| Objective: Severe hemorrhagic shock（SHS）has been one of the most challenging threats to global public health,commonly occurring in patients with trauma,perioperative bleeding,or gastrointestinal bleeding.In the ischemia-reperfusion（I/R）injury after hemorrhagic shock,the gut was often considered as a “motor” predisposing to the development of systemic inflammation response syndrome（SIRS）,multiple organ dysfunction syndromes（MODS）.Shenfu injection,as one of the Chinese medicine formulations frequently used for clinical management,is rich in several alkaloids and amino acids.The research about sepsis and cardiogenic shock has confirmed that Shenfu injection can effectively reduce the release of inflammatory factors,improve microcirculation status,and prevent intestinal mucosal epithelium injury.However,the effect of Shenfu injection on the intestinal I/R injury after severe hemorrhagic shock is unclear.Besides,the optimal dosing regimen of Shenfu injection also remains unknown.Thus,this study aims to assess whether Shenfu injection can protect the I/R injury after severe hemorrhagic shock by improving the intestinal microcirculation condition under the detection of sidestream dark-field（SDF）technology,and exploring the optimal dosing time.Methods: In a model of SHS,rats were randomly divided into four groups: 1)Sham group（n=6）;2)conventional resuscitation group（control group,n=6）;3)early resuscitation by using Shenfu injection（SF group,n=6）;4)delay resuscitation（after one hour）by using Shenfu injection（SF-delay,n=6）.During the shock phase,rats bled to a mean arterial pressure（MAP）of 40±5 mm Hg and maintained for 40 minutes.The rats in SF and SF-delay groups were resuscitated by blood and Shenfu injection while the dosing volume was 10ml/kg.The control group received a total blood draw volume combined with equal volumes of 0.9% saline.The duration of resuscitation was 40 min,then the rats were observed continuously for 4 hours.The intestinal microcirculation condition was assessed by SDF.Besides,the lactate,PH,and BE in the arterial blood were measured from each period.Pathological specimens were stained with hematoxylin-eosin and Periodic acid-Schiff to observe villi’s construction and the distribution and number of goblet cells.The I/R‐induced mucosal injury was evaluated according to the Chius scoring system.The levels of IL-6 and TNF-α in plasma and the concentration of MDA,DAO proteins in intestinal tissues at four hours after resuscitation completion were determined by Enzyme-linked immunosorbent assay（ELISA）.The expression levels of tight junction proteins,Claudin-1 and ZO-1 were analyzed using Western Blotting Immunoblotting（WB）.Results: 1.The change trending and comparison of blood gas indexes in each period:no statistical differences between groups for baseline weight,temperature,MAP,lactate,BE,and PH value（P>0.05）.After maintaining shock for 40 min,the lactate and BE value levels in the CON,SF,and SF-delay groups were significantly higher than that in the Sham group,while the PH level was significantly reduced.Furthermore,when comparing with the CON group,the BE（SF group: P=0.001;SF-delay group: P=0.003）and PH（SF group: P=0.02;SF-delay group: P=0.004）were significantly improved at two h post-resuscitation;levels of lactate tended to be reduce in the SF-delay group;however,the differences were statistically insignificant;in addition,significant differences were found in all blood gas indexes between SF and SF-delay group.At four hours post-resuscitation,compared with the CON group,the arterial lactate content in the SF group（P<0.001）and the SF-delay group（P<0.001）was significantly decreased,but the difference between the latter two groups was not statistically significant;the changing trend of BE was also similar to that of lactic acid（SF vs.CON,P<0.001;SFdelay vs.CON,P<0.001;SF vs.SF-delay,P=0.638）;however,there was no significant difference in PH between the SF group,the SF-delay group and the CON group.2.Change trend and comparison of intestinal microcirculation perfusion indexes in each period: by using SDF,the real-time monitoring of intestinal microcirculation results show that the intestinal microcirculation perfusion indexes in the CON group,SF group and SF-delay group were significantly deteriorated（P<0.001）after shock maintenance for 40 min.At two hours after resuscitation,the total vessel density（TVD）and the heterogeneity index（HI）of the SF group were significantly improved when compared with those of the CON group（TVD: P=0.044;HI: P=0.004）and SF-delay group（TVD:P=0.026;HI: P=0.004）;in addition,the microcirculation flow index（MFI）in the SF group was also significantly improved than that in the SF-delay group（P=0.021）.At four hours after resuscitation,the De Backer score（p<0.001）,perfused small vessel density（PSVD）（P=0.012）,TVD（P<0.001）,MFI（P=0.001）and HI（P<0.001）)in the SF group was significantly better than those in CON group;meanwhile,SF-delay was also better than CON group in terms of PSVD（P=0.026）and HI（P=0.007）;Compared with the SF-delay group,the status of PSVD（P=0.012）,TVD（P=0.04）and MFI（P=0.042）in the SF group was significantly better than those in the SF-delay group.3.Comparison of intestinal pathology in each group: HE staining results showed that the small intestinal mucosa in the CON group was significantly damaged,which was manifested by the appearance of the subepithelial space of the intestinal mucosa in the central villus,shedding of the propria membrane,hemorrhage,and ulceration at four hours after resuscitation.Compared with the CON group,the intestinal mucosal damage in the SF and SF-delay groups was less severe,and epithelial shedding and subepithelial space expansion rarely occurred.In addition,the CHIUS scores of the SF group（4.67±0.52 vs.3.0±0.63,P<0.001）and the SF-delay group（2.5±0.55 vs.3.0±0.63,P<0.001）were lower than those of the CON group,and the difference was statistically significant,while there was no significant difference between the SF group and SFdelay（P=0.119）.The results of PAS staining showed that the number of goblet cells and mucopolysaccharides in the intestinal mucosal epithelium of the CON group were significantly reduced,and were distributed in isolation at four hours after resuscitation;compared with the CON group,the number of goblet cells and the content of mucopolysaccharide in the SF and SF-delay groups were significantly increased.4.Comparison of serum inflammatory factor levels and intestinal injury-related proteins DAO and MDA contents in each group: ELISA results showed serum IL-6,TNF-α,and DAO,MDA expression in the intestinal tissue of the CON group all increased significantly（all P<0.001）.In terms of the levels of pro-inflammatory factors,compared with the CON group,the serum levels of IL-6 and TNF-α in the SF group were significantly decreased（IL-6,P<0.001;TNF-α,P<0.001）;in addition,compared with the SF-delay group,the IL-6 levels in the SF group was also lower（P<0.001）.Compared with the CON group,the expressions of DAO and MDA in the SF group and the SF-delay group were significantly decreased（both P< 0.001）;the MDA and DAO concentration in the SF group were also lower than those in the SF-delay group（P<0.001）.5.Comparison of the content of tight junction proteins in the intestinal tract of each group: WB results showed that the contents of tight junction proteins ZO-1 and Claudin-1 in the CON group were significantly decreased at four hours after resuscitation（P<0.001）.Compared with the CON group,the expression levels of ZO-1and Claudin-1 in the SF group were significantly higher（ZO-1,P=0.045;Claudin-1,P=0.004）.Although the levels of ZO-1 and Claudin-1 in the SF-delay group were higher than those in the CON group,the differences were all insignificant（ZO-1,P=0.23;Claudin-1;P=0.06）.At the same time,there was no significant difference in the expression of the above two proteins with the SF-delay group and SF group（ZO-1,P=0.66;Claudin-1,P=0.27）.Conclusions:1.During the resuscitation stage after severe hemorrhagic shock,the state of rat systemic circulation is not consistent with the recovery of intestinal microcirculation perfusion;2.Compared with conventional fluid resuscitation,Shenfu injection can effectively improve the level of intestinal microcirculation after SHS,inhibit the release of proinflammatory factors,reduce the damage of intestinal villi and mucosa,and protect the tight junction protein between epithelial cells;3.Compared with the delayed administration of Shenfu injection,the early administration of resuscitation has a more significant protective effect on intestinal injury after SHS. |
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