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.

Correlation between drug-induced liver injury in rats caused by Xianling Gubao oral preparation and extraction process / 中国中药杂志

In light of the liver injury risk associated with the oral administration of Xianlin Gubao oral preparation, this study compared the differences in liver injury induced by two different extraction processes in rats and explored the correlation between hepatotoxicity and extraction process from the perspective of the differences in the content of the relevant components. Thirty male Sprague-Dawley(SD) rats were randomly divided into a normal group, tablet extract groups of different doses, and capsule extract groups of different doses, with 6 rats in each group. Each group received continuous oral administration for 4 weeks. The assessment of liver injury caused by different extracts was conducted by examining rat body weight, liver function blood biochemical indicators, liver coefficient, and liver pathological changes. In addition, a high-performance liquid chromatography(HPLC) method was established to simultaneously determine the content of icariin, baohuoside I, and bakuchiol in the extracts to compare the differences in the content of these three components under the two extraction processes. The results showed that both extracts caused liver injury in rats. Compared with the normal group, the tablet extract groups, at the studied dose, led to slow growth in body weight, a significant increase in triglyceride levels(P<0.05), a significant decrease in liver-to-brain ratio(P<0.05), and the appearance of hepatic steatosis. The capsule extract groups, at the studied dose, resulted in slow growth in body weight, a significant increase in aspartate aminotransferase levels(P<0.05), a significant decrease in body weight, liver weight, and liver-to-brain ratio(P<0.05), and the presence of hepatic steatosis and inflammatory cell infiltration. In comparison, the capsule extraction process had a higher risk of liver injury. Furthermore, based on the completion of the liquid chromatography method, the content of icariin and baohuoside Ⅰ in the capsule extract groups was 0.83 and 0.81 times that in the tablet extract groups, respectively, while the bakuchiol content in the capsule extract group was 29.80 times that in the tablet extract groups, suggesting that the higher risk of liver injury associated with the capsule extraction process may be due to its higher bakuchiol content. In summary, the differences in rat liver injury caused by the two extracts are closely related to the extraction process. This should be taken into consideration in the formulation production and clinical application.

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.

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 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.

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.

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.

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.

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.

Icariin improves myocardial fibrosis induced by isoproterenol via regulating expression of a-SMA and MMPs/TIMP / 中国药理学通报

Abstract; Aim To explore the effect of icariin (ISO) in mice. Methods C57BL/6 mice were ran- (ICA) on myocardial fibrosis induced by isoproterenol domly divided into control group, ISO group, low-dose (15 mg • kg"1), middle-dose (30 mg • kg"1) and high-dose (60 mg • kg"1) of ICA-treated group and Losartan-treated group ( 9 mg • kg"1 ). The control group was subcutaneously injected with normal saline, and the other groups were subcutaneously injected with ISO (5 mg • kg"1, qd) continuously 14 days to established the myocardial fibrosis model. The ICA-treated groups and Losartan-treated group were simultaneously intragastrically administered of ICA or Losartan, respectively. And the other groups received the same a- mount of double distilled water. The left ventricular e- jection fraction (LVEF) and the left ventricular fraction shortening rate ( LVFS) were evaluated by the small animal ultrasound. The heart mass index (HMI) was calculated. The left ventricular collagen deposition was detected by Masson staining. The protein expressions of a-SMA, MMP-2, MMP-9 and TIMP-1 in the left ventricular tissue were detected by Western blot. Results ICA (30, 60 mg • kg"1) and Losartan could inhibit the decreased LVEF and LVFS, the increased HMI and left ventricular collagen deposition, the up- regulated a-SMA and MMP-9 protein expression, the down-regulated MMP-2 and TIMP-1 protein expression in the left ventricular tissues induced by ISO. Conclusions ICA can improve myocardial fibrosis induced by ISO in mice, and the underlying mechanism may be related to the regulation of the protein expression of a- SMA and MMPs/TIMP-1.

Protective Mechanism of Icariin on Nephrotic Syndrome Rats Based on RhoA/ROCK Pathway / 中国实验方剂学杂志

Objective:To study the effect of icariin on renin homologous protein A (RhoA)/Rho-related kinase (ROCK) pathway in rats with nephrotic syndrome (NS) and its protective mechanism. Method:Totally 54 clean-grade male SD rats were tested and randomly divided into normal group, model group, RhoA inhibitor group (Rhosin, 40 mg·kg-1·d-1) and three doses of icariin groups (low, medium and high corresponding dose， 30, 60, 120 mg·kg-1·d-1). Adriamycin hydrochloride 6.5 mg·kg-1 was given in tail vein of rats to induce NS model in rats. After the model was established, peritoneal administration was carried out. The normal group and the model group were given saline 2.5 mL·d-1, and the inhibitor group and all of dose groups were given corresponding doses of Rhosin and icariin for intervention. Total urinary protein (Alb), creatinine (Cre), total urinary protein/creatinine ratio (A/C) kit were detected in rats, ultrastructure of kidney was identified by transmission electron microscopy (TEM), and Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to detect the mRNA and proteins expressions of RhoA, ROCK1, ROCK2. Result:TEM showed that the basement membrane was intact and the foot process was regular in the normal group, in model group, basement membrane was damaged seriously, foot process disappeared, and fusion was serious, in the low-dose group, the basement membrane injury was alleviated, the number and density of foot process were improved, and the fusion was obvious， in the middle-dose group and the inhibitor group, the basement membrane thickening was alleviated, and the foot process was slightly fused, in the high-dose group, the basement membrane structure was more complete, and podocytes were longer and arranged tightly. Compared with the normal group, the levels of Alb, Cre and A/C in urine, and RhoA, ROCK1 and ROCK2 mRNA and protein expressions in kidney tissue of rats of the model group were significantly higher (P<0.05). Compared with model group, the levels of Alb, A/C in urine and RhoA, ROCK1, ROCK2 mRNA and protein expressions in kidney tissue in the inhibitor group and low, medium and high-dose groups, and Cre in urine in inhibitor group and high-dose group decreased significantly (P<0.05). Compared with the inhibitor group, the levels of Alb, Cre in urine and RhoA protein in kidney tissue in the high-dose group were significantly decreased (P<0.05), the levels of Alb, Cre, A/C in urine and RhoA, ROCK1, ROCK2 mRNA and protein expressions in kidney tissue of the low-dose group, and the levels of RhoA, ROCK1 and ROCK2 mRNA expressions in kidney tissue of the middle-dose group were significantly increased (P<0.05). Compared with the low-dose group, the levels of Alb, A/C in urine, and RhoA, ROCK1, ROCK2 mRNA and protein expressions in kidney tissue in the middle and high-dose groups, Cre in urine of the high-dose group were significantly decreased (P<0.05). Compared with the middle-dose group, the levels of Alb, Cre in urine, and RhoA, ROCK1, ROCK2 mRNA and protein expressions, ROCK2 mRNA expression in kidney tissue in the high-dose group were significantly decreased (P<0.05). Conclusion:Icariin may protect glomerular endothelium and podocyte by affecting RhoA/ROCK pathway in the treatment of NS rats.

Effect of Icariin on Damaged Neurons Based on Endoplasmic Reticulum Stress / 中国实验方剂学杂志

Objective::To observe the effect of icariin on damaged neurons from the perspective of endoplasmic reticulum stress, in order to explore some mechanisms for repairing damaged neurons. Method::PC12 cells were induced by nerve growth factor (NGF) to differentiate into neurons, and the positive rate of microtubule associated protein-2 (MAP2) and neuron-specific enolase (NSE) expressions was determined by flow cytometry. The experiment was divided into 4 groups, blank control group: PC12 induced differentiation into neuronal cells, solvent control group: PC12 induced differentiation into neurons+ 0.1% dimethyl sulfoxide (DMSO), thapsigargin group: PC12 induced differentiation into nerves Yuan+ 2 μmol·L-1 thapsigargin, and icariin group: PC12 induced differentiation into neurons+ 2 μmol·L-1 thapsigargin+ 0.1 μmol·L-1 icariin. The proliferation of the cells was detected by using cell counting kit-8(CCK-8) method, the apoptosis of the cells was detected by flow cytometry, the protein expressions of CCAAT/enhace-binding protein homologous protein(CHOP) and glucoseregulated protein 78(Grp78) were detected by Western blot, and the mRNA expressions of CHOP and Grp78 were detected by real-time quantitative PCR (Real-time PCR). Result::Compared with the solvent control group, the thapsigargin group inhibited the proliferation of neuron-like PC12 cells induced by NGF, promoted apoptosis, and up-regulated the expressions of CHOP and Grp78 (P<0.05, P<0.01). Compared with the thapsigargin group, the icariin group can alleviate the inhibition of neurotrophic activity by thapsigargin, reduce neuronal apoptosis, and down-regulate the expressions of CHOP and Grp78 (P<0.05, P<0.01). Conclusion::Icariin can inhibit endoplasmic reticulum stress by down-regulating the expressions of CHOP and Grp78 and promote the repair of damaged neurons.

Effect of Icariin on Neuroprotection of Cerebral Ischemia-reperfusion Rats and TLR4/NF-κB Pathway in Microglia / 中国实验方剂学杂志

Objective:To investigate the effect of icariin on neuroprotection in cerebral ischemia-reperfusion rats and microglia toll-like receptor 4 （TLR4）/nuclear transcription factor-κB （NF-κB） pathway. Method:In the blank group， blood vessels were only isolated but not ligated and blocked，and the rats were injected intraperitoneally with the same volume of normal saline. After successful modeling， they were randomly divided into model group， butyphthalide group （6 mg·kg-1）， and high， medium and low （40，20，10 mg·kg-1）-dose icariin group，and abdominally administered with drugs at 5，12， 24 h after ischemia， respectively. The nerve function scores were detected， 2，3，5-triphenyltetrazole chloride （TTC） staining was used to measure the cerebral infarction rate，immunohistochemical assay was performed to detect the expressions of microglial markers ionized calcium binding adapter molecule 1（Iba1） and TLR4 in the rat brain cortex， Western blot immunoassay was used to detect the expression of NF-κB p65 in the cerebral cortex， and enzyme-linked immunosorbent assay （ELISA） was used to detect interleukin-1α （IL-1α） and tumor necrosis factor-α （TNF-α） content. Result:Compared with the sham-operation group， the nerve score， the cerebral infarction rate， the activations of Iba1 and TLR4 in microglial cells， the protein expression of NF-κB p65（P<0.01）， and the contents of inflammatory factors IL-1α and TNF-α in the model group increased significantly（P<0.01）. After treatment with icariin， compared with the model group， the neurological function score and the cerebral infarction rate of rats were improved， whereas the activations of Iba1 and TLR4 in microglia， the protein expression of NF-κB p65， and the contents of inflammatory factors IL-1α and TNF-α decreased obviously（P<0.05，P<0.01）. Conclusion:Icariin may inhibit the activations of TLR4 and its downstream NF-κB signaling pathway and reduce the expression of relevant inflammatory factors IL-1α and TNF-α by regulating the activation of microglia， so as to play a protective role in the brain after stroke.

Effect of icaritin on ferroptosis of bone marrow mesenchymal stem cells and their differentiation into cardiomyocytes / 中国组织工程研究

BACKGROUND: Iron accumulation or iron overload is closely related to the proliferation and apoptosis of bone marrow mesenchymal stem cells, which may play an important role in the pathological process of cardiovascular diseases. OBJECTIVE: To investigate the regulatory effect of icariin on ferroptosis of bone marrow mesenchymal stem cells and its differentiation into cardiomyocytes. METHODS: Taking bone marrow mesenchymal stem cells as the research object, the blank group was cultured for 14 days. The model group was treated with 0.4 μmol/L Erastin for 14 days to establish ferroptosis model. The treatment group was treated with 80 nmol/L icariin and 0.4 μmol/L Erastin for 14 days. CCK-8 method was used to detect the proliferation of cells in each group, and the levels of malondialdehyde and superoxide dismutase were detected by related kits. Western blot assay was used to detect the expression of TFR1, FPN1, Bcl-2 and Bax protein. RT-PCR was used to detect the mRNA expression of GATA4 and cTnT. RESULTS AND CONCLUSION: (1) Compared with the model group, the cell proliferation ability of the treatment group was significantly improved (P < 0.05). (2) Compared with the model group, the level of malondialdehyde in the lysate of bone marrow mesenchymal stem cells in the treatment group was significantly lower (P < 0.05), while the level of superoxide dismutase was significantly increased (P < 0.05). (3) Compared with the model group, TFR1 protein expression was decreased (P < 0.05), FPN1 protein expression was increased (P < 0.05), and Bcl-2/Bax protein expression ratio was increased significantly (P < 0.05) in the treatment group. (4) Compared with the model group, the expression of GATA4 and cTnT mRNA was significantly increased in the treatment group (P < 0.05). (5) The results showed that icariin could inhibit the ferroptosis of bone marrow mesenchymal stem cells and improve the ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells.