Study On Pharmacodynamics And Molecular Mechanism Of Silkworm Feces Extract To Ameliorate Renal Anemia In Rats

Shengxuening tablets,with silkworm feces extract(SFE)as the main pharmacodynamic component,are commonly used to treat iron deficiency anemia in clinic.SFE,extracted and modified from silkworm feces,is a water-soluble iron complex,and is mainly composed of sodium chlorophyllin and chlorophyll derivatives.Sodium ferric chlorophyll and is similar with haem in chemical structure and physiological activities,such as anti-inflammatory and anti-anemia activities.In order to explore the therapeutic effect and molecular mechanism of SFE on renal anemia rats,a model of renal anemia rats was established in our research,and a cellular model of TNF-α induced HEK-293 T cell inflammation model inhibiting EPO expression was constructed in vitro.Pharmacodynamic indexes and relative molecular expression levels were detected by a series of methods,with emphasis on the promotion and molecular mechanism of SFE on EPO,and the inhibition and molecular mechanism of SFE on hepcidin,providing basic theoretical data for the clinical application of shengxuening tablets on renal anemia.Part One: Study on the pharmacodynamics of Silkworm feces extract to improve renal anemia in ratsIn this part of the experiment,the rat model of renal anemia was established by alternating feeding with 0.75% adenine in the interval of one week.At the same time,chronic kidney disease rats(CKD)were used as the model group,SFE(54mg/kg)was given by oral administration as the treatment group,and recombinant human erythropoietin(rh EPO,200 U/kg)was intraperitoneally injected as the positive control,and rats fed with normal feed were used as the normal control group(NC).After 6 weeks,the hamatology parameters of each group were detected.Compared with the CKD group,HGB,RBC,HCT and MCV in the SFE group were significantly increased,while those in the WBC group and the PLT group were significantly decreased,indicating that SFE effectively ameliorated renal anemia of the rats.The iron content of liver,spleen and serum was detected,and the results showed that the iron content of liver and spleen were significantly decreased in SFE group compared with CKD group,while the serum iron content was significantly higher than those in the CKD group.Also,the result of the serum TIBC and TS showed that SFE significantly increased TIBC and TS,and increased iron utilization.The effects of SFE on inflammation and renal function in rats with renal anemia were further investigated.Serum IL-6 and TNF-α levels were detected by ELISA,and the expressions of IL-6 and TNF-α were markably decreased in SFE group compared with CKD group.Meanwhile,the contents of serum creatinine(Cr)and blood urea nitrogen(Bun)were detected with the kit,and SFE significantly reduced the contents of Cr and Bun in the CKD rats.The appearance of the kidney and H&E sections were observed after dissection of rats.The results showed that SFE effectively alleviated the degree of renal inflammation and pathological microstructure in rats,indicating that SFE could significantly slow down renal failure in CKD rats.In summary,the rat model of renal anemia was successfully established.SFE significantly increased the HGB,RBC,HCT and MCV level of the rats and improved anemia,and meanwhile SFE increased the utilization rate of iron,which is effectively alleviated inflammation and slowed down kidney failure in the rats.Part Two: Study on the molecular mechanism of Silkworm feces extract to improve renal anemia in ratsIn this part,the molecular mechanism of SFE to improve renal anemia in rats was studied.First,serum EPO level was detected by ELISA kit,EPO protein content in the liver was detected by western blot,and EPO content in kidney was observed by immunohistochemistry.The results showed that SFE effectively promoted EPO expression in serum,liver and kidney.HIF-2α,PHD2,NF-κB and GATA2 were detected in different ways.As a result,HIF-2α in the SFE group was significantly higher and PHD2,NF-κB and GATA2 were significantly lower than those in the CKD group,indicating that SFE effectively inhibited the expression of NF-κB and GATA2 to increase EPO transcription,inhibited the expression of PHD2,promoted the expression of HIF-2α,increased the expression of EPO,further promoted the synthesis of red blood cells,and ameliorated anemia.At the same time,the hepcidin levels in serum and liver were detected,the result indicated that SFE could significantly inhibit the expression of hepcidin.Subsequently,western blot was used to detect the expression of p-STAT3,SMAD4,BMP6,TFR2,Ferroportin1 and Ferritin in the liver of the rats.The results showed that SFE promoted Ferroportin1 expression and inhibited Ferritin expression,indicating that Ferroportin1 promoted the transfer of iron from tissues to serum and increased the utilization rate of iron.The results showed that the expressions of p-STAT3,SMAD4,BMP6,TFR2 and Ferritin were significantly decreased in SFE group compared with CKD group.Also,SFE effectively reduced serum IL-6 levels.The results indicated that SFE inhibited hepcidin expression by effectively blocking the BMP6/SMAD4 and IL-6/STAT3 signaling pathways and inhibiting TFR2 expression,thereby increasing the expression of Ferroportin1.Combined with the above results,SFE not only effectively increased EPO expression by promoting HIF-2α expression,inhibiting PHD2 production,blocking transcription of NF-κB and GATA2,but also inhibited hepcidin expression by effectively blocking BMP6/SMAD4 and IL-6/STAT3 signaling pathways and inhibiting TFR2 expression,increasing iron utilization,regulating iron homeostasis in rats,and then improving renal anemia.Part Three: Study on promoting EPO expression of TNF-α induced HEK-293 T cell inflammation model in Silkworm feces extractIn this part,cell experiments were carried out to further verify the effect of SFE on EPO expression.The experimental conditions were selected by MTT experiment.The concentration of recombinant human TNF-α and SFE were 100ng/m L and 50μg/m L,respectively,the cell viability was satisfied,and subsequent cell experiments could be conducted.Subsequently,a TNF-α induced HEK-293 T cell inflammatory model was established.Western blot and Real-time PCR results showed that the expression of EPO and HIF-2α in the SFE group was significantly increased,while the expression of NF-κB was significantly decreased,compared with the model group.These results showed that SFE promoted the HIF-2α and EPO expression in cells while inhibited the expression of NF-κB in vitro,which was consistent with the results of animal experiments,proving that SFE effectively promoted EPO expression.