[The effects of activated protein C on the von Willebrand factor and von Willebrand factor cleaving protease of rat aortic endothelial cell induced by lipopolysaccharide].

OBJECTIVE: To investigate the activated protein C (APC) on the von Willebrand factor antigen (vWFAg) and von Willebrand factor cleaving protease (ADAMTS-13) protein expression in rat aortic endothelial cells (RAECs) induced by lipopolysaccharide (LPS). METHODS: RAECs from Wistar rats were cultured with the tissue explants adherence method. RAECs were cultured for one week, After one week culture, RAECs in 4-5 generations were divided into control group, LPS stimulation groups (1 mg/L) and APC intervention groups (0.1, 1 and 10 mg/L APC was added after LPS stimulation). The supernatants were obtained at 12, 24, 48, and 72 hours after LPS stimulated to determine the vWFAg and protein of ADAMTS-13 expression by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: In the control group, RAECs expressed little vWFAg and protein of ADAMTS-13. With stimulation of LPS, the vWFAg was significantly increased at 12 hours, and reached the peak at 48 hours [(285.45±30.13)%], and the level of ADAMTS-13 (µg/L) was gradually decreased, and reached the nadir at 72 hours (13.32±2.37), there was significant difference compared with control group [vWFAg: (94.53±7.83)%, ADAMTS-13: 115.76±2.36, both P<0.01). The effects on vWFAg promoting and ADAMTS-13 inhibition after LPS stimulation could be dose-dependently reversed by APC. 10 mg/L of APC could decrease the peak of vWFAg at 48 hours of LPS stimulation [(198.43±17.92)% vs. (285.45±30.13)%], and increase the minimize of ADAMTS-13 (µg/L) at 72 hours of LPS stimulation (125.25±2.70 vs. 13.32±2.37), with significant difference (both P<0.01). CONCLUSIONS: After stimulation with LPS, the level of vWFAg was time-dependent increased, as the protein of ADAMTS-13 was decreased. APC could attenuate the effect of LPS on vWFAg and protein of ADAMTS-13 with dose-dependent and time-dependent patterns.

[Induction of expression of von Willebrand factor cleaving protease of rat aortic endothelial cell by lipopolysaccharide challenge].

OBJECTIVE: To investigate the dose response effect and time response effect of lipopolysaccharide(LPS) on von Willebrand factor cleaving protease (ADAMTS-13, Vwf-cp) mRNA expression and protein in rat aortic endothelial cells (RAECs). METHODS: RAECs were grown by culturing of aortic tissue. When ARECs were cultured for one week, it was co-cultured by 1:3 to reach 4-5 generations. ARECs were randomly divided into five groups: control group and four LPS stimulation groups (0.01, 0.1, 1 and 5 µg/ml) . The RAECs and supernatants were obtained at 12, 24, 48, and 72 hours after being stimulated by LPS. ADAMTS-13 mRNA expression of RAECs was assessed by quantitation reverse transcription-polymerase chain reaction (RT-PCR), and protein of ADAMTS-13 in supernatants was determined by enzyme linked immunoadsorbent assay (ELISA). RESULTS: In the control group RAECs were shown to express ADAMTS-13 at both protein and mRNA levels. With the increase of concentration of LPS, or increase in stimulus duration, expression of ADAMTS-13 mRNA and protein were gradually lowered. Compared with the control group (25.22 ±1.41), the level of ADAMTS-13 mRNA in 0.01 µg/ml LPS stimulation group was markedly decreased at 48 hours (18.78±0.86, P<0.01). At 24 hours, the levels of ADAMTS-13 mRNA (23.43±0.63, 22.41±0.76) were markedly decreased in 0.1 µg/ml and 1 µg/ml LPS stimulation groups (P<0.05 and P<0.01). The level of ADAMTS-13 mRNA (20.01±2.47) in 5 µg/ml LPS stimulation group was markedly decreased at 12 hours (P<0.01). Compared with the control group [(115.76±2.36) ng/ml], protein level of ADAMTS-13 [(113.43±1.07) ng/ml] was markedly decreased at 12 hours in 0.01 µg/ml LPS stimulation group (P<0.05). The protein level of ADAMTS-13 [(7.63±2.64) ng/ml] was lowest in 5 µg/ml LPS stimulation group at 72 hours (P<0.01). CONCLUSION: Normal RAECs can express ADAMTS-13 at both mRNA and protein to certain extent. The expression of ADAMTS-13 mRNA and protein are decreased after LPS challenge in different concentrations for different duration in dose dependent and time dependent manner.

[Effect of Xuebijing injection on expression of endothelial protein C receptor and protease activated receptor 1 mRNA and protein in endothelial cells induced by lipopolysaccharide].

OBJECTIVE: To investigate the effect of Xuebijing injection on expression of endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1) mRNA and protein in rat aortic endothelial cells (RAECs) after lipopolysaccharide (LPS) challenge. METHODS: RAECs were cultured for one week, and the purity was determined with flow cytometry. ARECs were randomly divided into four groups: control group, LPS stimulation group (1 mg/L LPS), Xuebijing injection treatment group (LPS: 1 mg/L, Xuebijing injection ; 10 g/L) and activated protein C (APC) treatment group (LPS: 1 mg/L, APC: 0.1 mg/L). The RAECs were collected at 12, 24, 48, and 72 hours after being stimulated by LPS. EPCR and PAR1 mRNA of RAECs were assessed by reverse transcription-polymerase chain reaction (RT-PCR). EPCR and PAR1 protein were assessed by flow cytometry. RESULTS: At 12 hours, EPCR and PAR1 protein expressions were not significantly different among groups (all P>0.05). The level of EPCR and PAR1 mRNA were decreased in LPS stimulation group, but they were elevated in both APC and Xuebijing injection treatment groups (P<0.05 or P<0.01). From 24 to 72 hours, compared to control group, the levels of EPCR mRNA and protein expression were significantly decreased after LPS stimulation (P<0.05 or P<0.01). The levels of EPCR expression were decreased and negatively correlated with the time of LPS treatment. Also, compared to LPS stimulation group, treatment with Xuebijing markedly elevated the levels of EPCR (P<0.01). The levels of PAR1 expression were significantly decreased by LPS stimulation compared with those of control group (P<0.05 or P<0.01). After the treatment with Xuebijng, the expression of PAR1 was gradually increased (all P<0.01). Compared with APC treatment group, Xuebijing could increase the PAR1 expression better. CONCLUSION: Xuebijing could raise EPCR and PAR1 mRNA and protein expression of RAECs after LPS challenge, and it may be related to its protection of endothelial cell from undergoing apoptosis.

[Effects of Xuebijing injection on protein C and tumor necrosis factor-alpha mRNA in rats with sepsis].

OBJECTIVE: To investigate the effects of the integrated traditional Chinese medicine Xuebijing injection on protein C (PC) and tumor necrosis factor-alpha (TNF-alpha) mRNA in rats with sepsis. METHODS: Sepsis was induced in Wistar rats by cecal ligation and puncture (CLP). Ninety-six healthy animals were randomly divided into four groups: normal group, sham-operation group, CLP model group, and Xuebijing-treated group. The two latter groups were divided into 2, 8, 24, 48, and 72-hour subgroups with 8 rats in each subgroup. Platelet count of blood obtained from abdominal aorta was determined and tissue samples from liver and lungs were collected to measure tissue PC and TNF-alpha mRNA expression. RESULTS: PC gene expression levels in lung tissues were significantly lowered (all P<0.01), but they were dramatically raised by Xuebijing injection during 8-72 hours post-CLP (all P<0.01). Compared with normal group, TNF-alpha mRNA levels in liver and lungs were significantly elevated at 2 hours post-CLP (P<0.05 or P<0.01). However, treatment with Xuebijing injection markedly reduced TNF-alpha mRNA both in liver and lungs at 2-24 hours (P<0.05 or P<0.01). In CLP group, blood platelet count was significantly decreased to certain extent at different intervals within 8-72 hours, and it was markedly elevated in the Xuebijing-treated group (P<0.05 or P<0.01). CONCLUSION: The current study suggests that Xuebijing injection could exert preventing effect on the development of severe sepsis by suppressing PC and TNF-alpha mRNA.

[Effects of Xuebijing injection on thrombomodulin and endothelial cell protein C receptor in septic rats].

OBJECTIVE: To investigate effects of the integrated traditional Chinese medicine Xuebijing injection on thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) in septic rats. METHODS: Ninety-six healthy Wistar rats were randomly divided into four groups: control group, sham operation group, cecal ligation and puncture (CLP) model group, and Xuebijing-treated group. Sepsis was reproduced by CLP. The two latter groups were divided into five subgroups of 2, 8, 24, 48 and 72-hour with 8 rats in each subgroup. Tissue samples from liver and lung were collected to determine tissue TM and EPCR mRNA expression. RESULTS: TM and EPCR mRNA expressions were observed in liver and lung in control group and sham operation group, while with no significant differences at 2 hours post-CLP (both P>0.05). TM and EPCR gene expression levels in tissues were significantly increased to certain extent at 8-48 hours (all P<0.01), and were dramatically decreased following Xuebijing injection at 72 hours post-CLP (both P>0.05). Also, treatment with Xuebijing injection markedly decreased TM and EPCR mRNA levels to certain extents at 8 and 24 hours, and markedly increased at 48 and 72 hours compared with those of model group. CONCLUSION: These data suggest that Xuebijing injection could raise TM and EPCR mRNA expression, thereby it might be effective in prevention of development of severe sepsis.