AIM:To investigate the effect of dyes,Remazol BrOrange yellow(RBY)and erythrosine(ERY),on the outcomes of immunoblotting analysis when used for staining the concentrate gel in sodium dodecyl sulfate-polyacryl-amide gel electrophoresis(SDS-PAGE).METHODS:Polyacrylamide gels were divided into five groups:the control group(prepared according to the conventional kit protocol),the RBY-stained group with a final concentration of 0.08 g/L,the RBY-stained group with a final concentration of 0.16 g/L,the ERY-stained group with a final concentration of 0.08 g/L,and the ERY-stained group with a final concentration of 0.8 g/L.Gels were prepared and subjected to electro-phoresis,followed by coomassie brilliant blue staining to visualize protein bands.Subsequently,proteins were transferred to PVDF membranes,which were then blocked,incubated with primary and secondary antibodies,washed,and finally ex-posed for imaging to observe the target protein vinculin bands.RESULTS:Compared with the unstained concentrate gel,the loading wells of the RBY or ERY pre-stained concentrate gel were more clearly visible.Analysis of the gels stained with coomassie brilliant blue after electrophoresis and marker visualization showed no significant different in protein elec-trophoretic mobility between prestained and unstained gels.Comparative analysis of the immunoblotting also indicated that the detection of protein samples transferred to PVDF membranes was unaffected.CONCLUSION:Prestaining concen-trate gels with RBY or ERY can enhance the efficiency of gel-based electrophoresis and immunoblotting analysis.

AIM:To investigate the effect of dyes,Remazol BrOrange yellow(RBY)and erythrosine(ERY),on the outcomes of immunoblotting analysis when used for staining the concentrate gel in sodium dodecyl sulfate-polyacryl-amide gel electrophoresis(SDS-PAGE).METHODS:Polyacrylamide gels were divided into five groups:the control group(prepared according to the conventional kit protocol),the RBY-stained group with a final concentration of 0.08 g/L,the RBY-stained group with a final concentration of 0.16 g/L,the ERY-stained group with a final concentration of 0.08 g/L,and the ERY-stained group with a final concentration of 0.8 g/L.Gels were prepared and subjected to electro-phoresis,followed by coomassie brilliant blue staining to visualize protein bands.Subsequently,proteins were transferred to PVDF membranes,which were then blocked,incubated with primary and secondary antibodies,washed,and finally ex-posed for imaging to observe the target protein vinculin bands.RESULTS:Compared with the unstained concentrate gel,the loading wells of the RBY or ERY pre-stained concentrate gel were more clearly visible.Analysis of the gels stained with coomassie brilliant blue after electrophoresis and marker visualization showed no significant different in protein elec-trophoretic mobility between prestained and unstained gels.Comparative analysis of the immunoblotting also indicated that the detection of protein samples transferred to PVDF membranes was unaffected.CONCLUSION:Prestaining concen-trate gels with RBY or ERY can enhance the efficiency of gel-based electrophoresis and immunoblotting analysis.

AIM:To investigate the effect and mechanism of panax notoginseng saponins(PNS)inhibiting the proliferation of pulmonary artery smooth muscle cells(PASMCs)in rats under the ef-fect of monocrotaline(MCT).METHODS:PASMCs cultured in vitro were randomly divided into the normal control(Control)group,the monocrotaline(MCT)group,the panax notoginseng saponins(PNS)group,the knockdown(M+Si ADAM10)group,the knockdown postconditioning(M+P+Si ADAM10)group,the overexpression(M+OE AD-AM10)group,and the overexpression postcondi-tioning(M+P+OE ADAM10)group.After the model was constructed,the cell viability of each group was measured using the CCK-8 assay,along with Western blot utilized to detect the expression of proliferating cell nuclear antigen(PCNA),disinteg-rin metalloproteinase 10(ADAM10),and notch ho-mology protein-3(Notch3)at the cellular neurogen-ic locus,respectively.RESULTS:Under the effect of MCT,the viability of PASMCs was significantly en-hanced(P<0.05 or P<0.01);0-400 mg/L PNS was not toxic to the viability of normal cells,and 100 mg/L PNS could significantly inhibit the MCT-in-duced viability(P<0.01).After the knockdown of ADAM10,the viability of PASMCs significantly de-clined(P<0.01),and the expression of PCNA protein was significantly decreased(P<0.05),evidently in the M+P+Si ADAM10 group.Meanwhile,the ex-pression of ADAM10 and Notch3 protein was signif-icantly decreased(P<0.05 or P<0.01),evidently in the M+P+Si ADAM10 group.After overexpression of ADAM10,the viability of PASMCs was significant-ly enhanced(P<0.01),the expression of PCNA pro-tein was significantly increased(P<0.01),the PCNA value was slightly higher(P>0.05),and the expres-sion of ADAM10 and Notch3 protein was signifi-cantly elevated(P<0.05)in the M+P+OE ADAM10 group.Additionally,PASMCs overexpressing AD-AM10 with concomitant PNS exhibited a significant decrease in the expression of PCNA protein com-pared with PASMCs knocking down ADAM10(P<0.01),and the expression of ADAM10 and Notch3 protein declined to varying degrees(P>0.05).CON-CLUSION:Panax notoginseng saponins can mitigate MCT-induced PASMCs proliferation in rats by inhib-iting the ADAM10/Notch3 signaling pathway.

Pulmonary hypertension(PH)is a serious cardiovascular condition that significantly impacts pa-tients'quality of life.Currently available clinical medications lack selectivity for pulmonary blood vessels,often produce substantial side effects,and are prohibitively expensive.Therefore,it is crucial to explore the mechanisms underlying the onset and progression of PH and to develop new,effective treatments.Ubiquitination is a key form of protein post-transla-tional modification in which specific E3 enzymes recognize substrate proteins and induce ubiquitination,leading to chang-es in their activity or stability.During the onset of PH,the activities of ubiquitin ligases and deubiquitinases undergo vari-ous changes,resulting in altered ubiquitination levels of different proteins.These variations primarily influence the degra-dation rates of substrate proteins within cells,thereby regulating essential physiological processes.Proteasomes play a vi-tal role in the degradation of ubiquitinated proteins,and inhibitors targeting these complexes have been developed,demon-strating therapeutic efficacy in experimental settings of PH.However,their low specificity presents significant challenges for practical applications.In this context,we summarize the relevant mechanisms of ubiquitination regulation in the onset of PH and highlight its practical significance for future therapeutic strategies.

AIM:To investigate the effect and mechanism of panax notoginseng saponins(PNS)inhibiting the proliferation of pulmonary artery smooth muscle cells(PASMCs)in rats under the ef-fect of monocrotaline(MCT).METHODS:PASMCs cultured in vitro were randomly divided into the normal control(Control)group,the monocrotaline(MCT)group,the panax notoginseng saponins(PNS)group,the knockdown(M+Si ADAM10)group,the knockdown postconditioning(M+P+Si ADAM10)group,the overexpression(M+OE AD-AM10)group,and the overexpression postcondi-tioning(M+P+OE ADAM10)group.After the model was constructed,the cell viability of each group was measured using the CCK-8 assay,along with Western blot utilized to detect the expression of proliferating cell nuclear antigen(PCNA),disinteg-rin metalloproteinase 10(ADAM10),and notch ho-mology protein-3(Notch3)at the cellular neurogen-ic locus,respectively.RESULTS:Under the effect of MCT,the viability of PASMCs was significantly en-hanced(P<0.05 or P<0.01);0-400 mg/L PNS was not toxic to the viability of normal cells,and 100 mg/L PNS could significantly inhibit the MCT-in-duced viability(P<0.01).After the knockdown of ADAM10,the viability of PASMCs significantly de-clined(P<0.01),and the expression of PCNA protein was significantly decreased(P<0.05),evidently in the M+P+Si ADAM10 group.Meanwhile,the ex-pression of ADAM10 and Notch3 protein was signif-icantly decreased(P<0.05 or P<0.01),evidently in the M+P+Si ADAM10 group.After overexpression of ADAM10,the viability of PASMCs was significant-ly enhanced(P<0.01),the expression of PCNA pro-tein was significantly increased(P<0.01),the PCNA value was slightly higher(P>0.05),and the expres-sion of ADAM10 and Notch3 protein was signifi-cantly elevated(P<0.05)in the M+P+OE ADAM10 group.Additionally,PASMCs overexpressing AD-AM10 with concomitant PNS exhibited a significant decrease in the expression of PCNA protein com-pared with PASMCs knocking down ADAM10(P<0.01),and the expression of ADAM10 and Notch3 protein declined to varying degrees(P>0.05).CON-CLUSION:Panax notoginseng saponins can mitigate MCT-induced PASMCs proliferation in rats by inhib-iting the ADAM10/Notch3 signaling pathway.

Pulmonary hypertension(PH)is a serious cardiovascular condition that significantly impacts pa-tients'quality of life.Currently available clinical medications lack selectivity for pulmonary blood vessels,often produce substantial side effects,and are prohibitively expensive.Therefore,it is crucial to explore the mechanisms underlying the onset and progression of PH and to develop new,effective treatments.Ubiquitination is a key form of protein post-transla-tional modification in which specific E3 enzymes recognize substrate proteins and induce ubiquitination,leading to chang-es in their activity or stability.During the onset of PH,the activities of ubiquitin ligases and deubiquitinases undergo vari-ous changes,resulting in altered ubiquitination levels of different proteins.These variations primarily influence the degra-dation rates of substrate proteins within cells,thereby regulating essential physiological processes.Proteasomes play a vi-tal role in the degradation of ubiquitinated proteins,and inhibitors targeting these complexes have been developed,demon-strating therapeutic efficacy in experimental settings of PH.However,their low specificity presents significant challenges for practical applications.In this context,we summarize the relevant mechanisms of ubiquitination regulation in the onset of PH and highlight its practical significance for future therapeutic strategies.

Stroke flaccid paralysis is stroke patients with abnormal physical movement function and muscle tone decline as the main performance and is a kind of common pathological state after apoplectic stroke. The longer the flaccid paralysis is, the worse the prognosis. The theory of TCM holds that stroke is mainly due to "deficiency, wind, fire, phlegm, stasis, qi", and when the pathogenic factor accumulate and block the meridians, which would cause blood stagnation, muscle and tendon damage and flaccidity, resulting in stroke paralysis. Therefore, it is necessary to set up the "Tongjing Roujin" (stimulating the muscle and nourishing the tendon) as its main treatment. Fire-needling has the effect of stimulating muscle, warming yang, nourishing tendon, and relieving pain in the treatment of stroke flaccid paralysis. It can warm yang and dissipate cold, replenish and nourish meridian qi, release muscle nodules, promote the circulation of qi and blood, and nourish all limbs and bones. Fire-needling therapy can promote the recovery of neural pathway, strengthen local metabolism, improve local muscle tension, and thus restore limb function. The high-quality clinical research, acupoint selection rules, and standardized operating techniques of fire-needling treatment for stroke flaccid paralysis need to be further deepened.

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.

OBJECTIVE: To assess the association of apolipoprotein (apo) C1 (APOC1) gene rs4420638A/G and -317H1/H2 polymorphisms with the risk of pre-eclampsia (PE) and the influence of their genotypes on the clinical and metabolic indexes among Chinese women. METHODS: In total 289 PE patients and 824 women with uncomplicated pregnancies were included. The rs4420638A/G genotype was determined by a Taqman real-time PCR allelic discrimination assay. The -317H1/H2 genotype was measured through PCR and restriction fragment length polymorphism analysis. Serum lipid and apo levels were measured by an enzymatic kit and a PEG-enhanced immunoturbidimetric assay. RESULTS: Allelic and genotypic frequencies of the APOC1 gene rs4420638A/G and -317H1/H2 were not significantly different between the two groups (all P> 0.05). However, patients carrying the G allele of the rs4420638A/G locus had higher serum levels of triglyceride, non-HDL-C and apoB, and a higher apoB/apoA1 ratio compared with those with an AA genotype (all P< 0.05). Patients carrying the H2 allele of the -317H1/H2 polymorphism had smaller delivery gestational weeks compared with those with the H1H1 genotype (P< 0.05). CONCLUSION Polymorphisms of the APOC1 gene rs4420638 and -317H1/H2 sites may be associated with abnormal lipoprotein metabolism among Chinese patients with PE, though no association was found between variants of the APOC1 gene and the risk of PE among them.