Study On The Mechanism Of Oxytropis Cunea Extracts In Early Diabetic Nephropathy Mice Based On SOCS/JAK/STAT Pathway

Aim:Through a research of the Tibetan medicine---Oxytropis falcate Bunge extractive's(flavonoids compounds)regulating effect on the rats'(with DN)kidney's pathological feature,the protein expression in their tissues and SOCS3/JAK2/STAT3 signaling pathway,this passage discusses the Oxytropis falcate Bunge's protective functions on the kidney of animal model with DN and the questions of inflammatory pathway and pathogenesis of DN,further enriching the research on DN,offering scientific reference for developing national medicine and providing theoretical basis for conquering DN.Methods:KK-Ay rats' boulimia was caused by leptin's restraint,thus leading to obesity and hyperglycemia.KK-Ay rats are internationally recognized as the animal model for diabetes.Sixty 12-week-old SPF male rats were chosen in the experiment and randomly divided into model group,Chinese medicine group(subdivided into low-dose flavonoids,medium-dose flavonoids and high-dose flavonoids groups)and Western medicine(pioglitazone)positive control group,12 rats for each group.12C57BL/6J male rats(homologous with KK-Ay rats)were divided into 6 groups and chosen as the normal control groups.Detect blood glucose before Pharmacological intervention and make the blood glucose level reach 16.7 mmol/L.Collect rats' 24-hour urine with appropriative metabolism cage and detect the content of urinary albumin.When the content exceeds 0.4mg,the rats will be confirmed as DN model.Lavage administration(low-dose flavonoids,medium-dose flavonoids and high-dose flavonoids groups)were given a dosage of 100 mg,200mg,and400 mg respectively;The same volume of normal saline was given to the normal control groups and model group;Give the positive control group a doage of 10 mg pioglitazone.All the rats were given by gavage 0.1 ml/10 g per day.During the experiment,KK-Ay rats fed on HFSD while the C57BL/6J at the same age fed on common animal food,both drinking water freely.After 4 weeks,fasting but not water deprivation,collect 24-hour urine,record urine volume and keep it at minus 20 degrees to be tested.Pick the eyeballs to draw blood,separate serum and keep it at minus 20 degrees.Kill the rats after blood sampling and cut up the kidney vertically.According to different testing methods,put the kidney pieces into triformol,glutaraldehyde and liquid nitrogen.Then,do the nephridial tissue specimens HE staining.TestJAK2?SOCS3?STAT3's expression of protein by using Western blot method.Test JAK2?SOCS3?STAT3's gene pathway expression by using RT-qPCR.Result:1.Rats' s general condition:12-week-old KK-Ay rats shows obvious symptoms of diabetes.The symptoms include high water intake,rising food intake,rough fur,drooping spirits,inactivity,dirty straw mattress,stinky smell.2.the effect of Oxytropis falcate Bunge on KK-Ay rats with DN;Compared with the model group,the rats' general condition in the high,medium and low-dose flavonoids groups improved and their weight reduced.After 4-week medicine intake,rats' fasting blood glucose index went lower than the model group(P<0.05),creatinine decreased(P<0.05),urea nitrogen improved(P<0.05).Compared with the model group,the content of carbamide lessened,renal pathology HE staining pathologic sections showed that renal corpuscle and kidney tubules have different-leveled protection and repair functions,and the basilar membrance of enal corpuscle's incrassation slowed down.3.The effect of Oxytropis falcate Bunge on KK-Ay rats with DN and JAK2?STAT3?SOCS3 ?IL-6 protain:Compared with normal control groups,there is no difference in SOCS3?JAK2?STAT3's expression of protein between the pioglitazone positive control group and the high-dose flavonoids group(P>0.05).The differences in the rest of groups make a lot of sense(P<0.05);Compared with the model group,the differences between each group have a statistical significance(P<0.05).There is no difference in JAK2's expression of protain between the flavonoids groups(P>0.05).Compared with the normal control groups,there is no difference in STAT3's expression of protein between the pioglitazone positive control group and the high-dose flavonoids group(P>0.05).The differences in the rest of groups make a lot of sense(P<0.001).Compared with the model group,the differences between each group have a statistical significance(P<0.001).There is a dose-effect relationship between the flavonoids groups with the high-dose flavonoids groups' expression of protein being the most obvious.The difference has a statistical significance(P<0.05).There is no difference between the low and medium-dose flavonoids groups(P>0.05).4.The effect of Oxytropis falcate Bunge on KK-Ay rats with DN and SOCS3? JAK2?STAT3 ? IL-6's gene expression:Compared with the normal control groups,there is nodifference in SOCS3's gene expression between the pioglitazone positive control group and the high-dose flavonoids group(P>0.05).The differences in the rest of groups make a lot of sense(P<0.05).Compared with the model group,except the the flavonoids groups(P>0.05),the differences in SOCS3's gene expression between the rest of groups have a statistical significance(P<0.001).There is no difference in SOCS3's gene expression between the high-dose flavonoids group and the low-dose flavonoids group(P>0.05)while there exists a obvious difference in the medium-dose flavonoids group(P < 0.05).Compared with normal control groups,there is no difference in JAK2' gene expression between the pioglitazone positive control group and the high-dose flavonoids group(P>0.05).The differences in the rest of groups make a lot of sense(P<0.001).Compared with the model group,the differences between each group have a statistical significance(P<0.001).There is no difference in JAK2's gene expression between the medium-dose flavonoids group and the low-dose flavonoids group(P>0.05).There exists a obvious difference in the high-dose flavonoids group(P<0.001).Compared with the normal control groups,there are obvious differences in STAT-3's gene expression between each group(P<0.05).Compared with the model group,there is no difference(P<0.05)in the low-dose flavonoids group and the differences between the rest of groups have a statistical significance(P<0.001).There is no difference between the high and medium-dose flavonoids groups(P>0.05)and there are obvious differences in the low-dose flavonoids groups(P<0.05).Conclusion:1.Oxytropis falcate Bunge 's extractive---flavonoids compounds can reduce DN rats' blood glucose and the level of serum creatinine,urea nitrogen and uric acid,improve renal pathological damage and thus slow down the process of DN.2.By restraining renal tissues SOCS3?JAK2?STAT3 's gene and inflammatory pathway expression,Oxytropis falcate Bunge 's extractive---flavonoids compounds can also protect DN rats' renal tissues which could be one of the important effects on restraining DN.