Study On The Mechanism Of Intestinal Gluconeogenesis In The Prevention And Treatment Of Phlegm-damp Constitution Susceptible Type 2 Diabetes With Shenling Baizhu Powder Based On Gut Microbiota

Phlegm-damp physique is a physique state characterized by viscous and heavy turbidity due to the stagnation of water and dampness,which is characterized by obesity,plump and soft abdomen.Its formation is related to the imbalance of the five internal organs.Accompanied by phlegm and dampness,symptoms such as thirst,stroke,chest numbness and other diseases are high incidence diseases.Type 2 diabetes in modern medicine has similarities with the "diarrhea"and "spleen depression" in traditional Chinese medicine.As early as the the pre-Qin period,it has been recognized that insufficient congenital endowment and acquired living habits are important internal factors for its onset.Modern research has also shown that phlegm-damp constitution is a biased constitution type that is prone to diabetes,and it also plays an important role in diabetesrelated complications.However,the mechanism of phlegm dampness physique susceptibility to type 2 diabetes is still unclear.Studies have shown that there is a causal link between intestinal flora in the occurrence and development of obesity and type 2 diabetes.Disorders of intestinal flora can increase the susceptibility of obesity and type 2 diabetes.The possible mechanism may be related to changes in its metabolites.At present,short-chain fatty acid theory,bile acid theory,endotoxin theory,etc.have been proposed.At the same time,the intestinal flora and metabolites will promote the development of phlegm-dampness individuals to type 2 diabetes.This trend can be reversed by adjusting the composition of the flora or improving the structure of metabolites.Therefore,through physical identification,discover the biomarkers related to the intestinal flora and its metabolites,determine the disease progression from obesity to type 2 diabetes,and timely adjust the bacteria through methods such as drugs,bacterial strain biologics,and fecal bacteria transplantation.Group and metabolic structure,improving the phlegm-damp physique state,will be an important way to make use of Chinese medicine to prevent diseases before they occur.Previous studies have recognized the close connection between the normal flora of the gastrointestinal tract and the "spleen" of traditional Chinese medicine.The intestinal flora may be an important biological basis for the physiological functions of the "spleen" of traditional Chinese medicine.More and more studies have proved that the Chinese medicine compound for strengthening the spleen and mobilizing the spleen helps to regulate the intestinal flora and maintain the balance of the intestinal microecological.Shenling Baizhu Powder(SLBZP)is derived from the "The Prescriptions of the Bureau of Taiping People’s Welfare Pharmacy" of the Song Dynasty.It is often used clinically to treat digestive system diseases.In recent years,with the change of the disease spectrum,according to the principle of treating different diseases,it is also widely used in obesity,hyperlipidemia,fatty liver,diabetes and its complications,etc.,are effective.Through the intervention of SLBZP in obese rats caused by high-fat diet,the intestinal gluconeogenesis function can be adjusted by propanoic acid to improve insulin resistance,improve phlegm and damp constitution,prevent obesity,and delay the development of type 2 diabetes.Abnormal changes in the intestinal flora are of great significance to intestinal gluconeogenesis,and it is an effective key target of SLBZP.SLBZP can be used as a drug that acts on the intestinal flora and its metabolites to regulate the balance of intestinal flora and improve insulin resistance,providing an effective strategy for the prevention of diabetes.1.Theoretical researchCollect related literature from spleen therapy,diminish thirst,spleen obliteration,phlegm constitution,diabetes,obesity,intestinal flora,intestinal gluconeogenesis,etc.in classical medical books and modern journals,summarize the symptom,mechanism of the onset of phlegmdampness constitution and thirst,and the relationship between the intestinal flora and intestinal gluconeogenesis,and the relationship between the rule of treatment from the spleen and the related theoretical discussion.The difference of constitution determines the individual susceptibility to disease.The common soil of many related diseases lies in its physical foundation.Physical factors are related to the susceptibility,development and return of diseases.It is helpful to identify early and early intervention and reduce the incidence rate of phlegm dampness constitution.Diabetes is a predisposing disease of phlegm wet constitution.The pathological center of obesity and type 2 diabetes is in spleen,stomach and intestine.SLBZP is a classic prescription for strengthening spleen and moistening.The whole recipe replenishes the Qi of moistening and moistening,restores the function of spleen and stomach,but has many diseases.There is a causal relationship between intestinal flora and human metabolism.Short chain fatty acids are produced by intestinal flora metabolism to activate intestinal glycogenesis.Malonate transmits central signals through portal vein receptor,reduces liver glycogenesis,promotes glycogen synthesis,improves the sensitivity of the body to insulin,maintains blood glucose stability and reaches the homeostasis of glucose metabolism.From the intestinal flora,the important idea of prevention and change of disease is embodied in the effective control of obesity,regulating phlegm and humidity constitution,preventing type 2 diabetes and delaying the progress of disease.2.Experimental researchObjective:To confirm that intestinal flora is an important factor of phlegm dampness constitution susceptible to type 2 diabetes.To experimentally screen the optimal dose of SLBZP for preventing and treating obese type 2 diabetes and its complications,and prove that SLBZP regulates the structure of intestinal flora,increases the secretion of the metabolite short-chain fatty acid propanoic acid,and enhances intestinal gluconeogenesis,improves insulin sensitivity,and explore whether it depends on the development of enterocerebral nerve transmission axis,and use this as an entry point to enrich the scientific connotation of the theory of "preventing disease" in Chinese medicine.Method:1.Intestinal flora transplantation changes the susceptibility of obese rats to type 2 diabetes mellitus:9-week-old spontaneous obese type 2 diabetic model ZDF rats and 9-week-old nondiabetic lean littermate LZ(all males)as a gut microbial donor.After euthanasia under isoflurane anesthesia,the contents of the cecum and colon were collected and combined and diluted in sterile PBS Centrifuge at 20 times at 188g for 5 minutes,filter the supernatant and aliquot for later use.Thirty male 5-week-old LZ recipient rats were randomly divided into LZ-LZg group and LZ-ZDFg group,LZ-PBS group;Thirty male 5-week-old ZDF recipient rats were randomly divided into ZDF-LZg group,ZDF-ZDFg group,ZDF-PBS group.After the rats were adaptively reared,the rats in the transplantation group were given continuous intragastric administration of 1 mL of a broad-spectrum antibiotic mixture for 10 days,and feces were collected to analyze intestinal microbes by 16S rRNA to ensure that the effects of antibiotics on the microbiota are similar between LZ rats and ZDF rats.Two days after the last antibiotic administration,750 μL of cecum/colon supernatant was administered to the recipient rat by gavage every day for 28 consecutive days.Cecum/colon supernatant from ZDF donors was intragastrically administered to ZDF and LZ rats.In a similar manner,the cecum/colon supernatant from the LZ donor was gavaged ZDF and LZ rats.In addition to the above-mentioned rats,rats in the ZDF and LZ groups were intragastrically administered with PBS instead of the antibiotic mixture and donor supernatant.After 28 days,OGTT and ITT tests were performed.Stool samples were tested for 16S rRNA and metabolomics,Western Blotting was used to determine the protein and gene expression of gluconeogenesis in liver and jejunum.2.SLBZP delays the development of obesity to type 2 diabetes in ZDF rats:select ZDF rats and their lean control LZ rats as models,and set up a control group,a diabetes group,low-dose SLBZP group,middle-dose SLBZP group,high-dose SLBZP group,n=10 per group.Measure changes in body weight,water,abdominal circumference,and blood glucose every week,and perform OGTT,ITT tests and open field tests at the end of administration.Automatic glycosylated hemoglobin analyzer measures glycosylated hemoglobin,and automatic serum biochemical analyzer measures triglycerides Ester,total cholestero,high-density lipoprotein,low-density lipoprotein,Elisa measures serum insulin and leptin content,and judges the diabetic status of rats and intervention effect of SLBZP.3.Effects of SLBZP on intestinal microflora and metabolites of ZDF rats:Cecal contents and fecal samples were collected,and 16S rRNA gene amplification and sequence analysis were used.The metabolites in samples were measured by ultra performance liquid chromatography tandem mass spectrometry.Western Blotting and q-PCR were used to determine the protein and gene expression of G6pc,PEPCK and Mut in jejunum,colon and liver.4.Effects of SLBZP on intestinal gluconeogenesis:SD rats were divided into control group,HFD group,sodium propionate group,sodium butyrate group,SLBZP group,HFD+capsaicin group,sodium propionate+capsaicin group,sodium butyrate+capsaicin group,SLBZP+capsaicin group.n=10 per group.The SLBZP group was given high-dose SLBZP,and the other groups were given the same volume of ultrapure water by gavage,1mL/100g body weight per day for 14 days.Measure body weight and water twice a week.Abdominal circumference and blood glucose were measured once a week.After adaptive feeding,dissolve capsaicin in a solution of 8:1:1 in water:ethanol:Tween at a ratio of 80:1,and place sterile cotton with a prepared capsaicin solution(10 mg/mL)around the portal vein for 15 minutes.The animals were allowed to recover from the operation for 1 week,with free eating and drinking.Observe the state of the rat to prevent the wound from splitting and infection.The corresponding feed of each group was changed,and the traditional Chinese medicine group was given intragastric administration for 2 weeks.Carry out OGTT/ITT test and open field test.Perfusion of 3 rats in each group was used to determine cFOS of hypothalamus;Western Blotting was used to determine the protein expression of gluconeogenesis in liver and jejunum.Caco-2 cells are planted on a 6-well culture dish with 106 cells/well and cultured at 37℃.After that,the culture medium was replaced with a culture medium containing 1 mM sodium propionate,1 mM sodium butyrate,and SLBZP medicated serum,and a culture medium containing 10 mM forskolin was used as a blank control group,each with 3 multiple holes.After 24h,cells were collected for analysis.Elisa quantifies cAMP in Caco-2 cell lysates.Western Blotting was used to determine the protein of gluconeogenesis.Result:1.The glucose and lipid metabolism phenotype,intestinal flora structure and metabolic profile of the genetically deficient mice with the deletion of the leptin receptor can be transferred with FMT.This transferable trait has not been realized in the control non-gene mutant mice.In other words,the intestinal flora of ZDF rats already suffering from T2DM can aggravate the susceptibility of early ZDF rats(appearing obesity and IR)to T2DM and accelerate its progress,while intestinal bacteria from healthy lean control mice The group can delay its occurrence and development.Worse symptoms correspond to negative changes in the microbial structure and metabolic profile,and the beneficial bacteria that produce SCFAs decreased,such as Lactobacillus,Roseburia,Clostridium,Coprococcus,Rothia,etc.Metabolites such as 3-hydroxybutyric acid,docosapentaenoic acid n6,3-indolepropionic acid,acetic acid,propionic acid,butyric acid,docosahexaenoic acid and caproic acid were significantly reduced,while the reverse group was the opposite.In short,in disease-susceptible individuals,the intestinal flora becomes a catalyst for the occurrence and development of diseases.In individuals with obesity and insulin resistance phenotype,due to the imbalance of intestinal flora due to external factors,the development of obesity to T2DM is significantly accelerated,and the symptoms are serious.As a hot spot and promising treatment method,FMT can affect the function and metabolism of the intestinal flora of individuals in the early stage of the disease,thereby reversing the disease process.2.SLBZP can effectively improve the abnormal phenotype of glycolipid metabolism in ZDF rats,reduce body weight,food intake and blood glucose,increase glucose tolerance and insulin tolerance,and improve insulin sensitivity,and improve the performance of phlegm wet constitution such as fatigue.In other words,SLBZP can alleviate obesity and improve type 2 diabetes to achieve the effect of disease prevention.And we found that high dose SLBZP has better effect.3.As the disease progresses,the composition of the intestinal flora and metabolites of ZDF rats have changed.The high-dose SLBZP group can effectively reverse these changes.During this process,12 genera including Acinetobacter,Brevundimonas,Burkholderia,Butyricionas,Christensenella,Clostridium,Dehalobacterium,Oscillospira,Phyllobacterium,Prevotella,Rothia,Ruminococcus decreased significantly in the ZDF group,and 3 genera including[Ruminococcus],Blautia,and Coprococcus significantly decreased Rising,SLBZP reversed the abundance of these genera.Twelve different metabolites such as propionic acid,butyric acid and caproic acid may play an important role in it.These may be the biomarkers of SLBZP for preventing and treating obesity-T2DM.Differential bacteria predictive metabolic pathways and metabolite-enriched pathways intersect in gluconeogenesis pathways,butyrate,propionate metabolism,etc.This may be the key pathway or biological process of SLBZP for preventing and treating obesity-T2DM.The gluconeogenesis-related gene and protein expression in the jejunum and colon were checked.After drug intervention,the gluconeogenesis function in the intestine was activated.At the same time,the expression of glycogen-related molecules was inhibited to a certain extent.It may be related to the reduction of insulin resistance and the regulation of glucose and lipid metabolism disorders.The improvement of hypothalamic insulin signaling pathway may play a key central regulatory role.4.After portal vein denervation,the degree of obesity,including body weight,abdominal circumference,blood lipid and fat index,increased in propionate group,but there was no significant change in butyrate group and SLBZP group.This change was related to the amount of food intake,and the change of food intake was related to the transmission of portal vein signal.The trend of insulin,insulin tolerance and open field test was similar.The c-Fos in all regions was increased after taking probutyrate and SLBZP,but capsaicin treated rats no longer showed such activation,and SLBZP group also decreased to a certain extent.It can be seen that SLBZP plays a role through the gut brain nerve axis,but it is not completely dependent on this pathway.The expressions of G6pc,PCK1,PCK2 were significantly increased in Caco-2 cells incubated with butyrate and SLBZP containing serum.cAMP could regulate drug-induced gluconeogenesis related protein expression in vivo and in vitro,suggested that SLBZP can also activate gluconeogenesis related signal pathway directly through cAMP.Conclusion:1.It proves that the intestinal flora plays a directional role in the process of obesity to T2DM,and is more involved in catalyzing the progress rather than making the disease "out of nothing".This work further emphasizes the importance of intestinal homeostasis and the interaction of intestinal flora and genetic defects.It is very important to explore the interaction between intestinal flora and intestinal flora metabolites in the development of obesity-T2DM.The regulation effect of intestinal gluconeogenesis in the body is clear,but the specific mechanism is still unclear.The key role of intestinal flora balance on health has been repeatedly emphasized.It is expected that the future can be expected to correct the body’s insulin resistance,control obesity,and delay its progression to T2DM through dietary structure adjustment or auxiliary therapeutic FMT.2.SLBZP can effectively improve obesity and lipid metabolism,delay the occurrence of T2DM,and regulate glucose metabolism.It can transform intestinal flora,regulate metabolites,increase intestinal gluconeogenesis substrates and energy,and induce IGN.Enhancement to achieve the purpose of improving glucose and lipid metabolism.3.Increased intestinal gluconeogenesis shows metabolic benefits,including the reduce of food intake,body weight,fat mass and liver glucose production,and the increase of insulin sensitivity.SLBZP can activate the expression of IGN related genes and proteins through the gut brain nerve transmission axis or cAMP.This is one of the important mechanisms of SLBZP in the prevention and treatment of obesity susceptible T2DM.To a certain extent,the effect of SLBZP is also closely related to the gut-liver-brain axis.