| Background:Over the past years,the incidence of type 2 diabetes(T2D)has greatly increased worldwide.Prediabetes is an intermediate category between normal glucose tolerance and T2 D,which is much more prevalent in the modern society.Many studies have shown that prediabetes were at a high risk to develop into T2 D afterwards.But its pathogenesis has not been fully understood.A lot of evidence indicates that the composition of gut microbiota,which may affect the body weight,bile acid metabolism,activation of inflammation,insulin sensitivity and regulation of gut hormones,plays a critical role in the pathogenesis of obesity and T2 D.Although the exact mechanism about how gut bacteria affecting host's metabolism is not clear,many studies have confirmed that the application of probiotics,prebiotics,antibiotics,fecal microbiota transplantation and other ways to alter the intestinal flora structure greatly affect the host metabolism and insulin sensitivity.Tea has been consumed by the human for thousands of years.In general,there are two categories of teas,i.e.green tea and black tea or unfermented tea and fermented tea.Puer tea is a kind of fermented black tea.Many studies have indicated that it has a protective effect on metabolic syndrome,but the mechanism underneath is unclear.It is well known that human gut harbors the largest number of microbes.At present,about 3.3 million microbial genes from human gut have been clarified.Human beings and micro-organisms have been benefited each other from a symbiotic relationship for a long time.Very recently,it has been reported that supplement of dietary fibers greatly affects the composition of gut microbiota,thereby improving the metabolic control in T2 D.However,whether the ground Puer tea leaves,which is a dietary component in common use of daily life and particularly rich in fermented fibers,affect the composition of gut microbiota has not yet been studied.Most importantly,the imbalanced gut microbiota,i.e.either deficiency of beneficial or enrichment of detrimental intestinal flora has recently been indicated to be one of the most important causes for the development of many diseases,including many metabolic diseases such as T2 D and obesity.In present study,we investigated whether consumption of the Puer tea powder affects the composition of gut microbiota,thus affecting the metabolism in the patients with prediabetes.We further examined the relevant mechanism associated with Puer tea treatment-induced patho-physiological changes.Methods:1.Subjects who met eligibility criteria were divided to one of two study arms: Control group(n=18)administered 200-300 ml warm water three times a day after meals for 12 weeks;Puer consumption group(n=13)(administered 1g Puer tea powder)with 300 ml warm water three times a day after meals for 12 weeks.The function of liver and kidney,blood lipid,uric acid,glycosylated hemoglobin,blood glucose and islet function were measured by immunoluminescence,and the changes of the indexes before and after treatment were observed.2.The expression of CD3 + CD4 + IL10 cells,CD3 + CD4 + IL17 cells,CD14 + TLR4 cells and CD3 + CD4 + Foxp3 cells were detected by flow cytometry.The changes of inflammation and anti-inflammatory factors were observed before and after treatment.3.The expression of IL-10,IL-17,LPS and ADP in peripheral blood were detected by ELISA method,so as to evaluate the inflammatory response before and after treatment.The quantitative changes of GLP-1 and GLP-2 were measured by ELISA to observe the improvement of insulin resistance and the change of intestinal permeability.4.The genomic changes of intestinal flora in pre-diabetic patients were evaluated by 16 SrRNA assay,and the changes of protective relative flora and obese relative flora before and after treatment were determined.5.The SCFAs of patients' peripheral blood and feces were measured by GC-MS,and the changes of the contents of acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid and valeric acid were observed before and after treatment.Meanwhile,the effects of Puer tea on intestinal and peripheral inflammatory reactions were evaluated.Result:1.Fasting blood glucose and postprandial blood glucose at 1hour after meal(P1BG)decreased slightly(p>0.05),the postprandial blood glucose at 2 hour after meal(P2BG)was significantly decreased than the baseline in Control group(p=0.012).The serum uric acid level was significantly increased in the Control group(p < 0.05),but no change was found in the Puer tea-treated group.The urine PH was increased in the Puer tea-treated group(p < 0.05),but this is not the case in the Control group during the study period.The blood cholesterol and low-density lipoprotein in puer group decreased significantly verse the control group(p=0.005,p=0.032).2.Puer tea consumption significantly improved postprandial serum insulin levels at the 1 and 2 hour after meal as shown by the D-value(the difference of the value of endpoint minus the value of baseline)(p < 0.05),though there was no significant difference in either basal level or in the fasting level of insulin between these two groups.The blood C peptide levels measured at the 1 and 2 hour after meal in the Puer tea-treated group were increased significantly(p < 0.05),but this was no different in the Control group.3.The D-values of levels of serum GLP-1 at both fasting status and the 2 hour after meal in the Puer tea-treated group were significantly(p< 0.05)higher than that of the Control group at the12 th week of study period.The D-value of the fasting serum GLP-2 level was significantly(p< 0.05)higher in the Puer tea-treated group compared to that of the Control group at the 12 th week of study period.4.The D-value of the serum LPS was obviously decreased after 12 weeks of treatment,and the change of serum LPS after the 12-week treatment in the Puer tea group was significantly different from that of the Control group(p< 0.05).The D-value of the serum adiponectin(ADP)level was significantly increased after the 12 week treatment of Puer tea,which was a startling contrast to that observed in the control group.The change of serum ADP level after 12-week treatment in the Puer tea group was significantly different from that of the control group(p < 0.05).5.We performed 16 SrRNA gene sequencing of patients' fecal bacteria by using third-generation sequencing technology.Among the 164 genera of gut bacteria analyzed,the abundances of 15 genera were significantly(p< 0.05)increased at the 12 th week of the treatment in the Puer tea group verse the baseline.These 15 genera were not changed by the treatment in the control group.By using Pearson's correlation test,we found that among these 15 genera,the Puer tea-induced increases of Frankiaceae,Ruminococcus,Rhodospirillaceae and Burkholderiaceae were positively correlated with the increase in postprandial serum levels of GLP-1(r=0.8161,p=0.0022;r=0.5742,p=0.05;r=0.7278,p=0.0073;r=0.7288,p=0.0072;respectively).Phyllobacteriaceae's increase was highly correlated with the increase in fasting serum level of GLP-2(r=0.9315,p=0.0003).Moreover,the increases of Rikenellaceae and 1-68 were positively correlated with the increase of serum ADP level(r=0.7523,p=0.0048;r=0.6423,p=0.0243;respectively).Lactobacillaceae's increase was negatively correlated with the serum level of LPS(r=-0.6427,p=0.0242).6.The concentrations of both fecal and serum SCFAs were not changed in the control group(p > 0.05 for all).The concentrations of both fecal and serum butyric acids were significantly increased at the 12 th week of consumption of Puer tea(p < 0.05 for both),but no significant change was found in the concentration of either acetic acid,propanoic acid or valeric acid after the 12 th week of Puer tea treatment in either feces or serum(p> 0.05 for all).7.Peripheral blood representation of CD3+ CD4+ IL-17 cells was slightly decreased in Puer group,the difference between the end and baseline of the two groups was significant(p< 0.05).CD3+ CD4+ IL-10 cells was significantly increased in the blood of Puer group(p< 0.05),but no changed in blood of the Control group subjects(p> 0.05),the difference between the end and baseline of the two groups was significant(p< 0.05).CD14 + TLR4 cells increased significantly in the Control group,while the number of CD3+ CD4+ Foxp3(Treg)cells increased significantly in the Puer tea group(p< 0.05).Conclusion:1.Compared with the basic information of the patients in the Puer group and the Control group,drinking puer tea powder for 12 weeks,the metabolism of blood glucose,blood lipid and uric acid in the patients were improved significantly.The level of blood insulin and c-peptide in the patients also had a corresponding increase.It suggested that drinking puer tea powder was related to the metabolism of the patients in the pre-diabetes period.2.The level of serum GLP-1 and GLP-2 of pre-diabetes patients were improved correspondingly by drinking puer tea powder,thus improving the insulin sensitivity of pre-diabetes patients.And the increasing of serum GLP-2 level affected the changes of intestinal flora by improving the permeability of intestinal wall.3.Evaluate the genomic changes of intestinal flora in patients with pre-diabetes mellitus by16 SrRNA.15 bacteria in the subjects dranking puer tea powder had significant changes.The changed bacteria were positively related to the levels of GLP-1,GLP-2 and SCFAs in serum and feces,which indicated that puer tea powder could affect blood and intestinal flora by increasing the level of GLP-2,so as to change the abundance of intestinal flora.The level of SCFAs in feces and blood improved the changes of blood glucose and blood lipid by promoting the level of GLP-1.4.Detecting the expression of CD3+ CD4+ IL-10 cells,CD3+ CD4+ IL-17 cells,CD14+ TLR4 cells and CD3+ CD4+ Foxp3 cells,we can prove that puer tea powder can regulate the changes of inflammation and anti-inflammatory factors,and then inhibit the low-grade inflammatory response of the body,so as to improve the obesity,blood glucose and lipid metabolism.The above results provided a solid theoretical basis for us to understand the mechanism of puer tea powder in improving glucose and lipid metabolism of pre-diabetes patients,and also provided a new method for the treatment of pre-diabetes. |
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