Study Of Mechanism Of Baicalin On Metabolic Inflammation In The Respect Of Intestinal Flora And Permeability

Objective:As economy developed rapidly in recent years, the living standard of people improved substantially. However many factors such as increasing life stress, sedentary habit, unhealthy diets lead to rising morbidity of hyperlipemia. The intake of nutrients and surplus of metabolism frequently result in increase of inflammatory factor and lipopolysaccharides(LPS) in human body, which gives rise to metaflammation eventually. The metaflammation has a significant effect on formation and development of hyperlipemia and complications.Considering the complex mechanism of metaflammation, the major factors are as follows:one is the change of structure and ratio of intestinal flora. The increasing of Gram-negative bacterium leads to large amount of LPS. The other is destruction of intestinal mucosal barrier result from the increase of LPS, which contrarily leads to more LPS getting into circulatory system via intestinal mucosa, thus a vicious circle forms.Baicalin is a monosome that extracted from a kind of clearing heat antitoxicant traditional Chinese medicine-scutellaria baicalensis. It has various pharmacologic actions especially in treatment of intestines problem and anti-inflammatory.Our preliminary experiments performed externally in cell level proved that baicalin could promote the expression of ZO-1 mRNA and corresponding Protein in IEC-6 induced by LPS, which can protect intestinal mucosa to a certain extent.This study established a hyperlipidemic mouse model and discussed the following effects of baicalin in animal level:(1) Role of anti-metaflammation(2) Role of interpretion and expression of tight junction protein and corresponding mRNA in hyperlipidemic mouse(3) The structure and abundance of intestinal flora in hyperlipidemic mouseMethodsThirty-two male C57BL/6J mice were randomly divided into 4 groups, namely normal group, model group, low-and high--ose baicalin groups (25, 50mg· kg-1 · d-1). The experiment conducted after a week of adaptative feeding. Mice of normal group were fed with ordinary forage and the other groups were fed with high-fat forage for 8 weeks. Mice of low-and high-dose group were given gastric gavage of different concentration of baicalin for last 5 weeks while the other two groups were treat with normal saline simultaneously. Recorded body mass, living and physical condition of mice. Killed the mice by anesthesia in the end of eighth week and collected their serum, excrement and intestinal tissues. Blood lipid, tumor necrosis factor (TNF-a), interleukin 6 (IL-6) and LPS levels were determined by enzyme-linked immunosorbent assays (ELISA). Effects of baicalin on expression of Occludin, ZO-1 mRNA and microRNA 212,122 in mice intestinal tissues were detected by qRT-PCR. While for tight junction protein Occludin and ZO-1, a method of Western blot was applied. Gene sequencing of intestinal flora was conducted by Miseq system.Results1. There was no significant difference in mass among all groups of mice in the beginning or after 3 weeks of high-fat feeding(p> 0.05). However the mass of model group mice was obviously higher than that of normal group after eight weeks of treatment (P< 0.05).2. The level of low density lipoprotein(LDL) and total cholesterol(TC) in model group increased notably (p< 0.01) compared with that of normal group while the level of high density lipoprotein(HDL) decreased remarkable (P< 0.01). However there was no statistical difference in Blood lipid among model group, low-and high-dose baicalin groups (P> 0.05) after intervention of different dose of baicalin.3. The level of IL-6, TNF-a and LPS in model group increased notably (P < 0.01) compared with that of normal group. The level of IL-6, TNF-a and LPS in high-dose baicalin group were decreased (P<0.05 or P<0.01), and LPS level of low-dose baicalin group was also decreased (P<0.01).4. The expression of ZO-1 and Occludin mRNA in model group decreased notably (P< 0.05) compared with that of normal group. However there was no significant difference in expression of ZO-1 and Occludin mRNA among model group, low-and high--ose baicalin groups (P> 0.05)5. There was no significant difference in expression of microRNA 122 and 212 between model group and normal group (P> 0.05). It also differed rarely among model group, low-and high-dose baicalin groups (P> 0.05)6. There was no significant difference in expression of protein ZO-1 and Occludin between model group and normal group (P>0.05). It also differed rarely among model group, low-and high-dose baicalin groups (P> 0.05)7. Results of gene sequencing of intestinal flora(1) Analysis of diversity showed that the number of OTUs in model group is higher than that in normal group (P< 0.05). The number of OTUs in high-dose baicalin group decreased notably compared with that in model group (P< 0.05). The Shannon index of model group was lower than that of normal group (P< 0.05) and low-dose baicalin group (P< 0.05) The ACE and Chaol index of model group was higher than that of normal group while differed rarely with low-and high-dose baicalin groups. The coverage indexs of normal, model and high-dose baicalin group were all above 0.9.(2) The abundance of Deferribacteres and Proteobacteria of model group was significantly higher at phylum level, while the abundance of Verrucomicrobia was significantly lower than that of the normal group. At Genus level, the abundance of Christensenella, uncultured_Peptococcaceae and Desulfovibrio of the model group was significantly higher, while the abundance of Coprococcus, Akkermansia and uncultured_Lachnospiraceae was significantly lower than that of the normal group. The abundance of Desulfovibrio of both low- and high-dose baicalin groups was lower than that of the model group. The ratio of gram-negative bacterium to gram-positive bacterium of the model group increased as compared with the normal group(P<0.05), while that of low-dose baicalin group was decreased significantly compared with the model group (P<0.05).Conclusion1. Baicalin can’t bring down the blood lipid level, but it has the capability to reduce IL-6, TNF-α and LPS level so that has therapeutic effect on metaflammation.2. Eight weeks of high fat feeding causes the decrease of expression of ZO-1 and Occludin mRNA. But the intervene of baicalin can’t improve it.3. Eight weeks of high fat feeding and the intervene of baicalin has no obvious impact on genetic expression of microRNA 122 and 212.4. High fat diet lead to the increase of Christensenella, uncultured_Peptococcaceae and Desulfovibrio and reduce the number of Coprococcus, Akkermansia and uncultured_Lachnospiraceae. The ratio of gram-negative bacterium to gram-positive bacterium of the model group also increases. The intervene of baicalin causes the decrease of Desulfovibrio and the ratio of gram-negative bacterium to gram-positiv ones.