Objective:To analyze the efficacy and safety of modified associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) in the treatment of patients with primary liver cancer.Methods:Clinical data of 83 patients with hepatocellular carcinoma (HCC) undergoing hemihepatectomy in the Department of Hepatobiliary and Pancreatic Tumor Surgery of Gansu Provincial Cancer Hospital between January 2022 and November 2023 were retrospectively analyzed, including 53 males and 30 females, aged (54.0±6.5) years. According to the treatment protocol, patients were divided into the control group ( n=41), in which patients underwent traditional ALPPS, and the observation group ( n=42), in which patients underwent modified ALPPS (occlusion of portal venous branch using vascular clips, combined with radiofrequency ablation for physical separation of the diseased lobe, without liver mobilization). The completion rate of staged surgery, interval between surgeries, future liver remnant (FLR) growth rate at 7 days after first-stage surgery, alanine transaminase (ALT) and aspartate transaminase (AST) levels at 5 days after fisrt-stage surgery, and postoperative complications (ascites, nausea, and vomiting, etc.) were compared between the groups. Results:The completion rate of staged surgery was 95.2% (40/42) in the observation group and 90.2% (37/41) in the control group ( χ2=0.62, P=0.431). The ALT and AST levels at 5 days after first-stage surgery were (550.4±86.0) U/L and (327.1±52.8) U/L in the observation group, respectively, which were significantly lower than those in the control group (861.6±106.3) U/L and (533.8±73.7) U/L, respectively ( t=13.13 and P<0.001, t=12.93 and P<0.001). The FLR growth rate were higher in the observation group than that in the control group [(80.4±10.3)% vs (49.3±5.7)%; t=13.13, P<0.001] and the interval between procedures were also shorter in the observation group (10.9±2.1 vs 22.4±4.8, d; t=9.65, P<0.001). The intraoperative blood loss of the first-stage surgery was lower in the observation group than that in the control group (350.5±45.2 vs 825.5±21.7, ml; t=21.43, P<0.001). The total complication rates after the first-stage surgery were 11.9% (5/42) in the observation group and 19.5% (8/41) in the control group, while after the second-stage surgery, the complication rates were 7.5% (3/40) and 18.9% (7/37), respectively, with no statistically significant differences ( χ2=0.65 and P=0.419, χ2=1.81 and 0.177, respectively). Conclusion:The modified ALPPS offers better postoperative liver function, reduced surgical trauma, accelerated FLR growth, and a shorter interval between procedures, demonstrating a favorable safety in the treatment of primary liver cancer.

Objective:To investigate the expression and methylation status of the SALL4 gene in placental tissues of fetal growth restriction (FGR) and its effects on trophoblast cell proliferation, migration, and invasion. Methods:Placental tissues were collected from 20 full-term FGR patients and 20 healthy term controls who underwent regular prenatal examination and cesarean section at the Third Affiliated Hospital, Zhengzhou University between July 2023 and February 2024. SALL4 mRNA and protein expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Methylation specific polymerase china reaction (MSP) assessed promoter methylation levels. HTR8/SVneo cells were transfected with SALL4-targeting small interfering RNA (si-SALL4) or negative control small interfering RNA (si-NC). HTR8/SVneo cells were treated with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dC) to inhibit gene methylation (5-Aza-dC group) or with 10% RPMI-1640 medium as a vehicle control. Transfection efficiency (for siRNA) and the efficacy of 5-Aza-dC-induced demethylation were assessed by qRT-PCR and Western blot. The functional effects of SALL4 knockdown and methylation inhibition on trophoblast cells were evaluated using proliferation assays, scratch wound healing assays, and Transwell invasion assays. Statistical analyses included independent t-tests and Chi-square test. Results:(1) Human tissues: FGR placentas showed lower SALL4 mRNA (0.802±0.194 vs. 1.015±0.186, t=3.55) and protein expression (0.445±0.114 vs. 0.701±0.113, t=3.19), alongside higher methylation rates of SALL4 [80% (16/20) vs. 15% (3/20), χ2=14.44] compared to controls (all P<0.05). (2) In vitro: si-SALL4 transfection reduced HTR8/SVneo proliferation (OD450 at 48 h: 0.653±0.021 vs. 0.827±0.040, t=6.60), migration [healing rate at 48 h: (24.317±2.637)% vs. (49.327±1.961)%, t=13.18], and invasion [counted invaded cells: (133.000±6.557) vs. (272.667±18.009) cells, t=12.62] versus si-NC (all P<0.05). Conversely, 5-Aza-dC treatment increased HTR8/SVneo proliferation (0.917±0.042 vs. 0.783±0.031, t=－4.47), migration [(71.097±3.354)% vs. (51.632±2.877)%, t=－7.63], and invasion [(384.000±12.166) vs. (202.833±7.095) cells, t=－13.69] versus vehicle control (all P<0.05). Conclusions:Hypermethylation of the SALL4 promoter in FGR placentas suppresses its expression, impairing trophoblast cell function. Demethylation restores SALL4 expression and enhances cellular proliferation, migration, and invasion, involving in the occurrence and development of FGR disease.

Objective:To analyze the efficacy and safety of modified associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) in the treatment of patients with primary liver cancer.Methods:Clinical data of 83 patients with hepatocellular carcinoma (HCC) undergoing hemihepatectomy in the Department of Hepatobiliary and Pancreatic Tumor Surgery of Gansu Provincial Cancer Hospital between January 2022 and November 2023 were retrospectively analyzed, including 53 males and 30 females, aged (54.0±6.5) years. According to the treatment protocol, patients were divided into the control group ( n=41), in which patients underwent traditional ALPPS, and the observation group ( n=42), in which patients underwent modified ALPPS (occlusion of portal venous branch using vascular clips, combined with radiofrequency ablation for physical separation of the diseased lobe, without liver mobilization). The completion rate of staged surgery, interval between surgeries, future liver remnant (FLR) growth rate at 7 days after first-stage surgery, alanine transaminase (ALT) and aspartate transaminase (AST) levels at 5 days after fisrt-stage surgery, and postoperative complications (ascites, nausea, and vomiting, etc.) were compared between the groups. Results:The completion rate of staged surgery was 95.2% (40/42) in the observation group and 90.2% (37/41) in the control group ( χ2=0.62, P=0.431). The ALT and AST levels at 5 days after first-stage surgery were (550.4±86.0) U/L and (327.1±52.8) U/L in the observation group, respectively, which were significantly lower than those in the control group (861.6±106.3) U/L and (533.8±73.7) U/L, respectively ( t=13.13 and P<0.001, t=12.93 and P<0.001). The FLR growth rate were higher in the observation group than that in the control group [(80.4±10.3)% vs (49.3±5.7)%; t=13.13, P<0.001] and the interval between procedures were also shorter in the observation group (10.9±2.1 vs 22.4±4.8, d; t=9.65, P<0.001). The intraoperative blood loss of the first-stage surgery was lower in the observation group than that in the control group (350.5±45.2 vs 825.5±21.7, ml; t=21.43, P<0.001). The total complication rates after the first-stage surgery were 11.9% (5/42) in the observation group and 19.5% (8/41) in the control group, while after the second-stage surgery, the complication rates were 7.5% (3/40) and 18.9% (7/37), respectively, with no statistically significant differences ( χ2=0.65 and P=0.419, χ2=1.81 and 0.177, respectively). Conclusion:The modified ALPPS offers better postoperative liver function, reduced surgical trauma, accelerated FLR growth, and a shorter interval between procedures, demonstrating a favorable safety in the treatment of primary liver cancer.

Objective:To investigate the expression and methylation status of the SALL4 gene in placental tissues of fetal growth restriction (FGR) and its effects on trophoblast cell proliferation, migration, and invasion. Methods:Placental tissues were collected from 20 full-term FGR patients and 20 healthy term controls who underwent regular prenatal examination and cesarean section at the Third Affiliated Hospital, Zhengzhou University between July 2023 and February 2024. SALL4 mRNA and protein expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Methylation specific polymerase china reaction (MSP) assessed promoter methylation levels. HTR8/SVneo cells were transfected with SALL4-targeting small interfering RNA (si-SALL4) or negative control small interfering RNA (si-NC). HTR8/SVneo cells were treated with the demethylating agent 5-aza-2′-deoxycytidine (5-Aza-dC) to inhibit gene methylation (5-Aza-dC group) or with 10% RPMI-1640 medium as a vehicle control. Transfection efficiency (for siRNA) and the efficacy of 5-Aza-dC-induced demethylation were assessed by qRT-PCR and Western blot. The functional effects of SALL4 knockdown and methylation inhibition on trophoblast cells were evaluated using proliferation assays, scratch wound healing assays, and Transwell invasion assays. Statistical analyses included independent t-tests and Chi-square test. Results:(1) Human tissues: FGR placentas showed lower SALL4 mRNA (0.802±0.194 vs. 1.015±0.186, t=3.55) and protein expression (0.445±0.114 vs. 0.701±0.113, t=3.19), alongside higher methylation rates of SALL4 [80% (16/20) vs. 15% (3/20), χ2=14.44] compared to controls (all P<0.05). (2) In vitro: si-SALL4 transfection reduced HTR8/SVneo proliferation (OD450 at 48 h: 0.653±0.021 vs. 0.827±0.040, t=6.60), migration [healing rate at 48 h: (24.317±2.637)% vs. (49.327±1.961)%, t=13.18], and invasion [counted invaded cells: (133.000±6.557) vs. (272.667±18.009) cells, t=12.62] versus si-NC (all P<0.05). Conversely, 5-Aza-dC treatment increased HTR8/SVneo proliferation (0.917±0.042 vs. 0.783±0.031, t=－4.47), migration [(71.097±3.354)% vs. (51.632±2.877)%, t=－7.63], and invasion [(384.000±12.166) vs. (202.833±7.095) cells, t=－13.69] versus vehicle control (all P<0.05). Conclusions:Hypermethylation of the SALL4 promoter in FGR placentas suppresses its expression, impairing trophoblast cell function. Demethylation restores SALL4 expression and enhances cellular proliferation, migration, and invasion, involving in the occurrence and development of FGR disease.

Objective To investigate the effect and mechanism of Panax notoginseng saponins(PNS)in inhibiting c-Jun N-terminal protein kinase(JNK)/c-Jun signaling pathway activation to alleviate calcific aortic valve disease(CAVD)in mice.Methods Twenty-one male ApoE-/-mice aged 6 to 8 weeks were randomly divided into the model,PNS high-dose(60 mg/kg),and PNS low-dose(30 mg/kg)groups using the random number table method,with seven mice per group.Nine male C57BL/6 mice aged 6 to 8 weeks were used as the control group.Mice in the control group were fed a normal diet,whereas ApoE-/-mice were fed a high-fat diet for 12 weeks.After 12 weeks,three C57BL/6 and three ApoE-/-mice(one ApoE-/-mice from each group)were randomly selected to evaluate the CAVD modeling effect.After confirming successful modeling,the PNS high-and low-dose groups received daily intragastric PNS administration.The control and model groups were administered an equal volume of stroke-physiological saline solution by gavage for 4 consecutive weeks.The valve annulus diameter and peak velocity of the mice in each group were then detected using ultrasound.The degree of aortic valve calcification was evaluated using von Kossa and Alizarin Red S staining.The serum triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Inflammatory factor interleukin-4(IL-4),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-10(IL-10)levels were determined using an enzyme-linked immunosorbent assay.The expressions of calcification markers,runt-related transcription factor 2(RUNX2),and bone morphogenetic protein 2(BMP2)were detected using immunohistochemistry.Aortic valve cell apoptosis was evaluated using TUNEL staining,and JNK/c-Jun signaling pathway-related mRNA and mean fluorescence intensity were detected using quantitative real-time PCR and immunofluorescence,respectively.Results Compared with the control group,the mice in the model group showed an increase in serum TC,TG,LDL-C,TNF-α,and IL-1β levels,a decrease in IL-4 and IL-10 levels,a decrease in annulus diameter,an increase in peak flow velocity,and an increase in von Kossa and Alizarin Red S staining-positive areas.Additionally,the model group showed an increase in aortic valve cell apoptosis rate,an increase in BMP2 and RUNX2-positive rates,and an increase in JNK and c-Jun mRNA expression levels and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Compared to the model group,the PNS low-dose group showed a decrease in serum TC,LDL-C,and TNF-α levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in positive area in Alizarin Red S staining.Furthermore,the PNS low-dose group showed a decrease in BMP2 and RUNX2-positive rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).The PNS high-dose group showed an increase in HDL-C,IL-4 and IL-10 levels,a decrease in serum TC,LDL-C,TNF-α,and IL-1β levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in von Kossa and Alizarin Red S staining-positive areas and cell apoptosis rate.The PNS high-dose group also showed a decrease in BMP2 and RUNX2 positive staining rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Conclusion PNS may reduce valvular cell apoptosis,alleviate inflammation,and protect against aortic valve calcification in mice by inhibiting the activation of JNK/c-Jun signaling pathway.

Objective To investigate the effect and mechanism of Panax notoginseng saponins(PNS)in inhibiting c-Jun N-terminal protein kinase(JNK)/c-Jun signaling pathway activation to alleviate calcific aortic valve disease(CAVD)in mice.Methods Twenty-one male ApoE-/-mice aged 6 to 8 weeks were randomly divided into the model,PNS high-dose(60 mg/kg),and PNS low-dose(30 mg/kg)groups using the random number table method,with seven mice per group.Nine male C57BL/6 mice aged 6 to 8 weeks were used as the control group.Mice in the control group were fed a normal diet,whereas ApoE-/-mice were fed a high-fat diet for 12 weeks.After 12 weeks,three C57BL/6 and three ApoE-/-mice(one ApoE-/-mice from each group)were randomly selected to evaluate the CAVD modeling effect.After confirming successful modeling,the PNS high-and low-dose groups received daily intragastric PNS administration.The control and model groups were administered an equal volume of stroke-physiological saline solution by gavage for 4 consecutive weeks.The valve annulus diameter and peak velocity of the mice in each group were then detected using ultrasound.The degree of aortic valve calcification was evaluated using von Kossa and Alizarin Red S staining.The serum triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Inflammatory factor interleukin-4(IL-4),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-10(IL-10)levels were determined using an enzyme-linked immunosorbent assay.The expressions of calcification markers,runt-related transcription factor 2(RUNX2),and bone morphogenetic protein 2(BMP2)were detected using immunohistochemistry.Aortic valve cell apoptosis was evaluated using TUNEL staining,and JNK/c-Jun signaling pathway-related mRNA and mean fluorescence intensity were detected using quantitative real-time PCR and immunofluorescence,respectively.Results Compared with the control group,the mice in the model group showed an increase in serum TC,TG,LDL-C,TNF-α,and IL-1β levels,a decrease in IL-4 and IL-10 levels,a decrease in annulus diameter,an increase in peak flow velocity,and an increase in von Kossa and Alizarin Red S staining-positive areas.Additionally,the model group showed an increase in aortic valve cell apoptosis rate,an increase in BMP2 and RUNX2-positive rates,and an increase in JNK and c-Jun mRNA expression levels and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Compared to the model group,the PNS low-dose group showed a decrease in serum TC,LDL-C,and TNF-α levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in positive area in Alizarin Red S staining.Furthermore,the PNS low-dose group showed a decrease in BMP2 and RUNX2-positive rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).The PNS high-dose group showed an increase in HDL-C,IL-4 and IL-10 levels,a decrease in serum TC,LDL-C,TNF-α,and IL-1β levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in von Kossa and Alizarin Red S staining-positive areas and cell apoptosis rate.The PNS high-dose group also showed a decrease in BMP2 and RUNX2 positive staining rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Conclusion PNS may reduce valvular cell apoptosis,alleviate inflammation,and protect against aortic valve calcification in mice by inhibiting the activation of JNK/c-Jun signaling pathway.

Objective To investigate the effect and mechanism of Panax notoginseng saponins(PNS)in inhibiting c-Jun N-terminal protein kinase(JNK)/c-Jun signaling pathway activation to alleviate calcific aortic valve disease(CAVD)in mice.Methods Twenty-one male ApoE-/-mice aged 6 to 8 weeks were randomly divided into the model,PNS high-dose(60 mg/kg),and PNS low-dose(30 mg/kg)groups using the random number table method,with seven mice per group.Nine male C57BL/6 mice aged 6 to 8 weeks were used as the control group.Mice in the control group were fed a normal diet,whereas ApoE-/-mice were fed a high-fat diet for 12 weeks.After 12 weeks,three C57BL/6 and three ApoE-/-mice(one ApoE-/-mice from each group)were randomly selected to evaluate the CAVD modeling effect.After confirming successful modeling,the PNS high-and low-dose groups received daily intragastric PNS administration.The control and model groups were administered an equal volume of stroke-physiological saline solution by gavage for 4 consecutive weeks.The valve annulus diameter and peak velocity of the mice in each group were then detected using ultrasound.The degree of aortic valve calcification was evaluated using von Kossa and Alizarin Red S staining.The serum triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Inflammatory factor interleukin-4(IL-4),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-10(IL-10)levels were determined using an enzyme-linked immunosorbent assay.The expressions of calcification markers,runt-related transcription factor 2(RUNX2),and bone morphogenetic protein 2(BMP2)were detected using immunohistochemistry.Aortic valve cell apoptosis was evaluated using TUNEL staining,and JNK/c-Jun signaling pathway-related mRNA and mean fluorescence intensity were detected using quantitative real-time PCR and immunofluorescence,respectively.Results Compared with the control group,the mice in the model group showed an increase in serum TC,TG,LDL-C,TNF-α,and IL-1β levels,a decrease in IL-4 and IL-10 levels,a decrease in annulus diameter,an increase in peak flow velocity,and an increase in von Kossa and Alizarin Red S staining-positive areas.Additionally,the model group showed an increase in aortic valve cell apoptosis rate,an increase in BMP2 and RUNX2-positive rates,and an increase in JNK and c-Jun mRNA expression levels and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Compared to the model group,the PNS low-dose group showed a decrease in serum TC,LDL-C,and TNF-α levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in positive area in Alizarin Red S staining.Furthermore,the PNS low-dose group showed a decrease in BMP2 and RUNX2-positive rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).The PNS high-dose group showed an increase in HDL-C,IL-4 and IL-10 levels,a decrease in serum TC,LDL-C,TNF-α,and IL-1β levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in von Kossa and Alizarin Red S staining-positive areas and cell apoptosis rate.The PNS high-dose group also showed a decrease in BMP2 and RUNX2 positive staining rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Conclusion PNS may reduce valvular cell apoptosis,alleviate inflammation,and protect against aortic valve calcification in mice by inhibiting the activation of JNK/c-Jun signaling pathway.

Objective To investigate the effect and mechanism of Panax notoginseng saponins(PNS)in inhibiting c-Jun N-terminal protein kinase(JNK)/c-Jun signaling pathway activation to alleviate calcific aortic valve disease(CAVD)in mice.Methods Twenty-one male ApoE-/-mice aged 6 to 8 weeks were randomly divided into the model,PNS high-dose(60 mg/kg),and PNS low-dose(30 mg/kg)groups using the random number table method,with seven mice per group.Nine male C57BL/6 mice aged 6 to 8 weeks were used as the control group.Mice in the control group were fed a normal diet,whereas ApoE-/-mice were fed a high-fat diet for 12 weeks.After 12 weeks,three C57BL/6 and three ApoE-/-mice(one ApoE-/-mice from each group)were randomly selected to evaluate the CAVD modeling effect.After confirming successful modeling,the PNS high-and low-dose groups received daily intragastric PNS administration.The control and model groups were administered an equal volume of stroke-physiological saline solution by gavage for 4 consecutive weeks.The valve annulus diameter and peak velocity of the mice in each group were then detected using ultrasound.The degree of aortic valve calcification was evaluated using von Kossa and Alizarin Red S staining.The serum triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Inflammatory factor interleukin-4(IL-4),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-10(IL-10)levels were determined using an enzyme-linked immunosorbent assay.The expressions of calcification markers,runt-related transcription factor 2(RUNX2),and bone morphogenetic protein 2(BMP2)were detected using immunohistochemistry.Aortic valve cell apoptosis was evaluated using TUNEL staining,and JNK/c-Jun signaling pathway-related mRNA and mean fluorescence intensity were detected using quantitative real-time PCR and immunofluorescence,respectively.Results Compared with the control group,the mice in the model group showed an increase in serum TC,TG,LDL-C,TNF-α,and IL-1β levels,a decrease in IL-4 and IL-10 levels,a decrease in annulus diameter,an increase in peak flow velocity,and an increase in von Kossa and Alizarin Red S staining-positive areas.Additionally,the model group showed an increase in aortic valve cell apoptosis rate,an increase in BMP2 and RUNX2-positive rates,and an increase in JNK and c-Jun mRNA expression levels and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Compared to the model group,the PNS low-dose group showed a decrease in serum TC,LDL-C,and TNF-α levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in positive area in Alizarin Red S staining.Furthermore,the PNS low-dose group showed a decrease in BMP2 and RUNX2-positive rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).The PNS high-dose group showed an increase in HDL-C,IL-4 and IL-10 levels,a decrease in serum TC,LDL-C,TNF-α,and IL-1β levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in von Kossa and Alizarin Red S staining-positive areas and cell apoptosis rate.The PNS high-dose group also showed a decrease in BMP2 and RUNX2 positive staining rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Conclusion PNS may reduce valvular cell apoptosis,alleviate inflammation,and protect against aortic valve calcification in mice by inhibiting the activation of JNK/c-Jun signaling pathway.

Objective To investigate the effect and mechanism of Panax notoginseng saponins(PNS)in inhibiting c-Jun N-terminal protein kinase(JNK)/c-Jun signaling pathway activation to alleviate calcific aortic valve disease(CAVD)in mice.Methods Twenty-one male ApoE-/-mice aged 6 to 8 weeks were randomly divided into the model,PNS high-dose(60 mg/kg),and PNS low-dose(30 mg/kg)groups using the random number table method,with seven mice per group.Nine male C57BL/6 mice aged 6 to 8 weeks were used as the control group.Mice in the control group were fed a normal diet,whereas ApoE-/-mice were fed a high-fat diet for 12 weeks.After 12 weeks,three C57BL/6 and three ApoE-/-mice(one ApoE-/-mice from each group)were randomly selected to evaluate the CAVD modeling effect.After confirming successful modeling,the PNS high-and low-dose groups received daily intragastric PNS administration.The control and model groups were administered an equal volume of stroke-physiological saline solution by gavage for 4 consecutive weeks.The valve annulus diameter and peak velocity of the mice in each group were then detected using ultrasound.The degree of aortic valve calcification was evaluated using von Kossa and Alizarin Red S staining.The serum triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Inflammatory factor interleukin-4(IL-4),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-10(IL-10)levels were determined using an enzyme-linked immunosorbent assay.The expressions of calcification markers,runt-related transcription factor 2(RUNX2),and bone morphogenetic protein 2(BMP2)were detected using immunohistochemistry.Aortic valve cell apoptosis was evaluated using TUNEL staining,and JNK/c-Jun signaling pathway-related mRNA and mean fluorescence intensity were detected using quantitative real-time PCR and immunofluorescence,respectively.Results Compared with the control group,the mice in the model group showed an increase in serum TC,TG,LDL-C,TNF-α,and IL-1β levels,a decrease in IL-4 and IL-10 levels,a decrease in annulus diameter,an increase in peak flow velocity,and an increase in von Kossa and Alizarin Red S staining-positive areas.Additionally,the model group showed an increase in aortic valve cell apoptosis rate,an increase in BMP2 and RUNX2-positive rates,and an increase in JNK and c-Jun mRNA expression levels and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Compared to the model group,the PNS low-dose group showed a decrease in serum TC,LDL-C,and TNF-α levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in positive area in Alizarin Red S staining.Furthermore,the PNS low-dose group showed a decrease in BMP2 and RUNX2-positive rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).The PNS high-dose group showed an increase in HDL-C,IL-4 and IL-10 levels,a decrease in serum TC,LDL-C,TNF-α,and IL-1β levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in von Kossa and Alizarin Red S staining-positive areas and cell apoptosis rate.The PNS high-dose group also showed a decrease in BMP2 and RUNX2 positive staining rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Conclusion PNS may reduce valvular cell apoptosis,alleviate inflammation,and protect against aortic valve calcification in mice by inhibiting the activation of JNK/c-Jun signaling pathway.

Objective To investigate the effect and mechanism of Panax notoginseng saponins(PNS)in inhibiting c-Jun N-terminal protein kinase(JNK)/c-Jun signaling pathway activation to alleviate calcific aortic valve disease(CAVD)in mice.Methods Twenty-one male ApoE-/-mice aged 6 to 8 weeks were randomly divided into the model,PNS high-dose(60 mg/kg),and PNS low-dose(30 mg/kg)groups using the random number table method,with seven mice per group.Nine male C57BL/6 mice aged 6 to 8 weeks were used as the control group.Mice in the control group were fed a normal diet,whereas ApoE-/-mice were fed a high-fat diet for 12 weeks.After 12 weeks,three C57BL/6 and three ApoE-/-mice(one ApoE-/-mice from each group)were randomly selected to evaluate the CAVD modeling effect.After confirming successful modeling,the PNS high-and low-dose groups received daily intragastric PNS administration.The control and model groups were administered an equal volume of stroke-physiological saline solution by gavage for 4 consecutive weeks.The valve annulus diameter and peak velocity of the mice in each group were then detected using ultrasound.The degree of aortic valve calcification was evaluated using von Kossa and Alizarin Red S staining.The serum triglycerides(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)were detected by biochemical method.Inflammatory factor interleukin-4(IL-4),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-10(IL-10)levels were determined using an enzyme-linked immunosorbent assay.The expressions of calcification markers,runt-related transcription factor 2(RUNX2),and bone morphogenetic protein 2(BMP2)were detected using immunohistochemistry.Aortic valve cell apoptosis was evaluated using TUNEL staining,and JNK/c-Jun signaling pathway-related mRNA and mean fluorescence intensity were detected using quantitative real-time PCR and immunofluorescence,respectively.Results Compared with the control group,the mice in the model group showed an increase in serum TC,TG,LDL-C,TNF-α,and IL-1β levels,a decrease in IL-4 and IL-10 levels,a decrease in annulus diameter,an increase in peak flow velocity,and an increase in von Kossa and Alizarin Red S staining-positive areas.Additionally,the model group showed an increase in aortic valve cell apoptosis rate,an increase in BMP2 and RUNX2-positive rates,and an increase in JNK and c-Jun mRNA expression levels and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Compared to the model group,the PNS low-dose group showed a decrease in serum TC,LDL-C,and TNF-α levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in positive area in Alizarin Red S staining.Furthermore,the PNS low-dose group showed a decrease in BMP2 and RUNX2-positive rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).The PNS high-dose group showed an increase in HDL-C,IL-4 and IL-10 levels,a decrease in serum TC,LDL-C,TNF-α,and IL-1β levels,an increase in annulus diameter,a decrease in peak flow velocity,and a decrease in von Kossa and Alizarin Red S staining-positive areas and cell apoptosis rate.The PNS high-dose group also showed a decrease in BMP2 and RUNX2 positive staining rates,JNK and c-Jun mRNA expression levels,and p-JNK/JNK and p-c-Jun/c-Jun(P<0.05).Conclusion PNS may reduce valvular cell apoptosis,alleviate inflammation,and protect against aortic valve calcification in mice by inhibiting the activation of JNK/c-Jun signaling pathway.