| Objective:To explore the anti-inflammatory mechanism of Liangxue Tongyu prescription in treating cerebral hemorrhage and study on the influence of intestinal flora.Methods:Improvement of autologous blood injection method is adopted to establish the cerebral hemorrhage rat model,observing after 24 h,72 h,120 h three time points.From ethology,brain water content,the permeability of blood brain barrier and cerebral hematoma content to observe the influence of Liangxue Tongyu prescription on cerebral hemorrhage rats damage index;From the aspects of defecation function,intestinal smooth muscle contraction force to observe the effects on intestinal dynamic index;Using western blot to detect NF-κB protein expression of brain tissue,Using ELISA method to detect the expression of TNF-a and IL-1β of brain tissue to research the effect on inflammatory factors;Using immunohistochemical network stations and ELISA method to detect the expression of CCK-8 of brain and intestinal tissues,Using western blot to detect NF-κB protein expression of intestinal tissue,Using western blot to detect PKA,P38MAPK,IκB protein expression of brain tissue to clarify the anti-inflammatory mechanism;Using Illumina miseq 2×300 bp high-throughput sequencing analysis of the structure of intestinal flora.Results;1(1)Ethology score:the neural function of Liangxue Tongyu prescription group improved significantly and defect score lower than the model group(P<0.05,P<0.01)in cerebral hemorrhage 72 h,120 h.Liangxue Tongyu prescription could improve cerebral hemorrhage rats nerve function defect.(2)Brain hematoma:the content of brain hematoma Liangxue Tongyu prescription group was(21.71 + 7.31)μl,significantly lower than that of model group(27.52 + 6.43)μl in 72 h;the content of brain hematoma Liangxue Tongyu prescription group was(16.53 + 6.28)μl,significantly lower than that of model group(24.17+ 7.21)μl in 120 h(P<0.01).It suggested this prescription could obviously promote the hematoma absorption;(3)Brain tissue water content:Liangxue Tongyu prescription group in 72 h,72 h brain tissue water content decreased significantly(P<0.01),and its water content was close to 120 h scores.It could significantly reduce brain edema;(4)Permeability of blood brain barrier:the EB content of Liangxue Tongyu prescription group at each time point decreased significantly(P<0.01).It could obviously improve the permeability of blood brain barrier after intracerebral hemorrhage.2(1)Defecation function:the faeces of model group were qualitative hard or forming,Liangxue Tongyu prescription group were soft,black faeces percentage were significantly higher than that of model group(P<0.01).It revealed Liangxue Tongyu prescription could enhance gastrointestinal dynamics and promote the faeces;(2)Intestinal smooth muscle contraction force:the bowels value of model group was lowest,intestinal contraction frequency,contraction amplitude,the bowels value increased significantly after treatment(P<0.05,P<0.01).It suggested that cerebral hemorrhage could lead to intestinal peristalsis slowed,intestinal contraction force decreased,and Liangxue Tongyu prescription could accelerate intestinal peristalsis,promote the intestinal smooth muscle contraction.3(1)Brain tissue NF-κB P65 protein expression:cerebral hemorrhage model group and Liangxue Tongyu prescription group for 24 h expressed increasingly(p<0.01,p<0.05),Liangxue Tongyu prescription group was significantly lower than model group in 120 h(p<0.01).It revealed Liangxue Tongyu prescription could reduce p-NF-KB P65 protein expression,antagonism inflammatory response after cerebral hemorrhage;(2)Brain tissue TNF-a and IL-1(3:each time point Liangxue Tongyu prescription group of TNF-a and IL-1p content was lower than that of model group(P<0.05,P<0.01).This prescription could reduce the TNF-a and IL-1β expression,playing the role of anti-inflammatory.4(1)Brain tissue around hematoma CCK-8:groups of rats expressed no statistical difference in 24 h,Liangxue Tongyu prescription group of brain tissue expression of CCK-8 was higher than the model group in 72 h and 120 h(P<0.05,P<0.01).It revealed this prescription could increase brain tissue expression of CCK-8;(2)Intestinal tissue expression of CCK-8:each group of rats for 24 h,72 h cool blood stasis group of intestinal tissue expression of CCK-8 were higher than that of control group,model group(P<0.01,P<0.05),in 120h it had no statistical differences.It revealed the rise in intestinal tissue expression of CCK-8 immediately after cerebral hemorrhage,and this prescription could rapidly increase of intestinal tissue expression of CCK-8;(3)Bowel tissue NF-κB P65 protein expression:each time point Liangxue Tongyu prescription group of intestinal tissue P-NF-κB P65 protein expression was significantly lower than model group(p<0.01).It could adersely affect intestinal inflammatory response after cerebral hemorrhage;(4)Brain PKA protein expression:the protein expression in 24 h model and Liangxue Tongyu prescription groups were significantly higher than normal group,control group(P<0.01);the protein expression in 72 h,120 h Liangxue Tongyu prescription group was significantly higher than that of model group(P<0.01).It suggested that this prescription could increase the PKA protein expression,consistent with brain tissue CCK-8 trends;(5)Brain P38MAPK protein expression:the protein expression of model group were significantly higher than normal group,control group in 24 h(P<0.01),while the model group and Liangxue Tongyu prescription group had no obvious difference;in 72 h,120 h the P38MAPK protein expression of Liangxue Tongyu prescription group was lower than the model group(P<0.05).It revealed that this prescription reduce tthe P38MAPK protein expression,in contrast to the brain tissue CCK-8 trends;(6)Brain IκB protein expression:in 24 h,72 h the IκB protein expression of model and Liangxue Tongyu prescription groups were reduced,no significant difference;in 120 h Liangxue Tongyu prescription group was significantly higher than model group(P<0.01),consistent with the normal group.It revealed that this prescription could inhibit the IκB degradation predominate.5(1)Alpha diversity analysis:Chao,Ace index,Shannon index,Simpson index belong grade figure curve showed that rat gut microbes of control group 5 d,Liangxue Tongyu prescription group 5 d and 1 d were highest species richness and evenness,rat gut microbes of model group 3 d,control group 1 d and model group 1 d were lower species richness and evenness.It reflected intracerebral hemorrhage could cause intestinal microbial abundance,microorganism distribution disordered,and Liangxue Tongyu prescription could restore intestinal microbial flora richness,prompte the uniform distribution of gut microbes;(2)Based on the information analysis of species groups of samples of colony structure:for example gut microbes phylum level species results,control group 1 d with Bacteroidetes,Firmicutes and Proteobacteri,in 3 d,5 d Bacteroidetes reduced,Firmicutes increased gradually;Model group 1 d Bacteroidetes were significantly lower than the control group,Firmicutes were higher than that of control group,in 3 d,5 d Bacteroidetes increased gradually,Firmicutes gradually reduce;Liangxue Tongyu prescription group 1 d Bacteroidetes were lower than control way,but higher than that of model group;in 3 d,5 d over time have fewer Bacteroidetes,Firmicutes,trends were similar to that of control group.It revealed Bacteroidetes gradually reduced and Firmicutes gradually increased after treatment of cerebral hemorrhage,these changes were similar to that of control group,and this prescription could restore normal intestinal microbial flora composition;(3)Beta diversity analysis:phylum level sample clustering tree combined with histogram analysis results showed that cold Liangxue Tongyu prescription group in 3 d,5 d Bacteroidetes,Firmicutes and Proteobacteri were similar;Control group in 3 d,5 d Bacteroidetes,Firmicutes were similar with Liangxue Tongyu prescription group 3 d,5 d;Liangxue Tongyu prescription group in 3 d,5 d and control group were similar.It revealed this prescription treatment of cerebral hemorrhage could restore the normal microbial flora.Conclusions:Liangxue Tongyu prescription could inhibit the expression of inflammatory factors such as NF-κB,TNF-a and IL-1β,alleviate cerebral hemorrhage inflammatory reaction,thus it promoted the hematoma absorption,reduced bleeding in the brain edema,improved the permeability of blood brain barrier and nerve function.This prescription could promote intestinal peristalsis and intestinal smooth muscle contraction,increase intestinal tissue,brain tissue CCK-8 levels.Brain tissue CCK-8 inhibited the brain tissue NF-κB p65 protein expression by increasing PKA,IkB protein expression,reducing P38MAPK protein expression,regulating brain gut interaction.Intracerebral hemorrhage could decrease gut microbes richness,disturb microorganism distribution;while Liangxue Tongyu prescription could restore intestinal microbial flora richness,prompt the uniform distribution of gut microbes,and restore the normal microbial flora. |
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