AIM:To investigate the effect and mechanism of the exogenous hydrogen sulfide donor GYY4137 on hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(PASMCs).METHODS:Rat PASMCs in optimal growth condition were used.Once the cell density reached 60%～70%,the cells were serum starved for 24 h.The cells were then randomly divided into four groups:normal group,normal+GYY4137 group,hypoxia group,and hypoxia+GYY4137 group.A CCK-8 assay was used to determine the safe concentration range of GYY4137 that exerted no adverse effects on normal cells,and the optimal concentration of GYY4137 to inhibit hypoxia-induced proliferation of PASMCs was identified.EdU staining was employed to assess PASMCs proliferation in each group.Western blot analysis was conducted to evaluate the expression of proliferating cell nuclear antigen(PCNA)and proteins related to glycolysis and pyroptosis in PASMCs.Lactic acid content was quantified using a lactic acid assay kit.Immunofluorescence was used to detect the pro-tein expression of hexokinase 2(HK2)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)in PASMCs,and the levels of interleukin-1β(IL-1β)and IL-18 in PASMCs were measured using ELISA.RESULTS:The effective concentration of GYY4137 in inhibiting hypoxia-induced viability of PAMSCs was 100 μmol/L(P＜0.05).Com-pared with the hypoxia group,the hypoxia+GYY4137 group showed a significant decrease in PCNA protein expression(P＜0.05),reduced PAMSCs proliferation(P＜0.01),decreased HK2 and pyruvate kinase isozyme type M2(PKM2)protein expression(P＜0.05,P＜0.01),increased pyruvate dehydrogenase(PDH)protein expression(P＜0.05),and reduced lac-tic acid content(P＜0.01).Additionally,the expression of pyroptosis-related proteins was significantly decreased(P＜0.015,P＜0.01),and immunofluorescence revealed a significant decrease in HK2 and NLRP3 expression(P＜0.01).ELISA results showed that IL-1β and IL-18 protein levels were significantly decreased(P＜0.05,P＜0.01).CONCLU-SION:GYY4137 inhibits hypoxia-induced proliferation of rat PASMCs by regulating glycolysis and pyroptosis.

AIM:To investigate the effect and mechanism of the exogenous hydrogen sulfide donor GYY4137 on hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells(PASMCs).METHODS:Rat PASMCs in optimal growth condition were used.Once the cell density reached 60%～70%,the cells were serum starved for 24 h.The cells were then randomly divided into four groups:normal group,normal+GYY4137 group,hypoxia group,and hypoxia+GYY4137 group.A CCK-8 assay was used to determine the safe concentration range of GYY4137 that exerted no adverse effects on normal cells,and the optimal concentration of GYY4137 to inhibit hypoxia-induced proliferation of PASMCs was identified.EdU staining was employed to assess PASMCs proliferation in each group.Western blot analysis was conducted to evaluate the expression of proliferating cell nuclear antigen(PCNA)and proteins related to glycolysis and pyroptosis in PASMCs.Lactic acid content was quantified using a lactic acid assay kit.Immunofluorescence was used to detect the pro-tein expression of hexokinase 2(HK2)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)in PASMCs,and the levels of interleukin-1β(IL-1β)and IL-18 in PASMCs were measured using ELISA.RESULTS:The effective concentration of GYY4137 in inhibiting hypoxia-induced viability of PAMSCs was 100 μmol/L(P＜0.05).Com-pared with the hypoxia group,the hypoxia+GYY4137 group showed a significant decrease in PCNA protein expression(P＜0.05),reduced PAMSCs proliferation(P＜0.01),decreased HK2 and pyruvate kinase isozyme type M2(PKM2)protein expression(P＜0.05,P＜0.01),increased pyruvate dehydrogenase(PDH)protein expression(P＜0.05),and reduced lac-tic acid content(P＜0.01).Additionally,the expression of pyroptosis-related proteins was significantly decreased(P＜0.015,P＜0.01),and immunofluorescence revealed a significant decrease in HK2 and NLRP3 expression(P＜0.01).ELISA results showed that IL-1β and IL-18 protein levels were significantly decreased(P＜0.05,P＜0.01).CONCLU-SION:GYY4137 inhibits hypoxia-induced proliferation of rat PASMCs by regulating glycolysis and pyroptosis.

AIM: To construct E. coli expression plasmid of recombinant human NDPK-A with a 6?His tag, optimize the expression condition and identify the activity of the product. METHODS: nm23-H1 was subcloned from plasmid pBVNMH1 to pQE40 which contain 6?His purification tag. The expression condition was modulated in grades to get the optimal expression. We purified protein with the Ni+-NTA affinity chromatography column, identified the immunogenicity of the product with Western blot, and measured the kinases activity with HPLC. In addition, angiogenesis inhibition activity of rhNDPK was identified by CAM. RESULTS: The sequence of nm23-H1 subclone in pQE40 was exactly correct. The expression rate of rhNDPK-A was 49 6%. Purified rhNDPK-A specially recognized the antiserum of NDPK-A. It also inhibited angiogenesis. CONCLUSION: PQE-nm23H1 containing 6?His can express target protein at high level. This purification method is simple than other methods, and the product has the same activity as natural human NDPK-A.