| Objective:Constipation is a complex symptom caused by a variety of common etiologies which is difficult to cure.Prolonged constipation not only affects the digestion and absorption of patients,but also leads to the disorder of intestinal flora,induces or aggravates enteritis,cardiovascular and cerebrovascular diseases,and even relates to the occurrence of cancer.Guided by the theory of syndrome differentiation and treatment,traditional Chinese medicine has different treatment methods for constipation caused by different causes.As for the treatment of asthenia constipation,often use "relaxing bowel "medicine of hemp seed,which can often be received very satisfied with the results.The author' s research group considered that "relaxing bowel" can be interpreted as "nourishing" and "purgative activity M double effects of hemp seed.Previous studies have shown that hemp seed for constipation or aging animal models can relieve constipation,improve blood circulation,regulate immunity,reduce free radical damage.And it can prolong the survival time of silkworm,which has certain anti-aging effect.It also plays an important role on regulating the growth of various intestinal bacteria or in vivo colonization.However,for the mechanism of hemp seed with " relaxing bowel "should be further clarified.Recent studies have shown that intestinal flora is critical for health,and take the method of adjusting microflora as an important way to prevent disease and aging.Further studies have shown that intestinal flora can affect health by interfering with the expression of TLRs pathways.Therefore,this study investigated the regulation of intestinal flora 'and its impact on the TLRs signaling pathway.Therefore,this study from the perspective of intestinal flora regulation and its effect on TLRs signaling pathway,to explore the mechanism of hemp seed with " relaxing bowel " to provide the basic information of the application and development and new ideas and reference for basic research of Chinese Medicine.Methods:1.Establishment of Wistar rats/ratted constipation model.20 SPF Wistar rats were randomly divided into two groups.According to the dose of 10mg/kg,model group rats were given by gavage,continuous administration of 20d.When the experiment was end,rats were detected the physiological indexes(changes in body weight changes,the proportion of intestinal contents and intestinal microflora;tension);index(test ratio of G+/G-in the cecum;use PCR-DGGE to detect advantages of bacteria changes in the cecum;24h feces,test the change of Bifidobacterium,Lactobacillus and Escherichia coli in fecal).HE staining of rat colon tissue.2.Effects of hemp seed oil on intestinal microflora and the TLRs receptor signaling pathway.48 SPF Wistar rats,sex in half,were randomly divided into control group,model group(l0mg/kg),hemp Seed Oil group(21.6g/kg),positive control group(2.7g/kg),except the blank group was given distilled water,the other groups used compound diphenoxylate.2h later,give the corresponding drug treatment.Detection of rat physiological index(the change of body weight,intestinal contents,defecation in 24h).Determine TLR4,MyD88,NF-kB and other related factors of TLRs signaling pathway in serum by ELISA.Intestinal flora detection index(bacteria change;G+/G-ratio in the cecum;the change of dominant microflora).Gas chromatography method for the determination of SCFAs in rat cecum);HE staining of rat coton tissue.3.Effects of the water extract of hemp seed on intestinal microflora and the TLRs receptor signaling pathway.48 SPF Wistar rats,sex in half,were randomly divided into control group,model group(10mg/kg),water extract group(21.6g/kg),positive control group(2.7g/kg),except the blank group was given distilled water,the other groups used compound diphenoxylate.2h later,give the corresponding drug treatment.Detection of rat physiological index(the change of body weight,intestinal contents,defecating in 24h).Determine TLR4,MyD88,NF-kB and other related factors of TLRs signaling.pathway in serum by ELISA.Intestinal flora detection index(bacteria change;G+/G-ratio in the cecum;the change of dominant microflora).Gas chromatography method for the determination of SCFAs in rat cecum).HE staining of rat colon tissue.Results:1.The rat model of Wistar constipation was established successfully.Compared with the blank group,the frequency of ileum in constipation rats decreased(P<0.05).Intestinal contents of the weight percentage of the whole intestine increased(P>0.05).The amount of defecation decreased 24h(P<0.01).The number of fresh fecal Escherichia coli and Bifidobacterium increased(P>0.05,P<0.05),Lactic acid bacteria decreased(P<0.01).The total number of bacteria in the cecum increased and the ratio of G+/G-decreased(P<0.05);PCR-DGGE results showed that the total amount of bacteria decreased in constipation rat cecum,some bacteria increased,some is lost or reduced to its abundance detection;structure of colon in rats were severely damaged(P<0.01).2.Hemp seed oil can reduce the intestinal contents of rats with constipation accumulation(P>0.05).Hemp seed oil can reduce the level of MyD88,TLR4,IL-6,IFN-a(P>0.05,P>0.05,P>0.05,P>0.05)and increase the level of NF-kB,Klotho protein,TNF-a(P>0.05,P>0.05,P>0.05).Hemp seed oil can promote defecation,increase fecal water content(P>0.05,P>0.05).Hemp seed oil adjust the level of Escherichia coli,Bifidobacterium and Lactobacillus in fecal(P>0.05,P>0.05,P>0.05).The ratio of G+/G" increased in the cecum(P<0.01);Hemp seed oil can reduce the level of Firmicutes bacteria in constipation rat and increase the level of Bacteroides bacteria.Hemp seed oil can increase the level of acetic acid,propionic acid,butyric acid in constipation rat caecal(P<0.01,P>0.05,P<0.01).Hemp seed oil reduced the damage of intestinal structure of rats with constipation(P>0.05).3.The water extract of hemp seed can reduce the intestinal contents of rats with constipation accumulation(P>0.05).The water extract can reduce the level of TLR4,MyD88,NF-kB,IFN-a,TNF-a(P>0.05,P>0.05,P>0.05,P>0.05,P>0.05)and increase the level of Klotho protein,IL-6(P>0.05,P>0.05).The water extract can promote defecation,increase the water content in fecal(P>0.05,P>0.05).The water extract adjust the level of Escherichia coli,Bifidobacterium and Lactobacillus in fecal(P>0.05,P>0.05,P>0.05).The ratio of G+/G-increased in the cecum(P<0.01);The water extract can reduce the level of Firmicutes bacteria in constipation rat and increase the level of Bacteroides bacteria also reduced the level of acetic acid,propionic acid,butyric acid in constipation rat caecal(P<0.05,P>0.05,P<0.01).The water extract reduced the damage of intestinal structure of rats with constipation(P>0.05).Conclusion:1.The intestinal flora of constipation rats is disorder,and it can aggravate the constipation.2.Hemp seed oil and the water extract can alleviate constipation,promote the disorder of intestinal flora balance,reduce recovery of animal models of colitis injury,regulating the expression of TLRs pathway.3.Hemp seed oil and the effect of water extract on animal models of constipation have certain differences,effects of different characteristics.4.Based on the previous hemp seed improve the level of blood lipid in ageing animal model,immunity and reduce oxidative damage.We speculated that the heep seed play play the role of moistening the intestines and freeing the stool is through the improvement of intestinal microflora and regulating TLRs pathway... |
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