The Effects Of Dendrobium Officinale Polysaccharide On Glucose Metabolism Disorder In HFD-fed Mice And Its Potential Mechanisms

Dendrobium officinale is a perennial herb of the genus Orchidaceae,a valuable traditional Chinese medicine.Since ancient times,Dendrobium officinale has been known as the “medicine of gold” and is the first of the nine major Chinese grasses.The records about it have been known for thousands of years.It has been used as a traditional Chinese medicine for the treatment of diabetes since ancient times.Dendrobium officinale polysaccharide(DOP),which is isolated from the herbs of Dendrobium officinale,has significant pharmacological activities.Modern medical researches have shown that DOP has anti-oxidant,anti-cancer,anti-fatigue,hypoglycemic and lipid,immune regulation and liver protection effects.However,the underlying mechanisms for the hypoglycemic effect of DOP remains unclear.This study aimed to investigate the therapeutic effects of DOP on glucose metabolism disorder in HFD-fed mice and its potential mechanisms.In this study,the herbs of Dendrobium officinale was used as the raw material to obtain high-purity DOP by extraction and purification.To establish a HFD-fed mice model in vivo and an insulin resistance Hep G2 cells model in vitro to explore the molecular mechanisms of DOP in improving glucose metabolism disorder,providing a theoretical basis and research basis for clinical application and health product development of DOP.Part One: The extraction,purification and identification of DOPIn this paper,the herbs of Dendrobium officinale was used as the raw material,crude DOP was obtained by the method of water extraction combined with alcohol precipitation,then the refined DOP was acquired after repeated freeze-thawing removal and ultrafiltration.The identifications of DOP were determined.The polysaccharide content of DOP was more than 98%,and the weight average molecular weight of DOP was 3.6×105 Da.The UV scan showed that there was almost no nucleic acid and protein.The results showed that DOP was mainly composed of Mannose and Glucose,with the ratio of 4.06:1.00.The experiments in this part showed that the polysaccharide extracted from Dendrobium officinale had high purity and uniform molecular weight,and it was almost free of impurities.The experiment was easy to repeat,the control was stable and the result was stable.The DOP could be used in the subsequent pharmacological study.Part Two: The effects of DOP on improving glucose metabolism disorder in HFD-fed miceFeeding BALB/c mice with HFD results in glucose and lipid metabolism disorders and insulin resistance,which are similar to the early symptoms of human T2 DM,and are consistent with the processes and mechanisms of diabetes caused by changes in human dietary structure.Then the mice were treated with low-,middle-and high-dose DOP for four weeks.In the experiment,body weight and fasting blood glucose were monitored regularly;Oral glucose tolerance test(OGTT)was performed before the mice were sacrificed;Liver index,serum insulin and liver TG contents were measured;H&E and ORO histological staining liver tissue slices were produced.The experimental results showed that FBG levels in HFD-fed mice were significantly reduced after DOP treatment.In addition,the OGTT test confirmed that DOP can significantly improve glucose tolerance in HFD-fed mice.Moreover,DOP treatment significantly increased serum insulin levels,decreased the homeostasis model assessment of insulin resistance(HOMA-IR)and increased the insulin sensitivity index(ISI),indicating that DOP can promote insulin secretion,ameliorate insulin resistance and increase insulin sensitivity in HFD-fed mice.Furthermore,DOP can significantly reduce liver index and liver TG levels in HFD-fed mice.H&E staining and oil red O staining showed that livers of HFD-fed mice had severe steatosis and fat accumulation.Similarly,DOP could significantly improve the above-mentioned conditions.This part of the experiment proved that DOP has significant effects in reducing blood glucose,improving glucose tolerance,relieving insulin resistance,and improving hepatic steatosis and fat accumulation.Part Three: The mechanisms of DOP on improving glucose metabolism disorder in HFD-fed miceThe molecular mechanisms of DOP to improve glucose metabolism disorder in HFD-fed mice were discussed from two aspects: glycogen synthesis and glucose metabolism.We detected hepatic glycogen contents;Liver PK,HK and PEPCK activities were measured;The expression of p-GSK-3β,GSK-3β,p-GS,GS,HIF-1α,p-m TOR,m TOR,p-IR-β,IR-β,p-IRS1,IRS1,p-PI3 K,PI3K,p-Akt and Akt in the livers of mice were detected by Western blot(WB);The m RNA levels of HIF-1α,GLUT1,HK-2,IR-β,PTK and PTP 1B in the livers of mice were measured by real-time PCR.The experimental results show that DOP can significantly increase liver glycogen contents,and significantly inhibit the activity of glycogen synthase kinase(GSK-3β)and increase glycogen synthase(GS)activity in HFD-fed mice.On the other hand,DOP can significantly increase the activity of PK and HK in the livers of HFD-fed mice,accelerate glycolysis,and significantly reduce the activity of PEPCK and inhibit gluconeogenesis.In addition,DOP significantly increased the protein expression level and m RNA level of HIF-1α in livers of HFD-fed mice,increased the transcription of downstream target genes GLUT1 and HK-2,and promoted glycolysis.The WB method detected the liver PI3K/Akt signaling pathway-related proteins in HFD-fed mice.The results showed that DOP can significantly up-regulate the expression of pathway-related proteins,indicating that DOP can stimulate the PI3K/Akt signaling pathway in the liver of HFD-fed mice,and thus play a role in improving the glucose metabolism disorder.In addition,we found that m RNA level of PTK was significantly decreased,whereas m RNA level of PTP 1B was significantly increased in the livers of HFD-fed mice.However,after four weeks of DOP treatment,m RNA level of PTK was significantly increased,while m RNA level of PTP 1B was significantly decreased.This finding suggests that the role of DOP in increasing insulin receptor phosphorylation levels may be related to the regulation of PTK and PTP 1B m RNA levels.This part of the experiment proved that DOP improves glucose metabolism disorder via the regulation of PI3K/Akt-mediated glycogen synthesis and glucose metabolism(including accelerate glycolysis and inhibit gluconeogenesis)in HFD-fed mice.Part Four: Study on the regulation of PI3K/Akt signaling pathway by DOP in vitroTo further clarify the activation of PI3K/Akt signaling pathway by DOP,we established an insulin resistant Hep G2 cell model and cultured with DOP intervention.After DOP intervention,the expression of PI3K/Akt signaling pathway-related proteins was significantly up-regulated,while PI3 K inhibitor LY294002 could offset this effect of DOP,once again confirming that DOP can up-regulate PI3K/Akt signaling pathway.