Effect of icariin on ubiquitination of amyloid precur-sor protein in Alzheimer's disease mice / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of icariin(ICA)on the ubiquitination modification of β-amy-loid precursor protein(APP)in Alzheimer's disease mice.METHODS In vitro,① HEK 293 cells stably overex-pressing human APP695(OE-hAPP)were treated with different concentrations of ICA(10-100 μmol·L-1)for 24 h and the cell viability was detected by MTT assay.②CHX(50 mg·L-1)was used to block protein synthesis and MG132(20 μmol·L-1)inhibits proteasome activity,then the level of APP in different time(0,0.5,1,2,3 and 4 h)and the ubiquitination were tested by Western blotting.③ E3 ubiquitin ligases HMG-CoA reductase degradation pro-tein 1(HRD1)protein expression in OE-hAPP was tested by Western blotting,as well as the level and ubiquitination of APP were tested under HRD1 silent condition by Co-IP and Western blotting.In vivo,① male APP/PS1 mice and wild type(WT)mice were randomly divided into 5 groups:WT,WT+ICA,APP/PS1,APP/PS1+ICA,and APP/PS1+donepezil(DPZ)groups.ICA(60 mg·kg-1·d-1)and DPZ(1 mg·kg-1·d-1)were treated for 3 months by gavage from 6 months of age,and WT mice were given equal volume of distilled water.②Morris water maze and Y-maze experiments were used to detect the alteration of spatial learning memory function.③ After then,the brain tissues were collected,total proteins were extracted,APP antibodies were subjected to Co-IP,and total ubiqui-tination(Ub),K48-linked polyubiquitination(UbK48)and K63-linked polyubiquitination of APP level,APP and HRD1 proteins were detected by Western blotting.RESULTS In vitro results showed that ICA significantly enhanced APP degradation(vs control,P＜0.01),up-reg-ulated HRD1 expression(vs control,P＜0.05;vs OE-hAPP,P＜0.05),elevated the level Ub and UbK48 of APP,as well as increased APP degradation.Moreover,silenced HRD1 gene abolished abovementioned effects of ICA(vs control-siRNA,P＜0.05;vs HRD1-siRNA,P＜0.05).In vivo results showed that ICA improved the spa-tial learning and memory function APP/PS1 mice by Mor-ris water maze and Y-maze tests,increased HRD1 expres-sion(vs APP/PS1 + vehicle,P＜0.05),enhanced APP ubiquitination and reduced APP protein level(vs APP/PS1 + vehicle,P＜0.01).CONCLUSION ICA promotes the ubiquitination and proteasome-dependent degrada-tion of APP by up-regulating HRD1,thereby improving the spatial learning and memory function of Alzheimer disease mice.

Icariin ameliorates behavioral deficits and neuropa-thology in a mouse model of multiple sclerosis / 中国药理学与毒理学杂志

Multiple sclerosis(MS)is a systemic inflammatory illness of the central nervous system that involves demyelinating lesions in the myelin-rich white matter and pathology in the grey matter.Despite signifi-cant advancements in drug research for MS,the dis-ease's complex pathophysiology makes it difficult to treat the progressive forms of the disease.In this study,we identified a natural flavonoid compound icariin(ICA)as a potent effective agent for MS in ameliorating the deterioration of symptoms including the neurological defi-cit score and the body weight in a murine experimental autoimmune encephalomyelitis(EAE)model.These improvements were associated with decreased demyelin-ation in the corpus callosum and neuron loss in the hippo-campus and cortex confirmed by immunohistochemistry analysis.Meanwhile,it was observed that the activation of microglia in cerebral cortex and hippocampus were inhibited followed by the neuroinflammatory cytokines downregulation such as IL-1β,IL-6 and TNF-α after ICA treatment,which was probably attributable to the sup-pression of microglial NLRP3 inflammasome activation.Additionally,molecular docking also revealed the binding force of ICA to NLRP3 inflammasome protein complexes in vitro.Taken together,our findings have demonstrated that ICA,as pleiotropic agent,prevents EAE-induced MS by improving demyelination and neuron loss,which inter-feres with the neuroinflammation via microglial NLRP3 inflammasome activation.

Protective effect of icariin on Aβ25-35-induced HT22 cell injury by ameliorating glycolytic dysfunction through activating Wnt/β-catenin signaling pathway / 中国药理学与毒理学杂志

OBJECTIVE To investigate whether icari-in(ICA)plays a neuroprotective role by improving glyco-lytic function through activating Wnt/β-catenin signaling pathway.METHODS HT22 cells were treated with Aβ25-35 for 24 h to establish AD cell model,ICA was added in 2 h before Aβ25-35 and the DKK1(a specific inhibitor of the Wnt signaling pathway)was added in 0.5 h before ICA.Pharmacodynamic study:HT22 cells were divided into control group,ICA group(ICA 10 μmol·L-1),model group(Aβ25-3520 μmol·L-1),model + ICA group(Aβ25-3520 μmol·L-1 +ICA 2.5,5,10 μmol·L-1);Mechanism study:HT22 cells were divided into control group,model group,Aβ25-35+ICA 10 μmol·L-1 group,Aβ25-35+DKK1 group,Aβ25-35+DKK1+ ICA group.The cell viability was detected by MTT assay and the cell morphology was obtained by microscope,the lactate content was detected by lactate assay,the ATP content was measured with the chemiluminescence method,the expression levels of HK1,PKM1 and the pro-tein expression of molecules related to the Wnt/β-catenin signaling pathway(Wnt3a,GSK3β,pGSK3β Try216,pGSK3β Ser9,β-catenin,pβ-catenin Ser33/37 Thr41,Active β-catenin and nuclear β-catenin)was assayed by Western blotting.The nuclear translocation of β-catenin was observed by immunofluorescent staining.RESULTS Compared with the control group,the viability of cells in the model group was reduced,the morphology of cells was significantly damaged,the ATP content and lactate content were significantly decreased,and the glycolytic key enzymes:the protein levels of HK1,PKM1 and the protein levels of Wnt3a,pGSK3β Ser9,active β-catenin and nuclear β-catenin were significantly reduced,and the phosphorylation levels of β-catenin Ser33/37 Thr41 were significantly increased.Compared with the model group,the cell morphology was significantly improved and the viability was significantly increased,the ATP and lactate content were significantly increased,the expressions of HK1,PKM1 and Wnt3a,pGSK3β Ser9,active β-catenin and nuclear β-catenin protein were significantly upregulat-ed,and the phosphorylation levels of β-catenin Ser33/37 Thr41 were significantly reduced after ICA treatment.However,when the canonical Wnt signaling was inhibited by DKK1,the above effects of ICA on glycolysis were abolished.CONCLUSION ICA exerts neuroprotective effects on Aβ25-35-induced HT22 cell injury by enhancing the glycolysis function through the activation of the Wnt/β-catenin signaling pathway.

Effects of icariin on cognitive function and astrocytic pyroptosis in hemorrhagic shock resuscitation model mice / 中华行为医学与脑科学杂志

Objective:To investigate the icariin on cognitive function and astrocytic pyroptosis in hemorrhagic shock resuscitation model mice.Methods:Forty-eight SPF grade C57BL/6 mice (male) were randomly divided into four groups ( n=12 in each group): Sham operation control group (Group C), hemorrhagic shock and resuscitation group (Group H), hemorrhagic shock and resuscitation plus icariin group (Group HI) and hemorrhagic shock resuscitation plus icariin and SSK1 group (Group HIS, SSK1 was a phosphorylation agonist of mitogen-activated protein kinase p38(p38MAPK). The mice in Group H, HI and HIS were subjected to hemorrhagic shock and resuscitation model by bleeding and retransfusion via left femoral vein; the mice in Group HI and HIS were administered with icariin (10 mg/kg) intragastrically for 7 days; the mice in Group C and H were administered with the same amount of normal saline containing dimethyl sulfoxide(DMSO). The mice in Group HIS were administered with SSK1 (0.5 mg/kg) intraperitoneally, but the mice in Group C, H and HI were only administered with the same amount of normal saline containing DMSO.At 15 days after resuscitation, novel objective recognition test and fear conditioning test were used to assess cognitive dysfunction of mice.Microtubule-associated protein 2(MAP2), a specific marker protein of neurons reflecting astrocytic pyroptosis in the hippocampus of mice, were detected by immunofluorescence assay so as to assess neuronal injury and astrocytic pyroptosis.The levels of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the hippocampus were evaluated by Western blot.SPSS 21.0 software was used for data analysis, multiple samples among groups were compared by one-way ANOVA, and SNK- q test was used for further pairwise comparison. Results:The results of new object recognition test showed that the difference of new object recognition index among the four groups was statistically significant ( F=50.75, P<0.05). The new object recognition indexes in H group(22.7±6.9), HI group(40.1±7.0) and HIS group (22.5±7.5) were significantly lower than that in C group (58.5±11.2). The index in HI group was higher than that in H group, while the index in HIS group was lower than that in HI group (all P<0.05). The results of the fear conditioning test showed that there was a statistically significant difference in the percentage of freezing time among the four groups of mice ( F=60.54, P<0.05). And the percentage of freezing time in H group((21.8±5.0)%), HI group ((38.4±7.4) %)and HIS group((21.3±4.2)%)were lower than that in C group((49.1±7.0)%), which in HI group was higher than that in H group ( P<0.05)and which in HIS group was lower than that in HI group(all P<0.05). The results of immunofluorescence showed that there were significant decreases of MAP2 intensity ((35.3±9.3)%, (63.3±6.1)%, (28.7±10.3)%) but increases of pyroptotic astrocytes ((24.5±4.2)%, (9.3±1.5)%, (22.1±3.3)%) in the H, HI and HIS groups compared with those of C group ((106.7±19.7) %, (3.4±2.0)%). There was an increase of MAP2 intensity but a decrease of pyroptotic astrocytes in the HI group compared with those in H group, and there was a decrease of MAP2 intensity but an increase of pyroptotic astrocytes in the HIS group compared with those of HI group (all P<0.05). The Western blot results showed that there were significant increases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the H, HI and HIS groups compared with C group, there were decreases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the HI group compared with H group, and there were increases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the HIS group compared with those in HI group (all P<0.05). Conclusion:Icariin alleviates hemorrhage shock and resuscitation-induced cognitive dysfunction and astrocytic pyroptosis in mice, and the mechanism may be associated with inhibition of phosphorylated p38MAPK.

Icariin Ameliorates Efferocytosis Dysfunction of Alveolar Macrophages Stimulated by Cigarette Smoke Extract via PPARγ Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of icariin in ameliorating efferocytosis dysfunction and inflammatory response of alveolar macrophages induced by cigarette smoke extract via the peroxisome proliferator-activated receptor gamma （PPARγ） signaling pathway. MethodThe untreated rat alveolar macrophages （NR8383） were taken as the blank group. The NR8383 cells treated with 10% cigarette smoke extract were divided into model， low-， medium-， and high-dose （10， 20， 40 μmol·L-1） icariin， PPARγ inhibitor， and PPARγ inhibitor + low-， medium-， and high-dose icariin groups. Alamar blue colorimetry was employed to examine the proliferation and toxicity of icariin on NR8383 cells. The efferocytosis rate of NR8383 cells was detected by flow cytometry. Enzyme-linked immunosorbent assay was employed to measure the levels of tumor necrosis factor-alpha （TNF-α）， transforming growth factor-β1 （TGF-β1）， and milk fat globule-epidermal growth factor 8 （MFG-E8）. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were employed to determine the protein and mRNA levels， respectively， of PPARγ， CD36， and RAS-related C3 botulinum toxin substrate 1 （Rac1）. ResultThe efferocytosis dysfunction model of NR8383 was established with the cigarette smoke extract. Compared with the blank control group， the model group showed decreased efferocytosis rate （P<0.05）， elevated TNF-α level （P<0.05）， lowered TGF-β1 and MFG-E8 levels （P<0.01）， and down-regulated mRNA and protein levels of PPARγ， CD36， and Rac1 （P<0.05， P<0.01）. Compared with the model group， the treatment with icariin increased the efferocytosis rate （P<0.05， P<0.01）， lowered the TNF-α level （P<0.01）， elevated TGF-β1 and MFG-E8 levels （P<0.05）， and up-regulated the protein and mRNA levels of PPARγ， CD36， and Rac1 （P<0.05， P<0.01）. Compared with icariin alone， PPARγ inhibitor + icariin decreased the efferocytosis rate （P<0.05） and down-regulated the protein and mRNA levels of PPARγ （P<0.05， P<0.01）. In addition， PPARγ inhibitor + low-dose icariin down-regulated the protein level of CD36 （P<0.01） and PPARγ inhibitor + low-/medium-dose icariin up-regulated the protein level of Rac1 （P<0.05）. ConclusionIcariin ameliorates the cigarette smoke extract-induced efferocytosis dysfunction of alveolar macrophage by regulating the PPARγ signaling pathway and cytoskeletal structure rearrangement.

Protective effects and mechanism of icariin against vascular function in diabetic mice / 中国药科大学学报

@#To explore the effects and molecular mechanism of icariin on the vascular function of mice with type 1 diabetes induced by alloxan, type 1 diabetic mice model was established by intraperitoneal injection with 200 mg/kg alloxan.After oral administration with icariin (60, 120 mg/kg) daily for 2 weeks, blood glucose, body weight, food intake and water intake were detected.To evaluate the impact of icariin on the function of isolated vascular ring contraction and relaxation, thoracic aortas of mice were removed and the Ach-induced vascular ring relaxation, Phe-induced vascular ring contraction, SNP-induced vascular ring relaxation and KCl-induced vascular ring contraction response were detected.To further confirm the mechanism of icariin to improve vascular function, human umbilical vein endothelial cells (HUVECs) were induced by high glucose (HG) in vitro.Western blot was used to detect the effect of icariin on eNOS, p-eNOS, p38 MAPK and p-p38 MAPK expressions in HG-induced human umbilical vein endothelial cells (HUVECs).The results indicated that icariin significantly ameliorated the weight loss and dampened the increase in water intake of the diabetic mice.Meanwhile, icariin had a certain ameliorative effect on blood glucose and food intake without significant difference.The results of isolated thoracic aortas vascular rings contraction and vasodilation function indicated that icariin significantly improved Phe-induced vascular contraction and Ach‐induced vascular relaxation.Meanwhile, icariin had a certain ameliorative effect on KCl-induced vascular contraction response without significant difference.However, no significant change was observed on endothelium‐independent vascular rings relaxation response induced by SNP after treatment with icariin.Results of Western blot showed that icariin inhibited the expression of p-p38 MAPK and induced expression of p-eNOS in the high glucose-induced HUVECs cell model.Therefore, icariin may attenuate alloxan-induced type 1 diabetic mice vascular diastolic function by inhibiting expression of p-p38 MAPK and inducing expression of p-eNOS.

Icariin improves renal interstitial fibrosis in a rat model of chronic renal failure by regulating mitochondrial dynamics / 中国中药杂志

This study aims to explore the effect of icariin(ICA) on mitochondrial dynamics in a rat model of chronic renal failure(CRF) and to investigate the molecular mechanism of ICA against renal interstitial fibrosis(RIF). CRF was induced in male Sprague-Dawley(SD) rats with 5/6(ablation and infarction, A/I) surgery(right kidney ablation and 2/3 infarction of the left kidney). Four weeks after surgery, the model rats were randomized into the following groups: 5/6(A/I) group, 5/6(A/I)+low-dose ICA group, and 5/6(A/I)+high-dose ICA group. Another 12 rats that received sham operation were randomly classified into 2 groups: sham group and sham+ICAH group. Eight weeks after treatment, the expression of collagen-Ⅰ(Col-Ⅰ), collagen-Ⅲ(Col-Ⅲ), mitochondrial dynamics-related proteins(p-Drp1 S616, p-Drp1 S637, Mfn1, Mfn2), and mitochondrial function-related proteins(TFAM, ATP6) in the remnant kidney tissues was detected by Western blot. The expression of α-smooth muscle actin(α-SMA) was examined by immunohistochemical(IHC) staining. The NRK-52 E cells, a rat proximal renal tubular epithelial cell line, were cultured in vitro and treated with ICA of different concentration. Cell viability was detected by CCK-8 assay. In NRK-52 E cells stimulated with 20 ng·mL~(-1) TGF-β1 for 24 h, the effect of ICA on fibronectin(Fn), connective tissue growth factor(CTGF), p-Drp1 S616, p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 was detected by Western blot, and the ATP content and the mitochondrial morphology were determined. The 20 ng·mL~(-1) TGF-β1-stimulated NRK-52 E cells were treated with or without 5 μmol·L~(-1) ICA+10 μmol·L~(-1) mitochondrial fusion promoter M1(MFP-M1) for 24 h and the expression of fibrosis markers Fn and CTGF was detected by Western blot. Western blot result showed that the levels of Col-Ⅰ, Col-Ⅲ, and p-Drp1 S616 were increased and the levels of p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 were decreased in 5/6(A/I) group compared with those in the sham group. The levels of Col-Ⅰ, Col-Ⅲ, and p-Drp1 S616 were significantly lower and the levels of p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6 were significantly higher in ICA groups than that in 5/6(A/I) group. IHC staining demonstrated that for the expression of α-SMA in the renal interstitium was higher in the 5/6(A/I) group than in the sham group and that the expression in the ICA groups was significantly lower than that in the 5/6(A/I) group. Furthermore, the improvement in the fibrosis, mitochondrial dynamics, and mitochondrial function were particularly prominent in rats receiving the high dose of ICA. The in vitro experiment revealed that ICA dose-dependently inhibited the increase of Fn, CTGF, and p-Drp1 S616, increased p-Drp1 S637, Mfn1, Mfn2, TFAM, and ATP6, elevated ATP content, and improved mitochondrial morphology of NRK-52 E cells stimulated by TGF-β1. ICA combined with MFP-M1 further down-regulated the expression of Fn and CTGF in NRK-52 E cells stimulated by TGF-β1 compared with ICA alone. In conclusion, ICA attenuated RIF of CRF by improving mitochondrial dynamics.

Mechanism of Icariin in Ameliorating Neuronal and Dendritic Damage in Alzheimer's Disease by Regulating RhoA/ROCK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of icariin on the recombinant Ras homolog family member A （RhoA）/Rho-associated coiled-coil forming protein kinase （ROCK） signaling pathway in rats with Alzheimer's disease （AD）， and to explore the mechanism of icariin in ameliorating the neuronal and dendritic damage. MethodThe β-amyloid 1-42 （Aβ1-42， 2.5 g·L-1） was used to induce AD in rats via lateral ventricle injection， and the rats were divided into a model group， a low-dose icariin group （0.03 g·kg-1）， a middle-dose icariin group （0.06 g·kg-1）， a high-dose icariin group （0.09 g·kg-1）， and a control group. The control group and the model group were given an equal volume of normal saline at a dose of 10 mL·kg-1. The cognitive function of rats was assessed by the Morris water maze. The pathological morphology of the rat hippocampal CA1 area was observed by Nissl staining. Dendritic spine density and dendritic length in the CA1 region of the hippocampus were observed by Golgi-Cox staining. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of tumor necrosis factor-α （TNF-α）， interleukin （IL）-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus. Western blot assay was used to detect the protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus. ResultAs compared with the control group， the escape latency of the rats in the model group was increased （P<0.01）， while the number of crossing the platform and the dwelling time in the target quadrant were decreased （P<0.01）. As compared with the model group， the escape latency of the rats in the middle and high-dose icariin groups was decreased （P<0.05， P<0.01）， while the number of crossing the platform and the dwelling time in the target quadrant were increased （P<0.05， P<0.01）. As compared with the control group， the number of neurons， dendritic spine density， and dendritic length in the hippocampal CA1 area of the rats in the model group were decreased （P<0.01）. As compared with the model group， the number of neurons， dendritic spine density， and dendritic length in the hippocampus of the rats in the middle and high-dose icariin groups were increased （P<0.05， P<0.01）. As compared with the control group， the mRNA and protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus of the rats in the model group were increased （P<0.01）. As compared with the model group， the mRNA and protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus of the rats in the middle and high-dose icariin groups were decreased （P<0.05， P<0.01）. ConclusionIcariin improves cognitive function and neuronal and dendritic damage in AD by inhibiting the RhoA/ROCK signaling pathway.

Icariin Inhibits Proliferation of CLC5 Hepatoma Cells via Akt/GSK3β/CDK Pathway / 中国实验方剂学杂志

ObjectiveTo study the effect of icariin on the proliferative capacity of hepatocellular carcinoma cell line CLC5 and the underlying mechanism. MethodThe targets of icariin were screened out by network pharmacology， and the target network and protein-protein interaction （PPI） network were constructed to predict the possible targets and pathways of icariin. CCK-8 assay was employed to explore the effects of different concentrations （0， 6.25， 12.5， 25， 50 μmol·L-1） of icariin on the viability of CLC5 cells. Further， CLC5 cells were treated with 0， 25， 50 μmol·L-1 icariin， and the effect of icariin on CLC5 cell proliferation was examined by Edu-488 assay and clone formation assay （CFA）. Western blot was employed to measure the expression levels of proteins in the protein kinase B （Akt）/glycogen synthase kinase 3β （GSK3β）/cell cycle-dependent kinase （CDK） pathway in the CLC5 cells exposed to different concentrations of icariin. ResultNetwork pharmacological analysis revealed that icariin may inhibit the hepatocellular carcinoma via cell cycle arrest and inhibition of tumor cell proliferation. Compared with the blank group， icariin decreased the viability of CLC5 cells in a time- and concentration-dependent manner （P<0.01） and reduced the positive rate of Edu-488 and the colonies in CFA （P<0.05， P<0.01）. Moreover， icariin down-regulated the protein levels of p-Akt， p-GSK3β， CDK4， and CyclinD1 （P<0.05， P<0.01）. ConclusionIcariin may block cell cycle to suppress the proliferation of CLC5 cells via inhibiting the Akt/GSK3β/CDK pathway.

Simultaneous Determination of the Contents of 6 Components in Fuzheng Guben Granules by QAMS / 中国药房

OBJECTIVE：To establish the method for content determination of 6 components in Fuzheng guben granules ，such as 2，3，5，4′-tetrahydroxystilbene glucoside ，baicalin，icariin，scutellarin，baicalein and wogonin. METHODS ：HPLC method was adopted. The determination was performed on Dikma Diamonsil C 18 column with mobile phase consisted of acetonitrile- 0.1％ phosphoric acid aqueous solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelengths were set at 275 nm （0-8 min），320 nm（8-9 min）and 275 nm（9-33 min）. The column temperature was set at 25 ℃，and sample size was 10 μL. With baicalin as reference material ，the relative corr ection factors （fk/s） of other five components were calculated by multi-point correction method and slope correction method ；the retention time difference method was used to locate the chromatographic peaks ； the calculation values obtained by above 2 QAMS were compared with measured values of external standard method. RESULTS ： The linear range of 2，3，5，4′-tetrahydroxystilbene glucoside ，baicalin，icariin，scutellarin，baicalein and wogonin were 0.053-2.12， 0.163-6.52，0.059-2.36，0.021 6-0.864，0.03-1.2，0.021-0.84 μg（r＞0.999），respectively. RSDs of precision ，stability（12 h）and reproducibility tests were all lower than 3％. Average recoveries were 98.72％-99.82％（RSDs were 0.89％-1.24％，n＝9）. Using baicalin as reference material ，fk/s of multi-point correction method for 2，3，5，4′-tetrahydroxystilbene glucoside ，icariin，scutellarin， baicalein and wogonin were 1.172，0.528，1.479，1.820 and 2.534，respectively；fk/s of slope correction method were 1.234， 0.550，1.559，1.939，2.664. RSDs of 6 components in 10 batches of Fuzheng guben granules by 3 methods were 0.29％-2.77％ （n＝10），respectively. Pearson correlation coefficient was not lower than 0.999 9（P＜0.001）in measured values between QAMS and external standard method. CONCLUSIONS ：QAMS method is established successfully for simultaneous determination of 6 components in Fuzheng guben granules.

Molecular mechanism of icariin effects on articular chondrocytes, subchondral bone and synovium in the treatment of osteoarthritis / 中国组织工程研究

BACKGROUND: Icariin is the main effective component of Epimedium, which functions to tonify the kidney, and strengthen tendons and bones. In recent years, a large number of studies have found that icariin plays a significant role in the treatment of osteoarthritis. OBJECTIVE: To review the research progress in the molecular mechanism of icariin in the treatment of osteoarthritis. METHODS: The first author used “Icariin, Osteoarthritis, Cartilage, Subchondral bone, Synovial membrane, synovium, Inflammation" as search words in English and Chinese to search PubMed, CNKI, WanFang, and VIP databases. According to the inclusion criteria and exclusion criteria, 42 articles were included for final analysis. RESULTS AND CONCLUSION: Icariin can promote the cartilage differentiation of bone marrow mesenchymal stem cells and enhance the proliferation of cartilage cells and osteoblasts, to inhibit the degradation of cartilage extracellular matrix, reduce the activity of osteoclasts and alleviate synovial inflammation caused by inflammatory factors. It is an effective treatment for osteoporosis. However, the optimal effective dose and concentration safety of icariin still need a large number of experimental studies. Currently, most of the experiments are still in animal and tissue cell experiments. Numerous clinical studies are needed to continue to explore its specific mechanism in order to provide evidence-based medical evidence for icariin in the treatment of osteoarthritis.

Study on Improvement Effects of Icariin on Cognitive Function in Schizophrenia Model Rats and Its Mechanism / 中国药房

OBJECTIVE：To study the improvement effects of ica riin（ICA）on cognitive function in schizophrenia model rats and its mechanism. METHODS ：SD rats were divided into blank control group ，model group ，ICA low-dose ，medium-dose and high-dose groups （15，30，60 mg/kg）. Except for blank control group ，other groups were given N-methyl-D-aspartate receptor antagonist MK- 801（0.2 mg/kg）intraperitoneally to induce schizophrenia rats models ，once a day ，for consecutive 14 days. After modeling，ICA groups were intragastrically administered with the corresponding drugs ，while blank control group and model group were intragastrically administered with the same volume of water ，once a day ，for consecutive 7 days. The behavioral com changes of rats were detected by Morris water maze test ，open field test ， forced swimming test and Y maze test pathological changes of hippocampus were observed by Nissl staining；the levels of cholinergic indexes [acetylcholine （Ach），choline acetyltransferase （ChAT） and acetylcholinesterase （AchE）] in cerebral tissues were detected by ELISA. The expression of BDNF ，ERK and CREB mRNA in cerebral tissue were detected by RT-PCR ；expression or phosphorylation level of BDNF ，ERK，CREB protein ，apoptosis related proteins （Bcl-2，Bax and Caspase- 3）were detected by Western blot. RESULTS ：Compared with blank control group ，escape latency ，distance at T 1-T3， cumulative immobility time and the expression of Caspase- 3 protein in cerebral tissues were significantly increased in model group （P＜0.05）；the times of crossing platform ，alternation rate ，the number of Nissl staining positive neurons in hippocampus tissues ， the levels of Ach and ChAT in cerebral tissues ，Bcl-2/Bax ratio ，mRNA and protein expression of BDNF ，mRNA expression of ERK and CREB ，the phosphorylation of ERK 1/2 and CREB were significantly decreased （P＜0.05）.Compared with model group ， escape latency ，distance at T 1-T3，cumulative immobility time ，the number of Nissl staining positive neurons ，AchE level in cerebral tissues and relative expression of Caspase- 3 protein were significantly decreased in ICA high-dose group （P＜0.05）；the times of crossing platform ，alternation rate ，levels of Ach and ChAT in cerebral tissues ，Bcl-2/Bax ratio ，mRNA and protein expression of BDNF ，mRNA expression of ERK and CREB ，the phosphorylation of ERK 1/2 and CREB were increased significantly（P＜0.05）. Above indexes in ICA low-dose and medium-dose groups were partially improved significantly than model group（P＜0.05）. CONCLUSIONS ：ICA can improve cognitive function in schizophrenia model rats.Its mechanism may be related to regulating cholinergic system ，inhibiting neuronal apoptosis ，and promoting the expression of BDNF/ERK/CREB signaling pathway.

Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells / 中国中药杂志

This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.

Effects and mechanism of epimedium polysaccharide on solubility of icariin and baohuoside Ⅰ / 中国中药杂志

This study evaluated the effects of epimedium polysaccharide(EPS) on the solubility of icariin and baohuoside Ⅰ so as to preliminary explore its solubilization function and the underlying mechanism. The solubility of these two insoluble flavonoids in water and polysaccharide solutions was compared by high performance liquid chromatography, and the mechanism was investigated by diffe-rential scanning calorimetry(DSC) and critical micelle concentration determination. The results indicated that their solubilization in crude EPS solutions was concentration-dependent. The solubility of icariin and baohuoside Ⅰ in 20 mg·mL~(-1) EPS-1-1 was 9.05 times and 5.76 times that in water, respectively; while their solubility in 20 mg·mL~(-1) EPS-2-1 was 10.55 and 8.39 times that in water, respectively. The change of the DSC thermograms suggested the formation of new complexes from icariin and baohuoside Ⅰ with polysaccharides. The critical micelle concentrations proved the micellar properties of both EPS-1-1 and EPS-2-1. In short, EPS can significantly increase the solubility of icariin and baohuoside Ⅰ, the mechanism of which may be related to the formation of micellar complexes between EPS and insoluble flavonoids.

Effect of icariin on early βdefensin2 and T cell subsets in rats after tracheotomy / 中南大学学报(医学版)

OBJECTIVES@#To investigate the effect of icariin (ICA) on early β-defensin-2 and T cell subsets in rats after tracheotomy.@*METHODS@#A total of 54 SPF male Sprague-Dawley rats were randomly divided into a normal control group (group A), a model group (group B), and a model+ICA treatment group (group C), with 18 rats in each group. A tracheotomy intubation model of the B and C group was prepared. After 6 h of surgery, ICA intervention was given to group C. Groups A and B were given the same amount of normal saline. Lung tissue, alveolar lavage fluid and peripheral blood were taken at 24 h, 72 h and 168 h, respectively. The expression of rat β-defensin-2 mRNA in lung tissue was detected by RT-PCR. The content of β-defensin-2 in alveolar lavage fluid and peripheral blood serum was detected by ELISA. The content of peripheral blood T cell subsets (CD3, CD4, CD8) was detected by flow cytometry, and the ratio of CD4/CD8 was calculated.@*RESULTS@#After tracheotomy, the levels of β-defensin-2 mRNA and β-defensin-2 in lung tissue from the group B were increased significantly at 24 h, then they were decreased gradually, and decreased most significantly at 168 h (0.05). The level of CD3 T cells in peripheral blood was significantly lower than that in the group A (0.05). After ICA intervention in group C: lung tissue, alveolar lavage fluid, peripheral blood serum β-defensin-2 content, and peripheral blood CD3 and CD4 T cell levels were gradually increased, significantly higher than those in the group B (<0.05). CD8 T cell level was significantly lower than that in the group A at 24 h (<0.05), the CD4/CD8 ratio was significantly higher at 168 h than those in the group A or B (both <0.01).@*CONCLUSIONS@#ICA can improve the early lung immune function in rats with tracheotomy, which might be related to up-regulation of β-defensin-2 in lung tissue and alveolar lavage fluid, concomitant with increases in CD3 and CD4 T cells and CD4/CD8 ratio in peripheral blood while reduction in CD8 cells.

Effects of light quality on growth and icariin flavonoid content of Epimedium pseudowushanense under different light intensity / 中国中药杂志

In this study, the growth index including plant height, compound leaf area, specific leaf area, leaf water content, number of branches, and leaf biomass per plant and the icariin flavonoids such as epimedin A, epimedin B, epimedin C and icariin of Epimedium pseudowushanense were determined on 30 d and 60 d under light intensity(18.2±2.5) μmol·m~(-2)·s~(-1)(L1) and(90.9 ±2.5) μmol·m~(-2)·s~(-1)(L2), and white light as control, red light, blue light and yellow light were used as three light quality treatments, to study the effect of light quality on the growth and flavonoids accumulation of E. pseudowushanense. The E. pseudowushanense was sui-table for growth under L1 light intensity, the blue light treatment significantly reduced the leaf area, but had little effect on the stem height, the red light treatment and the yellow light treatment had no obvious effect on the stem height and leaf area, but the yellow light treatment significantly increased the germination of new branches, and had a sustained promoting effect, and the biomass was significantly higher than the white light treatment at 60 d. The content of icariin flavonoids in red light, blue light and yellow light treatment was higher than that in white light treatment at 30 d and 60 d under L1 light intensity, while yellow light treatment promoted the synthesis of icariin flavonoids to the largest extent, which was 1.8 and 1.9 times of white light treatment(30 d and 60 d).Under L2 light intensity, the effect of strong light on promoting stem germination became the main factor, while the yellow light treatment showed no significant effect on promoting stem germination, and the red light treatment exhibited a significant effect on reducing leaf area. Icariin flavonoids under red light, blue light and yellow light treatment were all lower than that under white light treatment, that is, the effect of white light treatment on the synthesis of icariin flavonoids is better than red light, blue light and yellow light treatment. When the time of strong light treatment was longer, the degradation range of icariin flavonoids in other light treatment appeared, while red light treatment promotes the synthesis of icariin flavonoids. Therefore, the influence of light quality on E. pseudowushanense is quite different under different light intensity, no matter from growth index or flavonoid content index. The results support that the biomass and icariin flavonoid content can be increased by providing appropriate red and yellow light.

Protection of icariin on impairment of rat bone mesenchymal stem cells induced by 5-fluorouracil in vitro / 中国药理学通报

Aim To study the protective effect of icariin on the impairment of rat bone mesenchymal stem cells (BMSCs) induced by 5-fluorouracil (5-FU) and the underlying mechanism. Methods The proliferation of BMSCs was evaluated by MTT assay, the morphological change of apoptotic cells was tested by using the chromatin dye Hoechst 33258, and the apoptotic rate was analyzed by flow cytometry. The proteins expression of cleaved caspase-3 was detected with Western blotting, the activity of caspase-3 was detected by Activity Assay Kit; iNOS and TNOS were detected by nitric oxide synthase (NOS) assay kit. Results MTT detection showed that 5-FU could reduce the survival rate of BMSCs, 10, 20, 40 μmol · L-1ICA could promote the proliferation of BMSCs, and blocked the inhibition of BMSCs cells growth induced by 5-FU. When BMSCs cells were treated by both ICA (10, 20 p, mol · L-1) and 5-FU (2.5×l0-2g· L-1) for 48 hours, compared with model group, the early apoptotic rates of ICA test groups were significantly reduced (P < 0. 01), the expression of cleaved caspase-3 and the activity of caspase-3 were significantly reduced (P < 0. 01), and the activity of iNOS and TNOS was inhibited in a certain degree (P < 0. 05). Conclusions ICA could protect BMSCs against 5-FU-induced damage. The protective role of ICA is probably related to reducing the activity of iNOS and TNOS, inhibiting the activation of caspase-3, and inhibiting the occurrence of apoptosis.

Icariin promotes differentiation of MC3T3-E1 pre-osteoblasts through Hedgehog signaling pathway / 中国药理学通报

Aim To investigate the effect of icariin (ICA) on the proliferation and differentiation of MC3T3-E1 pre-osteoblast cell line. Methods CCK-8 assay was used to detect the proliferation activity of MC3T3-E1 cells. Alizarin red staining (AR-S) assay was used to detect osteoblast differentiation and the formation of calcium nodules. Western blot assay was used to examine the protein expression of osteocalcin, ALP and shh. ALP assay was used to detect its activity. Results The effect of CCK-8 assay showed that ICA (5 ∼ 40 μmol · L-1) significantly promoted MC3T3-E1 cell proliferation compared with control group. The activity and protein expression of ALP and the quantitative analysis of mineralization deposition significantly increased in ICA group compared with control group. The ALP activity, protein expression level of shh and the quantification of alizarin red staining showed a significant decrease in ICA + CYC group compared with ICA group. Conclusions ICA promotes osteoblast proliferation and differentiation through the Hedgehog signaling pathway.

Effect of icariin on early steroid-induced osteonecrosis of the femoral head in rabbits / 中国修复重建外科杂志

Objective: To explore the effect of icariin on early steroid-induced osteonecrosis of the femoral head in rabbits. Methods: Fifty mature New Zealand rabbits (weighing, 2.5-3.0 kg) were randomly divided into control group ( n=10), model group ( n=20), and experimental group ( n=20). The rabbits of model and experimental groups were injected with lipopolysaccharide and methylprednisolone to establish the animal model of early steroid-induced osteonecrosis of the femoral head. The rabbits of experimental group were feeded with icariin solution once a day for 6 weeks since the first injection of methylprednisolone, whereas the rabbits of control and model groups were given normal saline at the same time points. The left femoral heads were removed after 6 weeks and gross morphological features were evaluated. Micro-CT scan was performed to analyze the trabecular microstructure with the following parameters: trabecular bone volume to total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Tn), and trabecular separation (Tb.Sp). The Micro-CT scan was also converted to three-dimensional reconstruction images for observation. HE staining was applied to observe the trabecular structure and morphological changes of osteocytes and marrow adipocytes. It was also used to determine whether the samples of femoral heads occurred osteonecrosis based on the criteria for pathological diagnosis, and calculate the rate of empty lacunae. Results: Seven rabbits died during the study, and 9, 16, and 18 rabbits in the control, model, and experimental groups, respectively, enrolled the final analysis. Compared with control group, the femoral head collapse and trabecular breaks were more obvious, and the trabeculae were sparse with irregular arrangement in the model group according to the results of gross observation, Micro-CT scan, and three-dimensional reconstruction images. But in the experimental group, the surface of femoral head was slight shrinking without obvious collapse, and the degeneration of trabecular structure was mild. According to bone microstructures analysis, the Tb.N, Tb.Tn, and BV/TV of femoral head in model and experimental groups were lower than those in control group, while the Tb.Sp in the model and experimental groups were significantly higher. The Tb.N, Tb.Tn, and BV/TV of femoral head in experimental group were higher than those in model group, while the Tb.Sp in the experimental group was significantly lower. The differences between groups were all significant ( P<0.05). In the model group, HE staining showed that the number of osteocytes reduced, the number of empty lacunae increased, and the marrow adipocytes piled up in the space between femoral trabeculae, some even mashed together like a cyst. In the experimental group, the trabecular structure was still relatively complete compared with model group, no obvious apoptosis of osteocytes was observed, the size and number of adipocytes were basically normal. None of the animals in control group occurred osteonecrosis of the femoral head based on the criteria for pathological diagnosis, and the incidence of osteonecrosis were 81.3% (13/16) in the model group and 66.7% (12/18) in the experimental group, and the difference was not significant ( P=0.448). The rate of empty lacunae of osteonecrotic femoral heads in the model group was 33.1%±1.4%, which was higher than that in experimental group (18.9%±0.8%) and in control group (12.7%±1.5%), and the differences between groups were significant ( P<0.05). Conclusion: The icariin has a protective effect on the early steroid-induced osteonecrosis of the femoral head in rabbits, which can decrease osteocytes apoptosis, improve the bone microstructure, and delay such disease processes.

Comparative study on icariin and Bu-Shen-Gu-Chi-Wan for alveolar reconstruction in chronic periodontitis in rats / 口腔疾病防治

Objective@#To observe the effects of icariin (ICA) and Bu-Shen-Gu-Chi-Wan on the alveolar bone absorption of chronic periodontitis in rats, and to explore the effect and possible mechanism of ICA in the treatment of chronic periodontitis. @*Methods@#After the establishment of the periodontitis model, the rats were divided into the periodontitis group (group P), ICA high dose group (group H), ICA low dose group (group L) and Bu-Shen-Gu-Chi-Wan treatment group (group B). Each group received treatment for one month and two months, separately, and the serum osteocalcin (OCN) level was measured. Three-dimensional reconstruction was performed after micro computed tomography (micro CT) scanning to measure the bone parameters of specific points, and the distance between the enamel cementum boundary and alveolar crest (CEJ-ABC) was recorded as alveolar bone resorption value. Tissue sections were generated to evaluate the effect of ICA on alveolar bone repair and reconstruction in rats with experimental periodontitis. @*Results@# Compared with the periodontitis group (group P), OCN levels in the serum in treatment groups (groups H, L and B) were decreased significantly (P < 0.05); the values of bone volume/tissue volume (BV/TV) and trabecular number (Tb.N) in treatment groups (groups H, L and B) were significantly higher than that in group P (P < 0.05). Compared with group P, trabecular thickness (Tb.Th) values of groups H and B significantly increased, and trabecular separation (Tb.Sp) of groups H and B significantly decreased (P < 0.05). The changing trend of parameters in group L was the same as that in group H but only after two months of administration. The difference between Tb.Sp values in groups L and P was statistically significant (P < 0.05). Compared with group P, the CEJ-ABC distance significantly reduced in group L (1 month and 2 months after administration), group H (1 month and 2 months after administration), and group B (only 2 months after administration) (P < 0.05).@*Conclusion@#ICA and Bu-Shen-Gu-Chi-Wan improve the alveolar bone resorption in an experimental model of chronic periodontitis in rats, and the mechanism may be related to the regulation of osteocalcin serum levels.