Theoretical And Experimental Study Of "Activating Spleen Essence, Dredging Spleen Collaterals" Method In Treatmentof Diabetes(Type2Diabetes)

Diabetes (Type2diabetes mellitus, T2DM) is characterized by polydipsia, polyphagia, polyuria, or sweet urinary and weight loss. Why repeatedly drinking can’t quench your thirst, the drinking water go straightly to lower-jiao and lead to frequent urination? Why polyphagia excessive appetite can’t fill skin and muscle, food essence leak and more and more thinner? The contradiction pathological phenomena occur mainly due to the disorder and imbalance of metabolism and distribution of body’s water and transfusion and use of food essence. Spleen dysfunction of transporting is critical in the pathological process, and there is difference between the concept of "spleen" in Chinese medicine and modern medicine, its role of transporting food essence covers pancreatic function. Pancreatic islet microvascular is not only supply a microenvironment for islet cell survival, but also transport insulin, glucagon and other endocrine hormones secreted by islet cells via blood circulation to the appropriate target organ to regulate the body’s glucose metabolic balance, thus it has great value to protect microvascular in the islets for the prevention of T2DM. In this study, we put forward a novel academic viewpoint "activating spleen essence, dredging spleen collaterals" based on vessel-collateral doctrine to treat diabetes (T2DM) from the"spleen" in TCM, and display its scientific connotation and value through animals experiment and cells experiment, in order to provide a new idea for the prophylaxis and treatment of T2DM.Objective:The T2DM rat models were developed to observe diabetic rats pancreatic microvascular and injury in islet cells and the intervention effects of Chinese herbs preparation composition "JINLIDA granule(JLD)and TONGXINLUO capsule(TXL)", the representative composition under the guidance by the theory " activating spleen essence, dredging spleen collaterals". Additionally, the high concentration glucose was used to induce the damages in pancreatic microvascular endothelial cells. Then the effects and mechanism of composition were explored on the model in vitro. Through the above experiments to explore the important role of "collateral-microvascular" disease of "Spleen (pancreas)" in the pathogenesis ofdiabetes (T2DM), hoping to supply a promising treatment methods and formulas for clinic,and these data would give scientific explanation for the theory of treating diabetes (T2DM) from the" spleen" and ying-nutrient and wei-defense "unblocking collaterals with subsequent convergence and substance-qi transformation"guided by vessel-collateral doctrine.Methods:1. Intervention studyof "activating spleen essence, dredging spleen collaterals" method in T2DM ratsglucolipid metabolism and oxidative stress injury156male SD rats were randomly divided into normal group, model group,JLD group, TXL group, JLD+TXL group, RSG group. Except the normal group, the rest of groupswere fed withhigh-fat food and corresponding drug respectively.4weeks later, then45mg/kg streptozotocin was injected intraperitoneallytodevelop T2DM rats.After72h, the rats fasting-glucose exceeding13.9mmol/L were adopted to feed high-fat food and drugfor eight weeks. Then the blood serum was separatedto detect of index including blood glucose, lipid, and oxidative stress indicators.2. Intervention study of "activating spleen essence, dredging spleen collaterals" method in T2DM rat islet microvascular lesionAnimal models and grouping ditto, detection of serum nitric oxide, endothelin, angiotensin Ⅱ, aldosterone levels, non radioactive microsphere method to detectpancreas andislet blood flow change, pancreatic microvascular morphology and microvascular endothelial ultrastructurewere observedby D-PAS stain,3D imaging of laser confocal microscopeand transmission electron microscopy, endothelial cell markers Nestin mRNA expression of islet cells were detected by Real-time PCR.3. Intervention study of "activating spleen essence, dredging spleen collaterals" method in T2DM rat islet cell functionAnimal models and grouping ditto, detection of serum glucagon and growth hormone levels, islet morphology change was byHE dyeing, islet beta cell ultrastructure change was observedby transmission electron microscopy, endocrine hormone expression of islet tissue was detected byimmunohistochemical staining, islet cell apoptosis was detected byTUNEL assay; Akt, p-Akt protein expression of islet cell was was detectedby Western blot; islet cell proliferation gene PDX-1and Ngn3mRNA expression was detected byReal-time PCR.4. Intervention study of "activating spleen essence, dredging spleen collaterals " method in high concentration glucoseinduced pancreatic microvascular endothelial cells injuryPancreatic microvascular endothelial cells MS-1were seeded in96-well plates or dishes, cells grew to80%, discarded old media, cells were used in experiment after synchronizing by1%fetal bovine serum (5.6mM) DMEM culture medium24h. Cells were divided into normal group, model group, Jinlida group, Tongxinluo group and Jinlida+Tongxinluo group,parallel wells were arranged3to5, high concentration glucose(50mM) was used to induce injury model, and give drug intervention, after cultured48h,1w,2w,cell viability was detected by MTSassay,48h cell apoptosis rate was detected by flow cytometry, cell supernatants, cells and cell lysates were collected to detect NO, SOD, MDA, ET-1levels in cell supernatant and cell reactive oxygen species levels,inflammatory cytokines TNF-a, the receptor for advanced glycation end products (RAGE) and apoptosis related proteins Bax, Bcl-2, Caspase3expression were detected by Western blot.Results:1. The results of intervention study of "activating spleen essence*dredging spleen collaterals" method in T2DM rats glucolipid metabolism and oxidative stress injury:fasting glucose, glycosylated hemoglobin and glycosylated serum protein level were significantly reduced by JLD, JLD+TXL and RSG; triglyceride, total cholesterol, low-density lipoprotein cholesterol and free fatty acids were significantly reduced by TXL, JLD+TXL and RSG, triglyceride and total cholesterol levels were effectively reduced by JLD, HDL-C level waseffectively increased by JLD+TXL and RSG; serum SOD and GSH content was significantly increased, MDA content was significantly decreased by JLD, JLD+TXL and RSG, serum SOD content was significantly increased, MDA content was significantly reduced byJLD(P<0.05, P<0.01).2. The result of intervention study of "activating spleen essence, dredging spleen collaterals " method in T2DM rat islet microvascular lesion:serum ET-1, Ang II, ALD levels were significantly reduced and serum NO content was elevated in each group; ET-1content was significantly reduced by TXL, JLD+TXL and RSG compared withJLD group,serum ALD content was significantly reduced by TXL compared with JLD+TXL and RSG;pancreas blood flow was significantly increased in each group, islet blood flow wassignificantly increased by TXL, JLD+TXL and RSG, islet/pancreatic blood flow ratio was significantly increased by TXL; D-PAS stain, laser confocal microscope and electron microscope showed that pancreatic microvascular morphology and microvascular endothelial structure was effectively improved and pancreatic microvascular lesions was reduced byTXL, JLD+TXL, Nestin mRNA expression in islet was significantly raised by JLD+TXL, and its effect was superior to the other group (P<0.05, P<0.01).3. The result of intervention study of "activating spleen essence, dredging spleen collaterals" method inT2DM rat islet cell function:fasting blood and fasting insulin levels were significantly reduced by JLD, JLD+TXL and RSG, HOMA-%beta index was elevated by JLD+TXL and RSG; serum glucagon and growth hormone levels, insulin expression in islet was reduced and glucagon expression, was reduced by JLD, JLD+TXL and RSG, and morphology and ultrastructure of islet cell was improved, islet cell apoptosis was reduced, p-Akt expression andp-Akt/Akt ratio in islet waselevated by JLD, JLD+TXL and RSG, PDX-1and Ngn3mRNA expression was increased by JLD, JLD+TXL (p<0.05, p<0.01).4. The result of intervention study of "activating spleen essence, dredging spleen collaterals" method inhigh concentration glucose induced pancreatic microvascular endothelial cells injury:NO content in supernatant was significantly increased andET-1content was reduced byJLD, TXL and JLD+TXL to improve the function of glucose induced microvascular endothelial cell injury, cell survival was elevated, SOD content was increased, MDA content, cellular ROS levels, apoptosis rateand expression of inflammation factors TNF alpha proteinand RAGE proteinwere decreased byJLD, TXL and JLD+TXL, meanwhile the expression of apoptosispromoting protein Bax, Caspase3were reduced and apoptosis suppressor protein was increased (P<0.05, P< 0.01).Conclusion:1. This study is based on vessel-collateral doctrine and use the experience of the modern famous doctor Zhang Xichun’s academic thought about diabetes "commence in middle jiao, and develop to the upper and lower" for reference, and put forward a new point to treat diabetes (T2DM) from the" spleen" in TCM, and point that the key of disease onset is due to subtle distribution and metabolic disorder and imbalance caused by dysfunction of spleen in transportation, the damages of islet "collateral-micro vascular" caused by the disorder of ying-nutrient and wei-defense "unblocking collaterals with subsequent convergence and substance-qi transformation" is an important factor of the onset of T2DM, first puts forward a novel academic viewpoint "activating spleen essence, dredging spleen collaterals", it not only explains the scientific connotation of ying-nutrient and wei-defense "unblocking collaterals with subsequent convergence and substance-qi transformation", but also highlights the scientific value of "commence in middle jiao", and open up a new avenues for clinical prevention of diabetes (T2DM).2. Representative drug of "activating spleen essence", JLD granulewithefficacyofhypoglycemic, lipid-lowering and antioxidation, representative drug of "dredging spleen collaterals" TXL capsule has obvious advantage in the protection of islet capillaries and improve microcirculation perfusion, combined application of "activating spleen essence, dredging spleen collaterals" method is better than that of single use, have the effect of complementary advantages and bring out the best in each other.3. Representative drug of "activating spleen essence",JLD granule can effectively protect islet beta cell structure and function, reduce islet cell apoptosis and promote proliferation, representative drugof "dredging spleen collaterals"TXLcapsule can effectively protect islet "collateral-microvascular" structure and functional integrityin T2DM rat,"activating spleen essence, dredging spleen collaterals" method shows its unique features and advantages by improving islet survival environment to reduce beta cell apoptosis, promoting insulin secretion and improving glucolipid metabolism.