The qRT-PCR experiments have been repeated with different RNA preparations for 3 times independently. Data are represented as mean ± SD . ∗ P < 0.05 and ∗∗ P < 0.01. (B,C,D) Ulinastatin reduces RANKL-induced protein expression of uPAR, cathepsin K and Trap. BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL), and treated with or without ulinastatin (800 units/mL) for 2 or 4 days. Protein expression levels of uPAR, cathepsin K and Trap were examined by western blotting at the indicated times. The amount of loaded protein was 25 μg. The experiment was performed three times independently and GAPDH was used as a loading control. ∗ P < 0.05, ∗∗ P < 0.01 versus RANKL group.

Article Snippet: The serum level of uPAR was evaluated by mouse uPAR ELISA kit (Boster, Wuhan, China).

Techniques: Expressing, Marker, Control, Cell Culture, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

doi: 10.3389/fphar.2018.01016

Figure Lengend Snippet: Ulinastatin decreased actin ring formations and suppressed uPAR expression. C represents control group, R represents RANKL group, 100 ∼ 800 represents RANKL + 100 units/mL ulinastatin ∼ RANKL + 800 units/mL ulinastatin group. BMMs (1 × 10 4 cells/well) were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL), treated with four different concentrations of ulinastatin (100, 200, 400 and 800 units/mL) for 4 days. Then the cells were stained for actin ring assay and Immunofluorescence assay. Images were obtained by fluorescence microscopy. The experiment was performed three times independently.

Article Snippet: The serum level of uPAR was evaluated by mouse uPAR ELISA kit (Boster, Wuhan, China).

Techniques: Expressing, Control, Cell Culture, Staining, Immunofluorescence, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

doi: 10.3389/fphar.2018.01016

Figure Lengend Snippet: Knockdown of uPAR decreased RANKL-induced osteoclast and actin ring formation. NC represents siRNA negative control, Si-1, Si-2, and Si-3 represent siRNAs that silence three different fragment of uPAR. BMMs were seeded in 96-well plates and transfected with three siRNAs and NC siRNA, respectively, and cultured with M-CSF (30 ng/mL) and RANKL (50 ng/mL) for 5 days. (A) Immunofluorescence and (B) TRAP staining was performed then. The experiment was performed three times independently.

Article Snippet: The serum level of uPAR was evaluated by mouse uPAR ELISA kit (Boster, Wuhan, China).

Techniques: Knockdown, Negative Control, Transfection, Cell Culture, Immunofluorescence, Staining