Establishment Of Zebrafish Type ? Diabetes Mellitus With Osteoporosis Model And Its Application In Drug Evaluation

Purpose and significance Purpose: 1 ? Analytical method verification of Asiaticoside(AS),Leonurine(SCM)and Polygonatum sibiricum(SR)to obtain the stability of the dosing solution.2?Determine the dose-toxicity relationship of modelling agents(Streptozocin STZ and Prednisolone PN),positive drugs(Metformin Met and Etidronate Disodium ED),AS,SCM,SR and their combined drugs on zebrafish embryos.3?Using non-toxic doses of STZ and PN pretreatment to establish a model of type ? diabetes with osteoporosis in zebrafish embryos;Comparing the effects of each drug alone or in combination on the treatment of this disease in zebrafish embryos;On this basis,discussing the therapeutic effects of AS,SCM and SR on type ? diabetes with osteoporosis,and briefly describe their mechanism.Significance: Developing a zebrafish embryos type ? diabetes with osteoporosis model,using this model to evaluate the anti-osteoporosis activity,which is of great significance to investigate toxic effects of the three drugs.It provides a basis for studying the pathological mechanism of human bone diseases and mechanism of drugs on the disease,and provides a theoretical basis for finding new drugs and therapies for the treatment of type ? diabetes with osteoporosis.Method 1?The linearity,stability inspection and content determination of AS,SCM and SR.2?Early developmental toxicity test: different concentrations of AS,SCM,SR,AS+SCM,AS+SR,Met+AS,Met+SCM,Met+SR,Met+AS+SCM,Met+AS+SR were used to stimulated 2 dpf zebrafish embryos to observe the early development of zebrafish.3?We used 2 dpf zebrafish embryos,microinjected STZ and soak PN to establish a zebrafish embryos type ? diabetes with osteoporosis model.Grouping according to the pre-experimental settings: control group,positive group,model group,administration group,randomly placed in a 24-well plate,6 embryos/well,cultured a period of 5 days,to 8 dpf.Then zebrafish were stained with osteoalizarin red,we took pictures and analyzed the mineralized area and integrated optical density(IOD).4?qRT-PCR was used to detect and observe the effects of drugs on the mRNA expression levels of osteoporosis-related genes Runx2 b,Sp7,Col1a2,Sparc,and Vdrb.Results 1?AS,SCM and SR were linearly related in different concentration ranges,and had good stability.2?After AS,SCM,and SR were administered alone or in combination with Met,the toxicity increased with increasing concentration,the embryos had different degrees of pigmentation,pericardial cysts,yolk cysts,abnormal morphology and showed a dose-dependent trend,even death.The combination of Met+AS or Met+SR reduced the toxicity of AS or SR alone.On the contrary,the toxicity of Met+SCM was greater than that of SCM alone;the toxicity of AS+SCM or AS+SR was less than that of pairwise combined with Met.After adding Met to AS or SCM,respectively,the hatching of zebrafish embryos was slowed down,Met+SR promoted embryos hatching;Comparing with AS+SCM and AS+SR,Met+AS+SCM and Met+AS+SR promoted the hatching of embryos.3?The tissue sugar content of the normal group was 1.063 nmol/tail,while the model group was 3.086 nmol/tail;the bone mineralization area of the normal group was 27281.3,IOD=6356,while the model group was 2721.7,IOD=669.285.There was a significant difference between them,which proved that the model is successful.4?On the whole,each administration group could significantly improve type ? diabetes with osteoporosis.Comparing with other administration groups,the bone mineralization area of 240 ?g/mL SR was 24423.3,IOD=5522.59.Its anti-osteoporosis activity was most prominent,and it was higher than the positive group(23784,5343.71).5?Within 8 days,the expression levels of Runx2 b,Sp7,Col1a2,and Sparc were significantly up-regulated after treatment in most groups.SR 240 ?g/mL had the best effect in promoting up-regulation of gene expression,while Vdrb was not significant.Conclusion 1?2 dpf zebrafish embryos were chosen to microinject 200 ?g/mL STZ,then 4 dpf zebrafish embryos were chosen to soak 10 ?g/mL PN,the above method can be used as a reference for establishing type ? diabetes with osteoporosis model condition;Observing the morphological changes and skeletal alizarin red staining of zebrafish under the EVOS imaging system.These can be used as reference indicators for screening anti-type ? diabetes with osteoporosis drugs.2?Different administration groups had different toxicity to zebrafish embryos.Both single administration and combined administration had anti-osteoporosis activity;However,the effect of combination medication may not be better than that of single administration;Its mechanism of action may be related to promoting the expression of Runx2 b,Sp7,Col1a2,Sparc and Vdrb,promoting active mineralization in bone tissue,which accelerates bone formation and reconstruction.AS,SCM and SR can be used as candidate drugs for further research on anti-type ? diabetes with osteoporosis,and provide theoretical basis for rational medication and treatment of type ? diabetes with osteoporosis.