| The lymph circulation is an important component of circulatory system and the systolic and diastolic functions of lymphatic vessels are the major propellent power of lymph circulation. Previous studies had showed that lymphatic contractility underwent a biphase chang in vivo and in vitro, that is, being elevated in early stage and reduced in late stage. Moreover, the contractility of lymphatcs to vasoactive mediator norepinephrine (NE) or substance P (SP) had a similar change. These findings suggested that the hyporesponsiveness of lymphatics is an important mechanism of the reduced lymphatic contractile activity. As is known to all, lymph circulation dysfunction is an important factor in exacerbating the progress of irreversible shock. So, the role of reduced lymphatic contractile activity and its mechanisms in the pathogenesis of shock deserve further research.Research showed that small Rho GTPases Rac1and RhoA play very important roles in regulating the biphasic change of vascular reactivity induced by hemorrhagic shock, and the balance of RhoA and Rac1activity also regulates vascular reactivity after hemorrhagic shock (HS). It has been confirmed that Rho was responsible for the physiologic pump activity of lymphatics in vitro. However, the following questions are still unknown:What is the protein expression of RhoA and Racl in shocked lymphatics? Do RhoA and Rac1involve in the regulation of reduced contractile activity of lymphatics after shock? Which factors are its possible mechanisms related to? And is the lymphatic contractility affected by the interaction between RhoA and Racl? The elucidation of these questions will have important theoretical and practical value for further studying the biological modulation of lymphatic contractility and improvement of lymph microcirculation dysfunction during HS.Therefore, this paper, by duplicating the model of HS in rats, observed the expression of RhoA and Rac1in lymphatic vessel. Furthermore, a platform of technique for shock lymphatic investigation in vitro is adopted through a pressure myograph and the effects of RhoA and Racl on the isolated lymphatic contractility was observed by using the tool agents of the signaling pathways of RhoA-Rho kinase-myosin light chain phosphatase (MLCP) and Racl-PAK-myosin light chain kinase (MLCK). Meanwhile, the reactivity of isolated lymphatics to SP was observed in an attempt to provide evidence for the regulation of lymphatic contractile activity during the process of shock.126male Wistar rats were randomLy divided into control (only anesthetization and laparotomy) and shock groups (duplicating the model of HS according to the tranditional methods in our laboratory; rats in this group were further divided into shock0h, shock0.5h, shock1h, shock2h and shock3h subgroups). The thoracic ducts were isolated from sham-operated and shocked rats (n=21),9lymphatic vessels of each group were used to detect the protein expression of RhoA by western blotting and the remaining12lymphatic vessels of each group were used to detect the content of phosphorylated proteins of RhoA, Racl, PAK and MLCK (p-RhoA, p-Rac1, p-PAK and p-MLCK) by enzyme-linked immunosorbent assay (ELISA). These results showed that the protein expression of RhoA in lymphatics was biphasic change, that is, the level of RhoA was significantly elevated in shock0h and shock0.5h groups and reduced in shock2h and shock3h groups. Meanwhile, the levels of p-RhoA and p-MLCK obviously increased in shock0h and shock0.5h groups and decreased in shock1h, shock2h and shock3h groups. At the same time, the contents of p-Rac1and p-PAK decreased at shock0.5h and increased after shock1h. Subsequently, this study observed further that the effects of the signaling pathways of RhoA-Rho kinase-MLCP and Rac1-PAK-MLCK on the biphase change of lymphatic contractility and reactivity.Firstly, fifty-four male Wistar rats were randomized into control (n=6), shock0.5h group (n=18) and shock2h group (n=30). The thoracic duct was dissected free, transferred to the chamber of pressure myograph and equilibrated at3cmH2O. Once spontaneous phasic contractions were observed, the0.5h-shocked lymphatic vessels were incubated with RhoA antagonist C3exotransferase (C3,60μg·L-1, as shock0.5h+C3group, n=6) and Rho kinase antagonist Y-27632(6×10-6mol·L-1, as shock0.5h+Y-27632group, n=6), respectively. The2h-shocked lymphatic vessels were incubated with RhoA agonist U-46619(5×10-7mol·L-1), Y-27632+U-46619, myosin light-chain phosphatase (MLCP) antagonist Okadaic acid (OA,1×10-6mol·L-1, as shock2h+U-46619group), OA+U-46619, respectively (as shock0.5h+Y-27632group, shock2h+U-46619group, shock2h+Y-27632+U-46619group, shock2h+OA group, shock2h+OA+U-46619group, n=6, respectively). After the equilibration period, end systolic diameter (ESD), end diastolic diameter (EDD), contraction frequency (CF) and passive diameters (PD) were measured, from which the tonic index (TI=(PD-EDD)/PD×100%), contraction amplitude (CA=(EDD-ESD)/PD×100%) and fractional pump flow (FPF=EDD2-ESD2)/EDD2×CF as the evaluation index of lymphatic contractility were calculated. The indicators of contractile activity of control,0.5h-and2h-shocked untreated lymphatic vessels (n=6, respectively) served as control to assess the effects of RhoA on lymphatic contraction. The results suggested that Y-27632can remarkably decrease the CF, TI and FPF of0.5h-shocked lymphatics, CF and FPF were significantly lower than that in the control group. U-46619elevated the CF, TI and FPF of2h-shocked lymphatics to the control levels. Y-21632suppressed the effect of U-46619and declined the elevated CF, TI and FPF of2h-shocked lymphatics by U-46619. OA reduced the CF, TI and FPF of2h-shocked lymphatics, but had no significant difference compared with2h-shocked lymphatics. OA suppressed the effect of U-46619. These tool agents, except C3, have no significant influence on the CA of lymphatics. C3elevated the CA of0.5h-shocked lymphatics. These data suggested that RhoA plays a major regulating role in the biphasic change of lymphatic contraction after HS, its mechanism may be related to the Rho kinase. The effects of MLCP should be further proved.After the baseline values in each group and lymphatics treated by tool agents were registered, the response of lymphatics to SP (1×10-8mol·L-1,3×10-8mol·L-1,1×10-7mol·L-1,3×10-7mol·L-1) was measured. The indice of CF, EDD, ESD and PD within1minute after the administration of SP were recorded, from which the TI, CA and FPF were calculated. Taken the values of maximum difference of△CF,△TI,△CA and△FPF before and after administration of SP, the influence of RhoA on reactivity of lymphatics was assessed during the process of HS. The results suggested that both C3and Y-27632reduced the△CF of0.5h-shocked lymphatics at various concentration conditions of SP, also reduced the TI and△FPF at certain concentration conditions of SP. U-46619elevated the△CF,△TI,△FPF and reduced the△CA of2h-shocked lymphatics at various concentration conditions of SP. This effect of U-46619was suppressed by Y-27632. But there was no important difference between shock2h+U-46619group and shock2h+Y-27632+U-46619group. OA significantly elevated the△CF,△FPF and reduced△CA,△TI of2h-shocked lymphatics at various concentration conditions of SP, the statistical analysis indicated that there were significant differences compared with control and shock2h group. This effect of U-46619was suppressed by OA without significant difference compared with shock2h+U-46619group. The findings suggested that RhoA involves in the biphasic modulation of shocked lymphatics and its effect might be achieved by Rho kinase and MLCP. Secondly,36male Wistar rats were randomLy assigned to shock0.5h group (n=18) and shock2h group (n=18). According to the methods described earlier, the thoracic duct preparation was isolated at the corresponding time points from rats in each group. Once spontaneous phasic contractions were observed, the0.5h-shocked lymphatic vessels were incubated with Racl agonist PDGF (20μg·L-1), myosin light chain kinase (MLCK) inhibitor ML-7(1×10-5mol·L-1) and PDGF+MLCK agonist SP (1×10-7mol·L-1) respectively (as shock0.5h+PDGF group, shock0.5h+ML-7group, shock0.5h+PDGF+SP group, n=6, respectively). The2h-shocked lymphatic vessels were incubated with Racl inhibitor NSC23766(5×10-6mol·L-1), PAK inhibitor PAK-18(1×10-5mol·L-1) and SP, respectively (as shock2h+NSC23766group, shock2h+PAK-18group, shock2h+SP, n=6, respectively). After the equilibration period, the ESD, EDD, CF and PD were measured. The results suggested that PDGF reduced the CF, TI and FPF of0.5h-shocked, TI was significantly lower than that in the control group. The CF, TI and FPF of0.5h-shocked were reduced by ML-7; these indices were significantly lower than that in the control and shock0.5h groups. Moreover, the reduced CF, TI and FPF by PDGF were supressed by SP, significantly higher than that in the control group. The CF, TI and FPF of2h-shocked lymphatics were elevated by NSC23766, PAK-18or SP, higher than the control and shock2h groups. Furthermore, the CA of2h-shocked lymphatics were significantly elevated by PAK and reduced by SP, respectively, and have significant difference with the control group. These data suggested that Rac1plays a role in the biphasic change of lymphatics contraction after HS; its effect might be achieved by PAK and MLCK.After the baseline values of0.5h-and2h-shocked and tool agents treated lymphatics registered, the response of lymphatics to SP (1×108mol·L-1,3×10-8mol·L-1,1×10-7mol·L-1,3×10-7mol·L-1) was measured. The indices of CF, EDD, ESD and PD were recorded and the TI, CA and FPF were calculated according to the methods described above. Taken into consideration the values of△CF,△TI,△CA and△FPF obtained in the first part experiment as control, the influence of Racl on reactivity of lymphatics was assessed during the process of HS. The results suggested that PDGF reduced the△CF,△TI and△FPF of0.5h-shocked lymphatics at various concentration conditions of SP. At certain concentration conditions of SP, the△CF,△CA,△TI and△FPF of0.5h-shocked lymphatics were reduced by ML-7. Both NSC23766and PAK-18significantly elevated the△CF,△FPF and reduced△CA,△TI of2h-shocked lymphatics at various concentration conditions of SP, the△CA and△TI were significantly lower than the control and shock2h groups. These data indicated that Racl involves in the biphasic modulation of shocked lymphatics and its effect might be achieved by PAK and MLCK.To investigate the effects of interaction between RhoA and Rac1on lymphatic contractility and reactivity in HS rats,24male Wistar rats were subjected to HS for2h. The lymphatics preparation was isolated at the corresponding time points from rats in each group. Once spontaneous phasic contractions were observed, the2h-shocked lymphatic vessels were incubated with PDGF+U-46619, ML-7+U-46619, C3+NSC23766, NSC23766+Y-27632, respectively (as shock2h+PDGF+U-46619, shock2h+ML-7+U-46619, shock2h+C3+NSC23766, shock2h+Y-27632+NSC23766, n=6, respectively). After the equilibration period, the ESD, EDD, CF and PD were measured. The study showed that PDGF and ML-7suppressed the effect of U-46619and declined the elevated CF, TI and FPF of lymphatics treated by U-46619. C3and Y-27632inhibited the effect of NSC23766and significantly declined the elevated CF, TI and FPF of lymphatics treated by NSC23766. Moreover, the CF, TI and FPF of shock2h+Y-27632+NSC23766group were evidently lower than the control and shock2h group. After the baseline values of2h-shocked and tool agents treated lymphatics registered, the response of lymphatics to SP (1×10-8mol·L-1,3X10-8mol·L-1,1×10-7mol·L-1,3×10-7mol·L-1) was measured, and the changes in reactivity of lymphatics were observed during the process of HS. The results suggested that the effect of U-46619was suppressed by ML-7or PDGF obviously. At several concentration conditions of SP, the△CF and△FPF in shock2h+PDGF+U-46619and shock2h+ML-7+U-46619groups were decreased strikingly compared with the shock2h+U-46619group. Meanwhile, the promotion effect of NSC23766was supressed significantly by C3or Y-27632, and the△CF and△FPF were reduced and the△CA and△TI were elevated significantly. These data indicated that RhoA and Rac1involve in the biphasic modulation of shocked lymphatics and the effect between RhoA and Racl is mutual antagonism.In summary, both RhoA and Rac1are involved in the biphasic modulation of shocked lymphatics. The effect of RhoA might be achieved by Rho kinase-MLCP and Rac1achieved by PAK and MLCK. Moreover, the effect between RhoA and Racl is mutual antagonism. Targeting RhoA and Rac1, the contractile activity of shocked lymphatics can be modulated, which provides a novel insight to advancing our understanding of the contractile mechanisms of lymphatics. These results of research provided a very important foundation on intervention of lymphatics dysfunction caused by irreversible shock. |
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