Cysteine protease

Papain is a peptidase C1 family cysteine protease that finds application in the culinary, pharmaceutical, textile, and cosmetic sectors.

Murine papain-induced emphysema is a model that reproduces many of the features found in patients. Bone marrow-derived mononuclear cells (BMMC) have already been used to repair the alveolar epithelium in respiratory diseases, but not in the papain model. Thus, we hypothesized that BMMC could prevent the pathophysiological processes in papain-induced experimental emphysema. Female BALB/c mice received intratracheal instillation of 50 μL of saline (S groups) or papain (P groups, 10 IU/50 μl of saline) on days 1 and 7 of the experimental protocol. On the 14th day, 2 × 106 BMMC of male BALB/c mice (SC21 and PC21) or saline (SS21 and PS21) were injected by the jugular vein. Analyses were done on days 14 (S14 and P14) and 21 (SS21, PS21, SC21, and PC21) of the protocol. qPCR evaluated the presence of the Y chromosome in the lungs of BMMC recipient animals. Functional residual capacity (FRC), alveolar diameter, cellularity, elastic fiber content, concentrations of TNF-α, IL-1β, IL-6, MIP-2, KC, IFN-γ, apoptosis, mRNA expression of the dual oxidase (DUOX1 and DUOX2), production of H2O2 and DUOX activity were evaluated in lung tissue. We did not detect the Y chromosome in recipients' lungs. FRC, alveolar diameter, polymorphonuclear cells (PMN) and levels of KC, MIP-2, and IFN-γ increased in P14 and PS21 groups; the changes in the latter were reverted by BMMC. TNF-α, IL-1β e IL-6 were similar in all groups. The amount of elastic fibers was smaller in P14 and PS21 than in other groups, and BMMC did not increase it in PC21 mice. PS21 animals showed increased DUOX activity and mRNA expression for DUOX1 and 2. Cell therapy reverted the activity of DUOX and mRNA expression of DUOX1. BMMC reduced mRNA expression of DUOX2. Apoptosis index was elevated in PS21 mice, which was reduced by cell therapy in PC21. Static compliance, viscoelastic component of elastance and pressure to overcome viscoelasticity were increased in P14 and PS21 groups. These changes and the high resistive pressure found on day 21 were reverted by BMMC. In conclusion, BMMC showed potent anti-inflammatory, antiapoptotic, antioxidant, and restorative roles in papain-triggered pulmonary emphysema.[1]

The animals were sedated by inhalation of sevoflurane, weighed (model BR), and saline or papain was injected into the trachea using an insulin syringe. This procedure last about 3 min.[1]
Fifteen male BALB/c mice (20–25 g) were quickly euthanized by cervical dislocation. BMMC were aspirated from their femur and tibia by flushing the bone marrow cavity with Dulbecco's modified Eagle's medium (DMEM). After a homogeneous cell suspension was obtained, the cells were centrifuged (4,000 g for 10 min), re-suspended in DMEM and added to Ficoll-Hypaque (Histopaque 1083), centrifuged again (5,000 g, 30 min) and supplemented with sterile PBS. Cells were counted in a Neubauer chamber with Trypan Blue for evaluation of viability. Cell characterization was performed by flow cytometry using specific antibodies (Conget and Minguell, 1999; Maron-Gutierrez et al., 2011).[1]
Figure ​Figure11 shows that 60 female BALB/c mice (20–25 g) were randomly divided into six groups. In S14 (n = 10), SS21 (n = 10), and SC21 (n = 10) groups, mice were intratracheally (i.t.) injected with 50 μL of sterile saline solution (0.9% NaCl) on days 1 and 7 of the experimental protocol. In P14 (n = 10), PS21 (n = 10), and PC21 (n = 10) groups, mice were i.t. injected with 50 μL of sterile saline solution (0.9% NaCl) containing 10 IU of papain (0.2 IU/μL) on days 0 and 7 of the experimental protocol. Papain (USP 225310) had been previously activated in 0.1 M sodium phosphate buffer containing 10 mM EDTA, 0.4 NaCl and 5 mM dithiothreitol for 10 min at 40°C (Machado et al., 2014). On the 14th day, 2 × 106 BMMC from male BALB/c mice suspended in 50 μL of sterile saline solution (SC21 and PC21 groups) or 50 μL of sterile saline solution (0.9% NaCl) (SS21 and PS21 groups) were injected into the jugular vein. Histopathological parameters and pulmonary mechanics were analyzed on the 14th (S14 and P14 groups) and 21st (SS21, PS21, SC21, and PC21 groups) days of the protocol.[1]

[1]. Bone Marrow-Derived Mononuclear Cell Therapy in Papain-Induced Experimental Pulmonary Emphysema. Front Physiol. 2018 Feb 20;9:121.

Type II alveolar cells express DUOX1 and DUOX2 enzymes, which, like those in airway cells, are located at the apical level. Despite the presence of DUOX1 and DUOX2 enzymes in the alveoli, information regarding their involvement in H₂O₂ production at this histological level remains scarce (Fischer, 2009). The amount of H₂O₂ produced by these enzymes at the alveolar level is considered lower compared to that produced in airway epithelial cells (Fischer et al., 2007). The exact role of DUOX enzymes in the pathogenesis of emphysema remains controversial and requires further investigation. We assessed the mRNA expression of DUOX1 and DUOX2 and observed increased mRNA expression of both DUOX1 and DUOX2 in lung tissues of animals exposed to papain (Figure 5), consistent with results from other research groups (Ameziane-El-Hassani et al., 2005; Harper et al., 2005; Rigutto et al., 2009). Administration of 2 × 10⁶ bone marrow-derived monocytes (BMMCs) attenuated the mRNA expression of DUOX1 and DUOX2 in lung tissue. Furthermore, compared to the control group, we observed increased H₂O₂ production and enhanced calcium-stimulated DUOX activity in the lung tissue of the papain-treated group, further confirming the potential antioxidant effect of 2 × 10⁶ BMMCs (Figure 6). NADPH oxidases play different roles in the airways (Fischer, 2009). Although the two DUOX isoenzymes are structurally highly similar, DUOX2 produces more H₂O₂ than DUOX1 in lung airway epithelial cells (Ameziane-El-Hassani et al., 2005; Rigutto et al., 2009). However, the expression level of DUOX1 is higher than that of DUOX2 in normal airway epithelial cells (Schwarzer et al., 2004; Harper et al., 2005). Therefore, in the airways of normal individuals, the ability of DUOX1 and DUOX2 to release H₂O₂ is likely similar. Several cytokines selectively regulate the expression levels of DUOX1 and DUOX2; for example, IFN-γ positively regulates DUOX2 expression (Harper et al., 2005). We found that papain exposure increased IFN-γ levels in mice, while BMMCs decreased IFN-γ levels (Table 2), consistent with changes in DUOX activity (Figure 6). In short, we emphasize the novelty of our findings, as there have been no reports to date on the effects of cell therapy on the NADPH oxidase pathway (particularly the regulation of DUOX enzymes) in a papain-induced mouse emphysema model. In conclusion, 2 × 10⁶ BMMCs exhibited significant anti-inflammatory, anti-apoptotic, antioxidant, and repair effects in a papain-induced emphysema model, likely through inhibition of DUOX1 and reduction of DUOX2 activity.

C9H14N4O3

226.2325

451.217

9001-73-4

5249653

White to off-white solid powder

1.5±0.1 g/cm3

29 °C

1.652

-1.47

3

4

5

16

254

0

CQOVPNPJLQNMDC-UHFFFAOYSA-N

InChI=1S/C9H14N4O3/c10-2-1-8(14)13-7(9(15)16)3-6-4-11-5-12-6/h4-5,7H,1-3,10H2,(H,11,12)(H,13,14)(H,15,16)

2-(3-azaniumylpropanoylamino)-3-(1H-imidazol-5-yl)propanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL
DMSO : ~25 mg/mL

Solubility in Formulation 1: 50 mg/mL (Infinity mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)