| ObjectiveThrough overall animal experiments,the mechanism of PPARγ/NF-κB pathway mechanism of compound Huangbai liquid coating to control abdominal infection in rats,improve systemic inflammatory response,protect intestinal mucosal barrier,and intervene in intra-abdominal infection was discussed,in order to provide new ideas for the treatment of abdominal infection by external treatment of traditional Chinese medicine,and provide a good entry point for the elucidation of the mechanism of action of traditional Chinese medicine by modern scientific theory.MethodsPart I:Observation of compound Huangbai liquid coating for the control of abdominal infection and systemic inflammation in rats:The rat model of intraperitoneal infection was established by intraperitoneal injection of 2ml bacterial suspension containing 104Bacteroides fragile colony units and 105Escherichia coli colony units.After two weeks of continuous injection,the rats were treated at different time points,and were divided into four groups:blank control group,model group,antibiotic group and traditional Chinese medicine group.Blank control group:No special treatment was given.Model group:intraperitoneal injection of the same dose of normal saline as compound Phelloderma Phelloderma liquid coating under ultrasonic guidance.Antibiotic group:intraperitoneal injection of gentamicin sterile solution under ultrasound guidance,administration time was 1 week,once a day.Chinese medicine group:intraperitoneal injection of compound Phellodendrin liquid coating under ultrasonic guidance,administration time was 1 week,once a day.After sampling,the rats were killed successively on days 1,3,5 and 7.Observation and detection indicators:1.Observation on the control of abdominal infection by compound Phellodendrin liquid coating:（1）The minimum inhibitory concentration（MIC）of compound Phellodendrin liquid coating was recorded.（2）Bacterial survival rate（CFU）of compound Phelloderma Phelloderma liquid coating was recorded.（3）Complete the configuration of bacterial solution,count the bacterial solution,and record the rat modeling situation.（4）The peritoneal fluid seepage of rats was recorded.（5）The formation of abdominal abscess in rats was analyzed.（6）The number of bacterial colonies in rats’peritoneal lavage solution was calculated.（7）The pathological morphology of rats was observed,i.e.the intestinal mucosa was observed under macroscopic and microscopic observation.2.Observation on the control of systemic inflammation by compound Phellodenticum Phellodenticum liquid coating:（1）Observe the general situation and behavioral changes of rats.（2）Blood routine,liver function and kidney function of rats were tested.（3）HE staining was observed in pathological sections of rat kidney tissue.（4）Serum levels of TNF-α,IL-1,IL-6,PCT,CRP,IL-10,and IL-4 were detected by enzyme-linked immunosorbent assay（ELISA）.Part II:Conservation of intestinal mucosal barrier with compound Huangbai liquid coating and its prevention and treatment mechanism based on PPARγ/NF-κB signaling pathway on systemic inflammation in rats with abdominal i nfectionA rat abdominal infection model was established as described above and divided into four major groups for observation and detection of indicators:empty control group,model group,antibiotic group and Chinese medicine group.1.The role of compound cypress liquid coating to protect the intestinal mucosal barrier:（1）TUNEL method analyzed the improvement effect of compound yellow cypress liquid coating on apoptosis of intestinal epithelial cells in rats with abdominal infection.（2）The effect of compound Huangbai liquid coating on the activity of malondialdehyde（MDA）in colon tissue of rats with abdominal infection was observed.（3）The effect of compound Huangbai liquid coating on the activity of superoxidedismutase（SOD）in colon tissue of rats with abdominal infection was observed.2.Based on PPARγ/NF-κB signaling pathway,the prevention and treatment mechanism of compound Huangbai liquid coating on systemic inflammation in rats with abdominal infection was studied:（1）The expression and tissue localization of key proteins PPARγ,p-p65 and p65 in the PPARγ/NF-κB signaling pathway were observed by immunofluorescence method.（2）Western-blot detected the expression of PPARγ,p-p65 and p65 proteins in rat colon tissue.ResultsPart I:Observation of compound Huangbai liquid coating for the control of abdominal infection and systemic inflammation in rats3.1 Observation of compound yellow cypress liquid coating to control abdominal infection:（1）Minimum inhibitory concentration of compound Huangbai liquid coating:The minimum inhibitory concentration（MIC）of compound Huangbai liquid coating inhibiting Bacteroides fragilis and Escherichia coli in vitro was 460μg/m L and128μg/m L,respectively.（2）Determination of bacterial CFU:When the OD600value of the bacterial suspension is 1,the CFU on the plate medium can be counted visually after dilution of103in the Bacteroides fragilisus suspension,and CFU on the plate medium can be counted visually after the E.coli suspension is diluted 104.（3）Abdominal exudate of rats with abdominal infection with compound Huangbai liquid coating:exudate formation in rats in the model group,antibiotic group and traditional Chinese medicine group.1 day after molding,model group,antibiotic group,There was no significant difference in the amount of peritoneal exudate between the model group and the antibiotic group（P>0.05）,3-5 days after modeling,there was no significant difference between the model group and the antibiotic group（P>0.05）,and the difference was statistically significant compared to that between the model group and the antibiotic group（P<0.05）,and 7 days after modeling,the intraperitoneal exudate of rats in the antibiotic group was lower than that in the model group（P<0.05）,but the rats in the traditional Chinese medicine group were lower than those in the antibiotic group（P<0.05）,the difference was statistically significant.（4）The formation of intra-abdominal abscess in rats with abdominal infection with compound Huangbai liquid coating:1 day after molding,there was no significant difference between the model group,antibiotic group and traditional Chinese medicine group（P>0.05）;3-5 days after molding,the area of intra-abdominal abscess in rats in the traditional Chinese medicine group was lower than that in the model group（P<0.05）;Seven days after molding,the area of intra-abdominal abscess in the Chinese medicine group was significantly reduced,and it was smaller than that in the antibiotic group,and the difference was statistically significant.（5）The effect of compound Huangbai liquid coating on bacteria in the peritoneal lavage solution of rats with abdominal infection:1 day after modeling,there was no significant difference in the colony count of intraperitoneal bacteria in the model group,antibiotic group and traditional Chinese medicine group（P>0.05）;the colony count of the model group was significantly higher than that in the traditional Chinese medicine group and the antibiotic group（P<0.05）3 days after modeling;5 and 7 days after molding,the colony count of the Chinese medicine group and the antibiotic group was significantly lower than that in the model group,and the decrease was more significant in the traditional Chinese medicine group.Seven days after molding,the traditional Chinese medicine group was significantly lower than that in the antibiotic group.（6）Effect of compound Huangbai liquid coating on the pathological morphology of rats:（1）Visual observation:1-7 days after molding,the model group rats had fibrinous purulent inflammation with bleeding in the abdominal cavity,peritoneal omentum hyperemia and edema,purulent fibrous exudate attached to the surface of organs such as liver,spleen,intestinal tube,etc.,and turbid abdominal fluid with odor.The antibiotic group was not much different from the model group for 1-5 days,and the abdominal bleeding decreased 7 days after modeling,and the purulent fibrous exudate decreased.In the Chinese medicine group,the fibrinous exudate in the abdominal cavity decreased3 days after molding,and the turbidity of the abdominal fluid was improved.Seven days after modeling,the rats had abdominal hemorrhage disappeared,the fibrinous exudate was reduced,and the naked eye was significantly less visible than that of the antibiotic group,and the intraperitoneal fluid was clearer and the odor was not obvious.In summary,it can be seen that compound yellow cypress liquid coating can reduce peritonitis in rats and effectively control abdominal infection.（2）Micropathological observation of rat colon tissue:Compared with the blank control group,the intestinal mucosal structure of rats in the model group was significantly damaged,and the intestinal mucosal structure was incomplete under microscopy,the mucosal layer was missing to varying degrees,the gland damage was serious,and the infiltration of generalized inflammatory cells.Compared with the model group,the inflammatory infiltration of the colon tissue of rats in the antibiotic group was improved 5 days after modeling,which was manifested by reduced intestinal mucosal damage and regular glandular morphology.Compared with the antibiotic group,the colon tissue of rats in the traditional Chinese medicine group improved significantly 3 days after modeling,and the intestinal mucosa of rats in the traditional Chinese medicine group had a complete intestinal mucosa structure and abundant goblet cells 7 days after modeling,and no obvious inflammatory cell infiltration was seen.3.2 Observation of compound Huangbai liquid coating to control systemic inflammation:（1）Effect of compound Huangbai liquid coating on the behavior of rats with abdominal infection:rats in the model group and various drug treatment groups showed obvious inflammatory reactions and systemic symptoms after modeling.There were no abnormalities in the normal group,and after treatment with compound Huangbai liquid coating and gentamicin,the intestinal inflammation-like reaction and other symptoms and signs of rats were improved to varying degrees compared with the model group,and the recovery was more obvious in the traditional Chinese medicine group than in the antibiotic group.Effects of compound Huangbai liquid coating on the weight of rats with intraperitoneal infection model:there was no significant difference in body weight between rats in the model group 1 day after modeling,and the weight and weight of rats in the model group,antibiotic group and traditional Chinese medicine group gradually decreased after 3 days,and the weight of rats in the traditional Chinese medicine group gradually increased after 5 days,and the weight of rats in the traditional Chinese medicine group gradually returned to the level of blank control group after 7 days of modeling,and was significantly higher than that of model group and antibiotic group（P<0.05）,which showed that the weight improvement of compound cypress liquid on rats with intra-abdominal infection was better than that of antibiotic group.（2）Effect of compound Huangbai liquid coating on blood routine in rats with abdominal infection:WBS and NEU counts at each time point in the model group were significantly higher than those in the blank control group（P<0.05）;3-7 days after modeling,serum WBS and NEU counts in the antibiotic group and the traditional Chinese medicine group decreased significantly（P<0.05）,and the traditional Chinese medicine group was lower than that in the antibiotic group,and the difference was statistically significant（P<0.05）.Effect of compound Huangbai liquid coating on liver function in rats with abdominal infection:the ALT count of rats in the model group was higher than that in the blank control group at all time points,and 7 days after molding,the ALT level in the traditional Chinese medicine group was significantly lower than that in the model group and the antibiotic group,and the difference was statistically significant（P<0.05）.The ALB count of rats in the model group was lower than that in the blank control group at all time points,and 7 days after modeling,the ALB level in the traditional Chinese medicine group increased（P<0.05）,which was significantly higher than that in the model group and the antibiotic group.Effects of compound Huangbai liquid coating on renal function in rats with abdominal infection:there were no significant differences in BUN and SCr levels at various time points in the blank control group,model group and traditional Chinese medicine group（P>0.05）,while both of them showed a continuous upward trend in the antibiotic group,and compared with the other three groups at the same time,they increased significantly（P<0.05）.（3）Effect of compound yellow cypress liquid on kidney histopathology of rats:the kidney tissue structure of rats in the blank control group,model group and traditional Chinese medicine group was clear,and no abnormalities were found.The kidney damage of rats in the antibiotic group was obvious,and the damage gradually worsened1,3,5 and 7 days after molding,mainly manifested as glomerular atrophy and infiltration of a large number of inflammatory cells.（4）Serum expression of inflammatory factors was detected by enzyme-linked immunosorbent assay（ELISA）:compared to control rats,the values of inflammatory markers TNF-α,IL-1,IL-6,PCT and CRP in serum were significantly higher in the model group（P<0.05）;the expression of anti-inflammatory markers IL-10 and IL-4 was significantly lower（P<0.05）;compared to the model group,the serum expression of inflammatory markers TNF-α,IL-1,IL-6 and PCT were significantly lower in the antibiotic group and the Chinese medicine group;the expression of CRP was lower and the expression of IL-10 and IL-4 was higher,with statistically significant differences（P<0.05）.The expression of serum inflammatory markers TNF-α,IL-1,IL-6 and PCT were significantly decreased;the expression of CRP was decreased and the expression of IL-10 and IL-4 was increased,and the differences were statistically significant.Part II Study on the protection of intestinal mucosal barrier by compound Huangbai liquid coating and its prevention and treatment mechanism based on PPARγ/NF-κB signaling pathway on systemic inflammatory response in rats with abdominal infection.3.3 The role of compound yellow cypress liquid coating to protect the intestinal mucosal barrier:（1）TUNEL method analyzed the improvement effect of compound Huangbai liquid coating on intestinal epithelial cell apoptosis in rats with abdominal infection:compared with the blank control group,the apoptosis rate of intestinal epithelial cells in the model group increased significantly（P<0.05）,compared with the model group,there was no significant difference in the intestinal epithelial apoptosis rate of rats in the antibiotic group（P>0.05）,and the apoptosis of intestinal epithelial cells in the traditional Chinese medicine group was significantly reduced.（2）Effect of compound Huangbai liquid coating on the activity of malondialdehyde（MDA）in colon tissue of rats with abdominal infection:compared with the blank control group,the content of MDA in the colon tissue of rats in the model group was significantly increased（P<0.05）;Compared with the model group,the level of MDA in the traditional Chinese medicine group was close to normal after 7 days of modeling,but there was no significant difference in the serum MDA content in the antibiotic group compared with the model group（P>0.05）.（3）Effect of compound Huangbai liquid coating on the activity of superoxidedismutase（SOD）in colon tissue of rats with abdominal infection:Compared with the blank control group,the activity level of SOD in the colon tissue of rats in the model group decreased significantly（P<0.05）;Compared with the model group,the level of SOD activity in the colon tissues of rats in the antibiotic group and the traditional Chinese medicine group showed an upward trend,and the increase in the traditional Chinese medicine group increased significantly（P<0.05）.3.4 Based on PPARγ/NF-κB signaling pathway,the prevention and treatment mechanism of compound Huangbai liquid coating on systemic inflammatory response in rats with abdominal infection was studied.（1）Effect of compound Huangbai liquid coating on key proteins of PPARγ/NF-κB signaling pathway in colon tissues of rats with abdominal infection:Compared with the blank control group,the content of PPARγprotein in colon tissue of rats in the model group was significantly reduced（P<0.05）,the expression level of PPARγprotein in the traditional Chinese medicine group was significantly increased（P<0.05）,and the antibiotic group did not change much（P>0.05）.Compared with the blank control group,the expression levels of p65 and p-p65 proteins in the rat colon tissue in the model group were significantly increased,and the protein expression of p65 and p-p65 in both the traditional Chinese medicine group and the antibiotic group were effective,but the decrease was more obvious in the traditional Chinese medicine group than in the antibiotic group（P<0.05）.（2）Expression and tissue localization of key proteins of PPARγ/NF-κB signaling pathway in colon tissues of rats with abdominal infection with compound Huangbai liquid coating:positive expression of PPARγ,p65 and p-p65 was observed in rat colonic epithelial cells in all groups,and compared with rat colonic epithelial cells in the blank control group,the expression of PPARγin the model group was significantly reduced,while the expression of p65 and p-p65 was significantly increased,compared with the rat colonic epithelial cells in the model group.The positive expression of PPARγin rats in the compound Huangbai liquid coating group was significantly increased,and the positive expression of p65 and p-p65 was significantly reduced.Conclusions1.Compound yellow cypress liquid coating can control abdominal infection,thereby reducing the systemic inflammatory response in rats with abdominal infection,and has obvious anti-inflammatory effect.2.Compound Huangbai liquid coating can upregulate the expression of PPARγin colon tissue of rats with abdominal infection,downregulate the expression of p65 and p-p65 in colon tissue,inhibit the activation of NF-κB signaling pathway,thereby protecting the intestinal mucosal barrier,preventing the transfer of bacteria and endotoxins into the blood,reducing the systemic inflammatory response of rats,and providing a reference for the clinical treatment of abdominal infection. |
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