| Diabetes mellitus(DM)is a systemic metabolic disease.According to the data of the World Health Organization in 2011,the number of diabetic patients in the world has reached 366 million,seriously endangering human life and health.Diabetes causes numerous complications and damages the kidneys,eyes,heart and other organs.Among them,Diabetic retinopathy(DR)is one of the most common Diabetic microvascular complications and also an important cause of blindness in the working population of developed countries.The fundamental cause of DR is disorder of glucose metabolism,which is closely related to hyperglycemia and hyperlipidemia.At present,the main treatment methods of DR include laser therapy,surgical therapy and drug therapy.Laser and surgical treatments have good clinical effects,but they are costly and often associated with many side effects.Therefore,seeking safer and more effective drugs from natural plants is a research hotspot at home and abroad.Breviscapine(BVP)is a flavonoid glycoside compound with Breviscapine as the main component.It has the effect of promoting blood circulation and removing blood stasis,promoting meridians and activating collatuses,and has a good clinical effect on coronary heart disease,cerebral thrombosis and diabetes complications,with little toxic and side effects,and has broad clinical application value.However,the efficacy and clinical dose of breviscapine on DR remain to be further explored.In addition,breviscapine has many components and its quality control is complicated.Therefore,the quality control of breviscapine was carried out by High Performance Liquid Chromatography(HPLC)and Optical coherence tomography(OCT),Fluorescein fundus angiography in vivo(FFA)were used to evaluate the efficacy of breviscapine on DR in db/db mice.In order to provide more scientific basis for clinical application and improve its medicinal value.HPLC method was used to determine the fingerprint of breviscapine API.The similarity of chromatographic fingerprint of 10 batches of different samples was evaluated.Four chromatographic peaks were calibrated by standard substance to control the quality of breviscapine.Then 40 db/db mice with diabetic retinopathy Model were randomly divided into Model group(MODEL),Dobesilate 225mg/kg,brecapellin high-dose group(BVP-H 80mg/kg),brecapellin low-dose group(BVP-L 40mg/kg).The same amount of C57 mice was used as Control group.The treatment group was intragastrically given drugs,while the control group and model group were intragastrically given the same amount of normal saline.Body weight and HBALC were measured weekly.After 12 weeks of intragastric administration,ERG,FFA,OCT,HE staining,immunohistochemistry and TUNEL staining were used to evaluate the efficacy of breviscapine against diabetic retinopathy in db/db mice.Through the above research findings:(1)fingerprint study of breviscapine:by optimizing the HPLC conditions,establish the lamp,the HPLC chromatographic fingerprint,and the repeatability,stability and precision of methodology,10 batches of different batches of lamps,the chromatographic fingerprint similarity is ≥95.4%,is obtained by the standard calibration lamps shell element,lamp b element,celery and wild radix scutellariae four main material.(2)General situation of mice:db/db mice gained weight after feeding high fat diet for one week.The feeding condition,metabolism condition,mental state and movement sensitivity of the mice were far less than those of the control mice,and the fur gloss of the mice decreased and fell off.These conditions worsened as the mice gained weight.Compared with the Control group,the situation in the Model group was the most serious,and the state of mice in the administration group was better than that in the Model group.(3)Effect on body weight of mice:compared with Model group,body weight of mice in administration group had no significant change(P>0.05).(3)Effect on HBALC:Compared with Control group,the value of HBALC in Model group was significantly increased(P<0.05),and the modeling was successful;Compared with Model group,there was no significant change in HbAlc value in BVP-L,BVP-H and Dobesilate groups(P>0.05).(4)Effect on eye electrophysiology of mice:Compared with Control group,the amplitude of A wave(P<0.01,P<0.001)and B wave(P<0.05,P<0.01)of Dmax and Drod in Model group were significantly decreased;Compared with Model group,the amplitudes of A and B waves of Dmax and Drod in BVP-L and BVP-H groups were significantly increased(P<0.01).(5)Effect on fundus blood vessels in mice:compared with Control group,the fluorescence intensity of fundus blood vessels in Model group was significantly increased(P<0.05);Compared with Model group,the fluorescence intensity of fundus blood vessels in each administration group was significantly decreased(P<0.05).(6)Effect on the thickness of retinal fiber layer in mice:Compared with Control group,the thickness of inner layer of retina in Model group was increased,and the thickness of inner plexus layer,ganglion layer,outer nuclear layer,outer membrane layer,inner/outer segment junction of photoreceptor and pigment epithelium were significantly decreased(P<0.01),while the thickness of inner layer was significantly increased(P<0.01).Compared with Model group,the thickness of ganglion layer and outer membrane in BVP-L group was significantly increased(P<0.05),and the thickness of outer nuclear layer and the junction of inner/outer segment of photoreceptor in BVP-H group was significantly increased(P<0.05).(7)The effect of retinal histopathology on mice:compared with the Control group,the retinal tissue structure of mice in the Model group was incomplete,with disordered arrangement of fiber layer,sparse arrangement of cells and intertissue edema;Compared with the Model group,the retinal structure of BVP-L and BVP-H groups was complete,with orderly arrangement of fibrous layers and cells,and no edema between tissues.(8)Effects on the apoptosis of retinal pericytes and retinal neuronal cells in mice:Breviscapine administration group could reduce the apoptosis of retinal pericytes and retinal neuronal cells in mice.(9)Effect on retinal angiogenesis:Breviscapine administration group can reduce the expression of retinal VEGF,and further reduce retinal angiogenesis.In conclusion,the following conclusions can be drawn:(1)The quality of breviscapine API is up to standard,and four compounds of breviscapine API are identified:breviscapine,baicalein,apigenin,apigenin-7-O-glucuronidoside;The fingerprint similarity of 10 batches of samples from the same manufacturer is ≥ 95.4%,and the quality of the samples is qualified.(2)Breviscapine had no significant effect on body weight and HbAlc value of db/db mice;It can significantly improve the thickness of ocular fiber layer,the intensity of ocular fundus blood vessel fluorescence,ocular electrophysiology and retinal histopathology.Moreover,it can inhibit the apoptosis of retinal neurons and pericytes,and reduce the expression of retinal VEGF. |
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