Study On The Effect And Mechanism Of Couplet Medicines Salviae Miltiorrhizae Radix Et Rhizoma-Moutan Cortex And Its Active Compositions Compatibility In Improving Diabetic Retinal Disorders

BackgroundDiabetic retinopathy（DR）is a common neurovascular complication of diabetes mellitus.The global prevalence of DR among patients was over 1/3,and it has become the major cause of visual disturbance and blindness in working-age adults^[1-3].The current treatments for DR include laser photocoagulation,anti-vascular endothelial growth factor（VEGF）intravitreal injection and vitreoretinal surgery,which may be accompanied by certain adverse reactions such as decreased vision and visual field defect.Meanwhile,frequent eye clinics can impose a heavy economic burden on patients^[3-5].Traditional Chinese medicine is an important conventional medicine in China,and also a pivotal part of Traditional and Complementary Medicine of the World Health Organization^[6,7].Traditional Chinese medicine has a long history and rich experience in the prevention and treatment of DR.For the complicated characteristics of DR,traditional Chinese medicine can give full play to the advantages of systemically multi-link,multi-level and multi-target regulatory effects,which shows a bright application prospect and research value in clinical treatment.DR in traditional Chinese medicine is called"Xiaoke eye disease".The etiology and pathogenesis are mainly Yin deficiency and dryness heat,and blood stasis and collateral obstruction.Cooling blood and removing stasis is one of the main therapeutic principles.Clinically,Shuangdanmingmu capsule,Zhixuequyumingmu tablet and Hexuemingmu tablet show significant therapeutic effects on DR,and play an important role in improving visual function and blood microcirculation^[8-10].Although these proprietary Chinese compound medicines have different prescriptions,they are all compatible with Salviae Miltiorrhizae Radix et Rhizoma and Moutan Cortex,a classic couplet medicines with the effect of cooling blood and removing blood stasis.However,the specific role and mechanism of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in the treatment of DR remain unclear.In addition,most of the previous studies focused on the proangiogenic effect of Salviae Miltiorrhizae Radix et Rhizoma and Moutan Cortex on myocardial ischemia injury,while the effect of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on DR angiogenic response has not been reported.The pathological mechanism of DR mainly involves two aspects:retinal capillary plexus（microvascular lesions）and nerve sensory layer（neuronal and glial cells damage）^[11].Müller glial cells are the functional link between neurons and blood vessels,and also the main source of VEGF in the progress of DR.Therefore,this study would firstly explore the overall pharmacological effect of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on diabetic retinal disorder,and analyze its chemical components in vitro and pharmacodynamic substance basis in vivo.Furthermore,the pharmacological effects of the ocular tissue targeting active compositions on inhibiting photoreceptor cells apoptosis and neovascularization response in the DR retina were elucidated,and the molecular mechanism on regulating Müller cells oxidative stress and inhibiting VEGFA was revealed.Therefore,this would help to explain the compatibility connotation of pharmacodynamic molecular components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex,provide scientific basis for the clinical application and secondary developing of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex related therapeutics in intervening DR,and inspire research ideas for exploring the mechanism of traditional Chinese couplet medicines and its active compositions on preventing and treating DR.ObjectiveThis study was to investigate the pharmacological effect of couplet medicines Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in attenuating diabetic retinal disorder,and analyze the transitional prototype effective components in blood serum and ocular tissue by tandem high performance liquid chromatography and Q-TOF mass spectrometry.And then,based on oxidative stress and neovascularization response,we intended to investigate the pharmacological effect and molecular mechanism of the compatibility of the ocular tissue targeting active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in vivo and vitro in DR.Methods1.The preliminary study of pharmacological effect and mechanism of couplet medicines Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on attenuating diabetic retinal disorderHealthy C57BL/6J male adult mice were selected and intraperitoneally injected with streptozotocin（50 mg/kg/d,5 d）to induce type 1 diabetes mellitus（DM）model.Then,diabetic mice were randomly divided into 9 groups:Normal group（CON,10 m L/kg/d）,normal+Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex group（CON+SCH,2.0g/kg/d）,diabetes mellitus group（DM,10 m L/kg/d）,diabetes mellitus+Calcium Dobesilate group（DM+Ca D,0.25 g/kg/d）,diabetes mellitus+Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex high-dose group（DM+SCH,2.0 g/kg/d）,diabetes mellitus+Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex medium-dose group（DM+SCM,1.0 g/kg/d）,diabetes mellitus+Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex low-dose group（DM+SCL,0.5 g/kg/d）,Salviae Miltiorrhizae Radix et Rhizoma group（DM+SM,1.2 g/kg/d）and Moutan Cortex group（DM+CM,3.6g/kg/d）.According to the literature and previous studies,electroretinogram examination was performed to confirm the occurrence of DR at 8 weeks after successful establishment of type 1 diabetes.Intragastric administration was then performed according to the dose indicated in the above grouping.After 8 weeks of intervention,the pharmacological effect of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on diabetic retinal disorder in mice were evaluated from three aspects including retinal function and morphology,blood circulation function and retinal neovascularization response.Thereinto,full field electroretinogram（ff ERG）,retinal coherence tomography（OCT）and HE staining of pathological sections were used to evaluate retinal functional and morphological damage.Hemorheology index was used to evaluate the systemic blood circulation function.The detections of m RNA levels（RT-PCR）and protein levels（immunofluorescence and Western blot）of VEGFA and HIF-1αwere used to evaluate retinal neovascularization response.2.The analyze of chemical components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in vitro and the transitional prototype active components in blood serum and ocular tissue in vivoSixteen healthy male C57BL/6J adult mice were divided into the control group（normal saline,10 m L/kg/d）and the experimental group（Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex,2.0 g/kg/d）.The mice were administered continuously for 7days.Blood and ocular tissue were collected from the anesthetized mice 1 h post the last administration.The blood was centrifuged and supernatant was obtained by methanol precipitation method.The ocular tissue was ground with methanol and centrifuged to obtain supernatant.After nitrogen blowing,the solid precipitation was obtained.The ACQUITY UPLC I-class tandem Xevo G2-XS QTOF mass spectrometer with a Waters HSS T3 UPLC column（2.1×100 mm,1.8μm）was applied.0.1%formic acid in water and acetonitrile were used as mobile phases.The flow rate was 0.30 m L/min and the column temperature was 35℃.Electrospray ion source（ESI）and reactive ion detection（SRM）mode were used.Based on liquid chromatography-mass spectrometry,we analyzed the chemical components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in vitro.The blood serum and ocular tissue transitional prototype active components were further identified in vivo.At the same time,high performance liquid chromatography（HPLC）was used to quantitatively analyze the prototype pharmacodynamic substances in Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex.3.The study of pharmacodynamics and mechanism of the compatibility of active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on attenuating diabetic retinal disorderHealthy C57BL/6J male adult mice were selected,and DR mice successfully modeled by STZ were randomly divided into the normal group（CON,10 m L/kg/d）,the diabetes mellitus group（DM,10 m L/kg/d）,the diabetes mellitus+Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex group（DM+SC,2.0 g/kg/d）,the diabetes mellitus+the compatibility of active compositions（targeting ocular tissue）of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex（cryptotanshinone,neocryptotanshinone,paeoniflorin and albiflorin）（DM+AC,175 mg/kg/d）.After 8 weeks of intragastric administration,retinal morphological and functional pathological changes were observed through the ff ERG,OCT,fundus fluorescein angiography and fundus photography and pathological HE staining,and photoreceptor cells apoptosis was assessed by TUNEL staining.Photoreceptor cells mitochondrial damage were evaluated by transmission electron microscope observation,mitochondrial DNA damage and mitochondrial ROS level detection.Then,through applying techniques of molecular biology（RT-PCR,immunofluorescence,Western blot,and ELISA）,retinal oxidative stress was evaluated by ALDH2,VHL,4-HNE and GPX4 expression levels,retinal angiogenic response was assessed by SIRT1,VEGFA and HIF-1αexpression levels,and apoptosis and inflammatory response were analyzed by Caspase 3,Bax,Bcl-2,p53,Cyt c,IL-6,and TNF-αexpression levels.Taken together,the effects and mechanism of the compatibility of active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on diabetic retina disorder were evaluated by four aspects,including retinal function and morphology,photoreceptor cells apoptosis,mitochondrial damage and retinal angiogenic response.4.The study of pharmacological effect and mechanism of the active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on high glucose-induced Müller cell damageThe Müller cells of mouse retina were isolated,identified and cultured,and Müller cell damage model was constructed by high glucose.The optimal concentration of cryptotanshinone,neocryptotanshinone,paeoniflorin and albiflorin were determined by CCK-8 method.TUNEL and AV/PI assay were used to investigate the effect of active compositions on Müller cell apoptosis.We used ALDH2 agonist Alda1 and inhibitor Daidzin,SIRT1 agonist CAY10602 and inhibitor EX527,and si RNA gene-silencing technique,and applied cellular and molecular biology methods（such as immunofluorescence,Western blot,RT-PCR,and fluorescence probe）to investigate whether the key active components could inhibit cellular oxidative stress through ALDH2/4-HNE signaling and impede angiogenic response through SIRT1/HIF-1α/VEGFA signaling,and simultaneously improve mitochondrial oxidative stress and mitochondrial function to protect the retinal functional and structural integrity.Results1.The preliminary study of pharmacological effect and mechanism of couplet medicines Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on attenuating diabetic retinal disorderAfter 8-week pharmacological intervention,there was no statistically significant difference in body weight and blood glucose between CON+SCH group and CON group.The results of hemorheology showed that Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could decrease the whole blood viscosity,Cassone viscosity and aggregation of erythrocytes,and improve the blood circulation function of DR mice.In vivo OCT and pathological sections HE staining showed that Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could increase the thickness and the number of cell layers of photoreceptors cell layer（IS/OS and ONL）,inner nuclear layer and the entire retina in DR mice.Visual electrophysiological examination suggested that Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could improve the retinal microvascular function,attenuate the condition of retinal ischemia and hypoxia,and maintain the integrity of retinal function in DR mice through increasing the amplitude of b wave of dark-adapted 0.01 response,a wave and b wave of dark-adapted 3.0 response,b wave of light-adapted 3.0 response,and wave of Flicker response.The results of immunofluorescence,Western blot and RT-PCR indicated that Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could inhibit angiogenic response via decreasing the expressions of VEGFA and HIF-1αin the retina of DR mice.2.The analyze of chemical components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in vitro and the transitional prototype active components in blood serum and ocular tissue in vivoApplying tandem high performance liquid chromatography and Q-TOF mass spectrometry method,the chemical compositions map of Salviae Miltiorrhizae Radix et Rhizoma and Moutan Cortex was successfully constructed.A total of 15 components including gallic acid,danshensu,protocatechuic acid,protocatechuic aldehyde,oxypaeoniflorin,paeoniflorin,salvianolic acid D,rosmarimic acid,salvianolic acid B,salvianolic acid A,benzoylpaeoniflorin,dihydrotanshinone I,tanshinone I,tanshinone IIA and albiflorin were identified in the negative mode.Paeonol,cryptotanshinone and neocryptotanshinone were identified in the positive mode.Among them,12 components were derived from Salviae Miltiorrhizae Radix et Rhizoma and 6 components were derived from Moutan Cortex.Serum transitional components analysis showed that 6prototype pharmacodynamic components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex,including tanshinone IIA（33.34 min）,cryptotanshinone（29.70min）,neocryptotanshinone（25.92 min）,albiflorin（9.94 min）,paeoniflorin（10.68 min）,and benzoylpaeoniflorin（17.11min）.Analysis of ocular tissue transitional components revealed 4 prototype active compositions,including neocryptotanshinone（25.92 min）and cryptotanshinone（29.70 min）from Salviae Miltiorrhizae Radix et Rhizoma,and albiflorin（9.94 min）and paeoniflorin（10.68 min）from Moutan Cortex.Based on high performance liquid chromatography quantitative analysis,it was found that the contents of serum transitional prototype pharmacodynamic components（including ocular tissue transitional active components）cryptotanshinone,cryptotanshinone,tanshinone IIA,paeoniflorin,albiflorin and benzoylpaeoniflorin in Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex were 0.21%,0.31%,0.26%,2.39%,5.76%and 0.31%,respectively.3.The study of pharmacodynamics and mechanism of the compatibility of active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on attenuating diabetic retinal disorderIn vivo OCT and HE staining of pathological sections indicated that the compatibility of the ocular tissue targeting active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could reduce the thinning of the photoreceptor layer（IS/OS and ONL）,inner nuclear layer and the entire retina.ff ERG showed that the compatibility of the active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could increase the amplitude of b wave of dark-adapted 0.01 response,b wave of dark-adapted3.0 response,OPs2 wave of dark-adapted response,b wave of light-adapted 3.0 response,and wave of light-adapted Flicker response in DR mice,and protect the function of retinal photoreceptor cells and Müller cells,and improve the state of retinal ischemia.Fundus fluorescein angiography showed that the compatibility of the active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could shorten the appearance time of fluorescein sodium in central retinal vein and increase the retinal perfusion area,which helped to improve retinal blood microcirculation function and remove blood stasis.Transmission electron microscopy indicated that the compatibility of the active compositions improved the retinal rods mitochondrial structure damage,and reduce retinal mitochondrial DNA damage and ROS level.Meanwhile,molecular biological detection results showed that the compatibility of the active compositions could improve the retinal oxidative stress level by up-regulating the expression of ALDH2,VHL and GPX4 and down-regulating the expression of 4-HNE.Through increasing SIRT1 expression and decreasing VEGFA and HIF-1αexpression,the retinal angiogenic response was inhibited.In addition,the compatibility of the active components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could depress cell apoptosis and inflammatory response via decreasing the levels of Caspase 3,Bax,p53,Cyt c,IL-6 and TNF-α.4.The study of pharmacological effect and mechanism of the active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on high glucose-induced Müller cell damageTUNEL results showed that the active components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could significantly inhibit Müller cell apoptosis induced by high glucose.Immunofluorescence and Western blot showed that Salviae Miltiorrhizae Radix et Rhizoma and Moutan Cortex could protect the Müller cell structural and functional homeostasis,up-regulate the expressions of GS,ALDH2 and SIRT1,and decrease the expressions of GFAP,VEGFA,HIF-1α,4-HNE,IL-1βand Caspase 3,which indicated that it could inhibit the reactive gliosis,proangiogenic response,inflammatory response and apoptosis in Müller cells.At the same time,the active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could improve mitochondrial oxidative stress and functional disorder,and reduce mitochondrial damage in Müller cells.In the compatibility of the active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex+ALDH2 inhibitor/si RNA group,the Müller cell apoptosis was increased,the expression of 4-HNE and Caspase 3 was up-regulated,and the levels of ROS and MDA were increased,suggesting that it could inhibit oxidative stress and cell apoptosis through ALDH2 signaling.In the compatibility of the active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex+SIRT1 inhibitor/si RNA group,the expressions of HIF-1αand VEGFA were up-regulated and the levels of inflammatory factors were increased,suggesting that it could inhibit proangiogenic response through SIRT1 signaling.In addition,there existed mutual feedback regulation between ALDH2and SIRT1.These results suggested that Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could improve Müller cell damage by activating SIRT1-ALDH2 axis.Conclusion1.The present study was the first to preliminarily clarify the pharmacological effect of classic"cooling blood and removing stasis"couplet medicines Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on improving diabetic retina disorderIt was found that couplet medicines Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could attenuate diabetic retinal disorder in a dose-dependent manner by protecting retinal function and structure,enhancing blood circulation and inhibiting retinal angiogenic response.Additionally,the intervention effect of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex was better than that of Salviae Miltiorrhizae Radix et Rhizoma or Moutan Cortex alone,indicating that the combination of Salviae Miltiorrhizae Radix et Rhizoma and Moutan Cortex exerted synergistic effect.Thus,it may provide theoretical and experimental basis for the expansion of clinical application of the classic"cooling blood and removing stasis"couplet medicines Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in the treatment of DR.2.The present study identified the serum transitional prototype pharmacodynamic components and the ocular tissue targeting effective substances of couplet medicines Salviae Miltiorrhizae Radix et Rhizoma-Moutan CortexAccording to three dimensions,including chemical composition map construction in vitro,and serum transitional prototype pharmacodynamic components identification and ocular tissue targeting effective substances analyzation in vivo,we confirmed that the ocular tissue targeting active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex comprising neocryptotanshinone,cryptotanshinone,albiflorin,and paeoniflorin could be the key pharmacodynamic substance of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in treating DR.Therefore,it is helpful to reveal the effective substance basis and components compatibility mechanism of couplet medicines Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex in improving diabetic retinal disorders.3.The present study demonstrated the pharmacological effects of the compatibility of the ocular tissue targeting active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex on inhibiting retinal photoreceptors cell apoptosis and angiogenic response in the diabetic retinaIt was found that the ocular tissue targeting active components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex comprising cryptotanshinone,neocryptotanshinone,albiflorin and paeoniflorin could attenuate diabetic retinal disorder through four aspects.Firstly,it could protect retinal electrophysiological function,microcirculation function,and retinal structure integrity.Secondly,it could inhibit the apoptosis of retinal photoreceptor cells.Thirdly,it could alleviate oxidative stress-induced retinal mitochondria damage.Fourthly,it could depress retinal angiogenic response.Thus,this study demonstrated that with the characteristics of tissue targeting,definite composition and controllable quality,the active components of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex exerted significant pharmacological effects in DR treatment,which laid a foundation for the further study of its pharmacological mechanism.4.The present study revealed that the compatibility of the ocular tissue targeting active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could regulate oxidative stress and inhibit VEGFA in Müller cells via activating ALDH2/SIRT1 signalingIt was found that the compatibility of the ocular tissue targeting active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex could reduce the Müller cell apoptosis and oxidative stress through Al DH2/4-HNE signaling pathway,and inhibit the production of VEGFA and angiogenic response through SIRT1/HIF-1αsignaling pathway.It revealed that the activation of ALDH2-SIRT1 signaling axis was an important molecular mechanism of the key active compositions of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex to regulate oxidative stress and depress VEGFA signaling in Müller cells.Thus,it may contribute to the explanation of the mechanism of molecular components compatibility of Salviae Miltiorrhizae Radix et Rhizoma-Moutan Cortex,provide scientific basis for the clinical application and secondary development of its related therapeutics,and inspire research ideas for the investigation of the mechanism of traditional Chinese couplet medicines and its effective components in the prevention and treatment of DR.