Objective:To evaluate the effect of cathepsin B(CTSB)/NOD-like receptor pyrin domain containing 3(NLRP3)pathway on the polarization of macrophages induced by LPS.Methods:The well-growing RAW264.7 mouse mononuclear macrophage lines were cultured in vitro and divided into 3 groups(n=6)according to the random number table method:control group(C group),LPS group(L group)and LPS+CA074-me(CTSB inhibitors)group(B group).C group was cultured normally for 24 h,L group was cultured with LPS concentration of 1 μg/ml medium for 24 h.B group was pretreated with CTSB inhibitor CA074-me 30 μmol/L for 1 h before LPS induction,and co-cultured with LPS concentration of 1 μg/ml medium for 24 h.After 24 hours,the morphological changes of the cells were observed by microscope,the concentrations of IL-1β and IL-18 in the supernatant were determined by ELISA.The ex-pressions of cathepsin B precursor(pro-CTSB),mature cathepsin B(mature-CTSB),NLRP3,apoptosis-related speck protein(ASC)and apoptosis-related speck protein-1(caspase-1)were detected by Western blot.The mRNA expression levels of CD32,inducible ni-tric oxide synthase(iNOS),arginase 1(Arg-1)and CD206 were detected by qRT-PCR.The positive expression rates of M1 macro-phage surface marker CD86 and M2 macrophage surface marker CD206 were detected by flow cytometry.Results:Compared with group C,the morphology of cells in groups L and B became larger and pseudopodia appeared.The concentrations of IL-1β and IL-18 in cell supernatant were increased,the expressions of pro-CTSB,mature-CTSB,NLRP3,ASC and caspase-1 were increased,and the expressions of CD32,iNOS mRNA were up-regulated and the positive rates of CD86 and CD206 were increased(P<0.01).Arg-1 and CD206 mRNA in group B were up-regulated(P<0.01).Compared with group L,the pseudopodia of group B were reduced,and the morphology was closer to group C.The concentration of IL-1β and IL-18 in the supernatant,the expression of mature-CTSB,NLRP3,ASC and caspase-1,CD32 and iNOS mRNA and the positive rate of CD86 were down-regulated in group B.The expression of pro-CTSB,Arg-1 and CD206 mRNA and the positive rate of CD206 were increased(P<0.01).Conclusion:Inhibition of CTSB/NLRP3 pathway can reduce the inflammatory response,reduce the LPS-induced polarization of RAW264.7 cells to M1 macrophages,and pro-mote their polarization to M2 macrophages.

Objective:To evaluate the effect of cathepsin B(CTSB)/NOD-like receptor pyrin domain containing 3(NLRP3)pathway on the polarization of macrophages induced by LPS.Methods:The well-growing RAW264.7 mouse mononuclear macrophage lines were cultured in vitro and divided into 3 groups(n=6)according to the random number table method:control group(C group),LPS group(L group)and LPS+CA074-me(CTSB inhibitors)group(B group).C group was cultured normally for 24 h,L group was cultured with LPS concentration of 1 μg/ml medium for 24 h.B group was pretreated with CTSB inhibitor CA074-me 30 μmol/L for 1 h before LPS induction,and co-cultured with LPS concentration of 1 μg/ml medium for 24 h.After 24 hours,the morphological changes of the cells were observed by microscope,the concentrations of IL-1β and IL-18 in the supernatant were determined by ELISA.The ex-pressions of cathepsin B precursor(pro-CTSB),mature cathepsin B(mature-CTSB),NLRP3,apoptosis-related speck protein(ASC)and apoptosis-related speck protein-1(caspase-1)were detected by Western blot.The mRNA expression levels of CD32,inducible ni-tric oxide synthase(iNOS),arginase 1(Arg-1)and CD206 were detected by qRT-PCR.The positive expression rates of M1 macro-phage surface marker CD86 and M2 macrophage surface marker CD206 were detected by flow cytometry.Results:Compared with group C,the morphology of cells in groups L and B became larger and pseudopodia appeared.The concentrations of IL-1β and IL-18 in cell supernatant were increased,the expressions of pro-CTSB,mature-CTSB,NLRP3,ASC and caspase-1 were increased,and the expressions of CD32,iNOS mRNA were up-regulated and the positive rates of CD86 and CD206 were increased(P<0.01).Arg-1 and CD206 mRNA in group B were up-regulated(P<0.01).Compared with group L,the pseudopodia of group B were reduced,and the morphology was closer to group C.The concentration of IL-1β and IL-18 in the supernatant,the expression of mature-CTSB,NLRP3,ASC and caspase-1,CD32 and iNOS mRNA and the positive rate of CD86 were down-regulated in group B.The expression of pro-CTSB,Arg-1 and CD206 mRNA and the positive rate of CD206 were increased(P<0.01).Conclusion:Inhibition of CTSB/NLRP3 pathway can reduce the inflammatory response,reduce the LPS-induced polarization of RAW264.7 cells to M1 macrophages,and pro-mote their polarization to M2 macrophages.

Objective:To evaluate the role of cathepsin B (CTSB) in mechanical ventilator-induced lung injury (VILI) in rats and the relationship with NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome.Methods:Thirty-six SPF-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 220-300 g, were divided into 3 groups ( n=12 each) by the random number table method: control group (group C), VILI group (group V) and VILI + CA074-me group (group Me). CA074-me 5 mg/kg was intraperitoneally injected in group Me, while the equal volume of normal saline was given instead in group C and group V. Group C kept spontaneous breathing for 4 h, and the animals were mechanically ventilated (tidal volume 20 ml/kg, respiratory rate 80 breaths/min, fraction of inspired oxygen 21%, PEEP 0 cmH 2O). Blood samples from femoral artery were collected for arterial blood gas analysis before tracheal intubation and after spontaneous breathing or ventilation, and PaO 2 was recorded.Rats were sacrificed, and bronchoalveolar lavage fluid (BALF) was collected and lung tissues were collected for determination of the wet/dry lung weight ratio (W/D ratio), serum interleukin-1beta (IL-1β) and IL-18 concentrations in BALF (by enzyme-linked immunosorbent assay), expression of CTSB, NLRP3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) and caspase-1 mRNA in lung tissues (quantitative real-time polymerase chain reaction), and expression of CTSB, NLRP3, ASC and caspase-1 in lung tissues (by Western blot) and for microscopic examination of the pathological changes (using HE staining). Lung injury was assessed and scored. Results:Compared with group C, PaO 2 was significantly decreased after the end of ventilation, the lung injury score, W/D ratio and concentrations of IL-1β and IL-18 in serum and BALF were increased, and the expression of CTSB, NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was up-regulated in group V and group Me ( P<0.01). Compared with group V, PaO 2 was significantly increased after the end of ventilation, the lung injury score, W/D ratio and concentrations of IL-1β and IL-18 in serum and BALF were decreased, and the expression of CTSB, NLRP3, ASC and caspase-1 protein and mRNA in lung tissues was down-regulated in group Me ( P<0.01). Conclusions:CTSB is involved in VILI in the rats, and the mechanism may be related to activation of NLRP3 inflammasomes.