| Circulatory failure is the main factors of refractory hypotension and multiple-organ injury after hemorrhagic shock (HS). As an important constituent part of circulatory system, the lymph circulation plays an important role in the regulation of body homeostasis. Some studies in vivo or in vitro have been documented that, the contractile activity of lymphatics during the process of HS presented a biphasic change, which is manifested with an increase at early shock and a decrease at late shock, and has a close relation with the development of shock. As is known, the pumping function of lymphatic vessels is dynamics basis of lymph circulation. So, a good modulation of pumping function is very important to prevent the severe shock.The pumping function of lymphatic can be regulated by lots of humor factors. As well as related to the modulation of contraction, relaxtion and tention of lymphatic under physiological status, the periodical changes of NO also involve in the regulation of pumping function of lymphatic in the process of HS; ATP-sensitive K+channel (KATP) regulated the pumping function by modulating the membrane potential of lymphatic smooth musle under physiological status. Foreign studies found that NO regulated the contractility of lymphatic by KATP through cGMP/cAMP dependent protein kinase under physiological status. So whether KATP involves in the NO on the regulation of pumping function of lymphatics after HS is still a problem to be solved. And make the KATP as a target spot will be important for the studies to regulate the lymphatics after HS. As to this reserch, the changes of cAMP, PKA, cGMP, PKG were studied in the process of HS. Then the technique of pressure myograph was applied to observe the effect of tool agents of KATP, cAMP/PKA, cGMP/PKG, NO/NOS on the contractility of shock lymphatics and the response to substance P (SP). At the end, the role and mechanisme of KATP in the modulation of NO on isolate lymphatics from HS rats was investigated to provide evidence for the modulation of lymphatic pumping function during the process of shock.In the first part of experiments,126male Wistar rats were randomized into control (only with anesthetization and laparotomy, n=21) and shock group (mean arterial pressure being maintained at40mmHg through reinfusing or withdrawing blood; rats in shock group being further divided into shock0h, shock0.5h, shock1h, shock2h and shock3h subgroups also with21cases in each group). The thoracic ducts were isolated from control group and shock group at different time points. Then the thoracic duct was dissected free for detecting. Then take out twelve thoracic ducts from each group to prepare the tissue homogenate, and detect the content of cAMP, cGMP, p-PKA, p-PKG by ELISA; and take out nine thoracic ducts tissues to extract protein from each group for detecting the PKG by western blot. The results indicated that the concentrations of cAMP, p-PKA are increased gradually along with the development of shock, and they both increased significantly at shock0.5h and later time. Similarly, the concentrations of cGMP, p-PKG also increased gradually along with the development of shock, they both increased significantly at shock2h and the later time, the concentration of PKG protein is increased significantly at shock oh, but with the development of shock, the content of PKG is decreased to the level of control group.Forty-two male Wistar rats, in the second part of experiments, were randomized into control, shock0.5h (n=6), after preparing the thoracic duct then transferred it into the chamber of pressure myograph, When spontaneous phasic contractions were observed, the lymphatic vessels were equilibrated at3cmH2O for30min. After this period, the contraction frequency (CF), end systolic diameter (ESD), end diastolic diameter (EDD) and passive diameters (PD) of lymphatics were measured and recorded, from these datas the tonic index (TI), fractional pump flow (FPF), contraction amplitude (CA) were calculated to assess the pumping function of lymphatics. And the others were incubated with L-Arg (NO doner), L-Arg+H89(PKA inhabitor), L-Arg+Glibenclaimde (KATP inhabitor),8-Br-cAMP (PKA doner),8-Br-cAMP+Gli separately after spontaneous phasic contractions were observed, then the CF, EDD and ESD were recorded to assess the effect of KATP on the signal way of NO-cAMP-PKA regulating the pumping function of isolate lymphatics from HS rats. The results showed that the CF, FPF, TI of Shock0.5h lymphatics increased significantly compared with control, and the CF, FPF, TI decreased significantly when incubated with L-Arg. Among them, the FPF and TI were lower than control, When the Shock0.5h lymphatics was done with H-89and L-Arg, the CF and FPF all higher than shock0.5h+L-Arg; When the Shock0.5h lymphatics incubated with8-Br-cAMP, the CF and FPF were all decreased significantly, and when the shock0.5h lymphatics incubated with8-Br-cAMP and Gli, the CF was significantly higher than control.To further evaluate the reactivity of shock lymphatic, the different values between pre-and post-administration of gradient SP (10-8mol·L-1,3×10-8mol·L-1,10-7mol·L-1,3×10-7mol·L-1) of CF, CA, TI and FPF were calculated and expressed these values as△CF,△TI, ACA and△FPF after the index of contractility records. The results suggedted that the△CF,△FPF and△TI of Shock0.5h increased significantly compared with the control at certain concentration conditions of SP; L-Arg decreased the△CF,△FPF and△TI of shock0.5h at certain concentration conditions of SP, Gli can inhabit the effect of L-Arg on the index of△CF,△FPF and△TI at certain concentration conditions of SP. Among them, the△CF was higher than the control at certain concentration conditions of SP;8-Br-cAMP can decrease the△CF,△CA,△FPF and△TI of Shock0.5h at certain concentration conditions of SP compared with control, Gli inhabit the effect of8-Br-cAMP, and increased the△CF,△FPF and△TI significantly. These data indicated that KATP involes in the NO regulating the pumping function of shock early lymphatics, and the effect may be related to the signal pathway of NO-cAMP-PKA.In the third part of experiments,42male Wistar rats were used to duplicate the model of shock to prepare the isolated of shock2h lymphatics, and the method mentioned above was used to record the contractility and reactivity (n=6). After spontaneous phasic contractions were observed, the other thirty-six lymphatics were incubated with L-NAME (NOS inhabitor), ODQ (sGC inhabitor), KT-5823(PKG inhabitor), Pinacidil (opener of KATP)10min, divided into groups of shock2h+L-NAME, shock2h+L-NAME+pinacidil, shock2h+ODQ, shock2h+ODQ+pinacidil, shock2h+KT-5823, shock2h+KT-5823+pinacidil (n=6) and the same method was applied to observe the changes of contractility and reactivity, then to discuss the role of KATP in the NO regulation of the pumping function of shock lymphatics. The results demonstrated that the CF, FPF, TI of shock2h decreased significantly compared with control, L-NAME can increase the CF, FPF, TI; ODQ can increase the CF, FPF; KT-5823can increase the CF, FPF; after incubated with Pinacidil respectively, the CF, FPF when incubated with L-NAME decreased; the CF, FPF when incubated with ODQ decreased. In addition, the CA was recovered; the CF, FPF when incubated with KT-5823decreased. At the aspect of reactivity, the results indicated that the△CF,△FPF,△CA of shock2h decreased compared with the control; L-NAME can increase the△CF,△FPF,△CA of shock2h; ODQ can increase the △CF,△TI, decrease the△FPF; KT-5823can increase the△CF,△FPF,△TI of shock2h; Pinacidil can decrease the△CF,△FPF,△TI which were elevated by L-NAME, decrease the△CF which was elevated by ODQ, in further increasing the△C A,△TI, also decrease the△CF which was elevated by KT-5823. The results indicated NO induced the hypo-contractility and hypo-reactivity of severe shock through the signal pathway of cGMP-PKG which may be related to KATP.In summary, in the process of shock, the contractility and reactivity of lymphatics both presented a biphasic change during the process of HS, which is manifested by an increase at early shock and decrease at late shock by using the technique for shock lymphatic investigation in vitro. KATP involves in the NO regulating the pumping function of isolated lymphatics, the signal pathway may be ralated to NO-cAMP-PKA, NO-cGMP-PKG. Targeting KATP, the pumping function of shock lymphatics can be regulated. |
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