Protective effect and mechanism of icariin against carbon tetrachloride-induced acute liver injury in mice / 中国组织工程研究

BACKGROUND:Icariin,with antiinflammatory,antioxygenatory and immunoregulatory effects,can be a potential drug for preventing and treating acute liver injury. OBJECTIVE:To investigate the protective effect and possible mechanism of icariin in mice with acute liver injury induced by carbon tetrachloride. METHODS:Thirty-two Kunming mice were equally and randomly divided into the following groups:normal,model,low-dose icariin and high-dose icariin groups.The low-and high-dose icariin groups were continuously gavaged with icariin(100 and 200 mg/kg,respectively)once a day for 7 continuous days.The normal group and model group were injected with physiological saline(10 mL/kg)at the same time point.After the last administration,all the groups except for the normal group were injected with carbon tetrachloride to induce acute liver injury.The mice were killed 24 hours later,and the liver index was detected.Serum levels of alanine aminotransferase and aspartate aminotransferase were detected by automated biochemical analysis.Tumor necrosis factor α and interleukin 6 levels in serum were detected using ELISA.The levels of superoxide dismutase,glutathione peroxidase and malondialdehyde in liver tissue were detected through a reagent kit.The histopathology changes of the liver were observed by hematoxylin-eosin staining.TUNEL method was used to detect the apoptosis in hepatocytes.Western blot was performed to detect the expression levels of glucose-regulated protein 78 kDa,endoplasmic reticulum stress-related protein(C/-EBP homologous protein),mixed lineage kinase domain-like protein and Caspase-3 in liver tissue. RESULTS AND CONCLUSION:Compared with the normal group,the liver index and serum levels of alanine aminotransferase,aspartate aminotransferase,tumor necrosis factor α and interleukin 6 were increased in the model group(P＜0.05).Compared with the model group,the above indexes were decreased in the low-dose and high-dose icariin groups(P＜0.05).Compared with the normal group,the activities of superoxide dismutase and glutathione peroxidase in the liver tissue of mice were decreased in the model group(P＜0.05)and the level of malondialdehyde was increased(P＜0.05).Compared with the model group,the activities of superoxide dismutase and glutathione peroxidase were increased in the low-and high-dose icariin groups(P＜0.05)and the level of malondialdehyde was decreased(P＜0.05).Hematoxylin-eosin and TUNEL staining showed that mice in the model group had severe structural destruction of liver tissue,extensive necrosis of hepatocytes and high apoptotic rate of hepatocytes,while the structural destruction of liver tissue and the area of necrosis of hepatocytes in the low-and high-dose icariin groups were significantly milder than those in the model group,and the apoptotic rate of hepatocytes was lower than that in the model group(P＜0.05).Western blot assay showed that the protein expression of glucose-regulated protein 78 kDa,C/-EBP homologous protein,mixed lineage kinase domain-like protein and Caspase-3 in liver tissue of mice in the model group was increased compared with that in the normal group(P＜0.05),while the expression levels of these proteins in liver tissue of mice were significantly reduced after low-and high-dose icariin intervention(P＜0.05).To conclude,icariin can produce a protective effect against carbon tetrachloride-induced acute liver injury,and its mechanism may be related to the regulation of endoplasmic reticulum stress and reduction of programmed necrosis.

Effects of icariin on proliferation and differentiation of MC3T3-E1 cells in an inflammatory environment / 中国组织工程研究

BACKGROUND:Studies have shown that chronic apical periodontitis is one of the common inflammatory bone destruction diseases.Icariin can promote osteogenic differentiation,inhibit bone resorption,and may play a protective role in bone destruction caused by chronic apical periodontitis. OBJECTIVE:To investigate the effect of icariin on the proliferation and differentiation of MC3T3-E1 cells in the inflammatory environment stimulated by lipopolysaccharides. METHODS:Lipopolysaccharides were used to stimulate MC3T3-E1 cells to establish an inflammatory environment in vitro,and cell counting kit-8 was used to detect the best concentration and optimal action time of lipopolysaccharides.Cell counting kit-8 was used to detect the optimal concentration of icariin under the stimulation of lipopolysaccharides at a concentration of 1 μg/mL.Alkaline phosphatase detection,Real-time PCR and western blot assay were used to detect the effect of icariin on osteogenic differentiation of MC3T3-E1 cells in the inflammatory environment.Real-time PCR and western blot were used to detect the effects of icariin on the expression of interleukin-1β and interleukin-6 in MC3T3-E1 cells in the lipopolysaccharide-stimulated inflammatory environment. RESULTS AND CONCLUSION:Cell counting kit-8 results showed that the optimal concentration of icariin was 0.1 μg/mL.In the inflammatory environment,icariin enhanced the expression of alkaline phosphatase and promoted osteoblast differentiation.Compared with the lipopolysaccharide group,the expression of osteogenesis-related factors alkaline phosphatase and Runx2 was increased in the lipopolysaccharide+icariin group.Compared with the lipopolysaccharide group,the expression levels of inflammation-related factors interleukin-1β and interleukin-6 decreased in the lipopolysaccharide+icariin group.To conclude,lipopolysaccharides weaken the osteogenic ability of MC3T3-E1 cells and aggravate the inflammatory response,but icariin has a protective effect on them.

Icariin regulates acidic microenvironment to alleviate pain caused by postmenopausal osteoporosis in the elderly / 中国组织工程研究

BACKGROUND:Previous studies have demonstrated that icariin has important roles in promoting bone formation and inhibiting bone resorption,but its effects on osteoporosis-mediated bone pain have not been reported. OBJECTIVE:To investigate the possible mechanism of icariin alleviating bone pain in postmenopausal senile osteoporosis. METHODS:(1)Animal experiment:200 C57BL/6 mice were randomly divided into 4 groups:sham group(n=50),model group(n=50),icariin treatment group(n=50),and carbonic anhydrase Ⅱ inhibitor(Brinzolamide)treatment group(n=50).Ovariectomy was performed on C57BL/6 mice to establish a postmenopausal osteoporosis model in all groups except the sham group.The icariin group was given icariin on the second day after modeling,and pain behavior tests(Von Frey,Hot Plate,and Tail Flick tests)were performed every 2 weeks for 20 weeks.After sampling,bone mass was detected by microCT,osteoclast activity was detected by hematoxylin-eosin and tartrate-resistant acid phosphatase staining,and neuronal morphology and related ion channel expression were detected by tissue immunofluorescence staining.(2)Cell experiment:Osteoclast precursor cells derived from mouse bone marrow were extracted and induced to differentiate into osteoclasts using the RANKL/M-CSF system in vitro and supplemented with icariin of different concentrations(1 and 10 μmol/L).Tartrate-resistant acid phosphatase staining was used to detect osteoclast differentiation,ghost pen cyclic peptide staining was used to detect osteoclast actin ring,bone plate absorption assay was used to detect osteoclast osteophagy function,pH value of the system was detected by pH meter,and expression of osteoclast differentiation-related proteins was detected by western blot.In addition,mouse dorsal root ganglion-derived nerve cells were extracted and treated with icariin.Cell counting kit-8 was used to detect neuronal activity and CGRP staining was used to detect neuronal morphology. RESULTS AND CONCLUSION:Compared with the model group,the icariin treatment group had higher bone mineral density,fewer tartrate-resistant acid phosphatase-positive osteoclasts in bone tissue,decreased neuronal activity,and decreased neuronal transient receptor potential vanilloid subtype 1 channel and carbonic anhydrase Ⅱ expression.Behavioral results showed that the icariin treatment group was less sensitive to pain than the model group.Icariin inhibited osteoclast differentiation and bone phagocytosis in vitro.Icariin enhanced the activity of dorsal root ganglion neurons and inhibited the expression of calcitonin gene-related peptide and transient receptor potential vanilloid subtype 1 channels in dorsal root ganglia in a relatively non-toxic pH range.To conclude,icariin alleviates bone pain caused by postmenopausal osteoporosis by regulating the acidic microenvironment through its effect on osteoclasts.

Effect of Icariin Regulating mTOR/Akt/CREB Pathway on High Glucose Induced Autophagy and Apoptosis in Podocytes / 医药导报

Objective To investigate the effects of icariin on high glucose-induced autophagy and apoptosis of podocytes,and the regulating effects on mammalian target of rapamycin(mTOR)/serine-threonine kinase(Akt)/cyclic adenosine monophosphate response element binding protein(CREB)pathway.Methods The mouse podocytes MPC5 were taken and divided into five groups:normal control group(5.5 mmol·L-1 glucose),high glucose group(30 mmol·L-1 glucose),icariin group(30 mmol·L-1glucose+5 μmol·L-1icariin),GDC-0349 group(30 mmol·L-1glucose+50 μmol·L-1 GDC-0349),icariin+GDC-0349 group(30 mmol·L-1 glucose+5 μmol·L-1 icariin+50 μmol·L-1 GDC-0349).Cultured for 48 hours,the tetramethylazozolium salt method was used to detect the viability of MPC5 cells;acridine orange staining was used to observe the autophagy of MPC5 cells;apoptosis of MPC5 cells was detected by flow cytometry;Western blotting was used to detect the expression of autophagy[microtubule associated protein one light chain 3(LC3)II,LC3Ⅰ,autophagy-related protein(Beclin-1)],apoptosis[Bcl-2 related X protein(Bax),B cell lymphoma-2(Bcl-2)]and mTOR/Akt/CREB pathway-related proteins of MPC5 cells.Results Compared with the normal control group,the cell viability,expression levels of Bcl-2,phosphorylated mTOR(p-mTOR)/mTOR,phosphorylated Akt(p-Akt)/Akt,phosphorylated CREB(p-CREB)/CREB protein of MPC5 cells in the high glucose group were significantly decreased(P＜0.05),the autophagy ability was enhanced,the autophagosome showed orange fluorescence,and the apoptosis rate,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bax protein expression levels were significantly increased(P＜0.05).Compared with the high glucose group,the cell viability,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bcl-2,p-mTOR/mTOR,p-Akt/Akt,p-CREB/CREB protein expression levels of MPC5 cells in icariin group were significantly increased,the autophagy ability was further enhanced,the number of autophagosomes was increased,the autophagosomes showed brick red fluorescence(P＜0.05),the apoptosis rate and Bax protein expression level were significantly decreased(P＜0.05),and the cell viability,LC3Ⅱ/LC3Ⅰ,Beclin-1,Bcl-2,p-mTOR/mTOR,p-Akt/Akt and p-CREB/CREB proteins expression levels of MPC5 cells in GDC-0349 group were significantly decreased,the autophagy ability was weakened,the number of autophagosomes was reduced,the autophagosomes showed orange fluorescence(P＜0.05),and the apoptosis rate and Bax protein expression level were significantly increased(P＜0.05);icariin+GDC-0349 could reverse the effect of icariin on high glucose induced MPC5 cells(P＜0.05).Conclusion Icariin promotes elevated glucose-induced podocyte autophagy and inhibits apoptosis by activating the mTOR/Akt/CREB pathway.

Pharmacological Effects of Icariin on Male Reproductive System:A Review and Literature Visualization / 医药导报

Icariin,which belongs to the class of flavonoids,is the main active ingredient of the traditional tonic Chinese herb Epimedii Folium.Modern studies have shown that icariin has a wide range of effects on the male reproductive system.It has various pharmacological activities such as regulating cell proliferation and apoptosis,antioxidants,promoting testosterone secretion,improving erectile function,inhibiting prostate cancer cell migration,invasion,and regulating cell cycle.It has research value and application prospects in the field of urology and assisted reproduction.Therefore,Icariin's pharmacological effects and molecular mechanisms on the male reproductive system are reviewed in this paper combined with literature visualization analysis.It is expected to provide a theoretical basis for the therapeutic value development and application of icariin in male reproductive health.

Effects of icariin-astragaloside IV-puerarin mixture on cognitive function and ferroptosis amino acid metabolism pathway in APP/PS1 HAMP-/-mice / 中国病理生理杂志

AIM:To observe the effect of icariin-astragaloside Ⅳ-puerarin mixture(Yin-Huang-Ge mixture,YHG)on cognitive function and ferroptosis amino acid metabolism pathway in hepcidin(HAMP)knockout APPswe/PS1dE9(APP/PS1 HAMP-/-)mice.METHODS:The mice were divided into 7 groups:negative control(C57BL/6 mice)group,APP/PS1 group,APP/PS1 HAMP-/-group,APP/PS1+YHG group,APP/PS1 HAMP-/-+YHG group,APP/PS1+de-ferasirox(DFX)group,and APP/PS1 HAMP-/-+DFX group,with 6 mice in each group.The YHG and DFX were adminis-tered intragastrically,while the mice in C57 group,APP/PS1 group and APP/PS1 HAMP-/-group were given intragastric administration of distilled water,once a day for 2 months.The iron content in mouse brain tissues was detected by tissue iron kit.The morphological changes of the mitochondria in hippocampal neurons were observed by transmission electron microscopy.Morris water maze was used to detect the learning and memory ability of the mice.The content of neuronal nu-clear antigen(NeuN)in mouse brain tissues was detected by immunofluorescence staining.The expression of glutathione(GSH)in mouse brain tissues was detected by biochemical kit.The expression levels of glutamate-cysteine ligase catalytic subunit(GCLC)and glutamatase 2(GLS2)in mouse brain tissues were detected by Western blot.RESULTS:Compared with C57BL/6 mice,the brain iron content of APP/PS1 mice was significantly increased(P＜0.01),the mitochondria were seriously damaged,the learning and memory ability was significantly decreased(P＜0.05),the brain neurons were seri-ously damaged(P＜0.01),and the expression levels of GSH,GCLC and GLS2 were significantly decreased(P＜0.01).Compared with APP/PS1 mice,the brain iron content of APP/PS1 HAMP-/-mice was significantly increased(P＜0.01),the mitochondria were seriously damaged,the learning and memory ability was significantly decreased(P＜0.05),the brain neurons were seriously damaged(P＜0.01),and the expression levels of GSH,GCLC and GLS2 were significantly decreased(P＜0.05).After treatment with YHG and DFX,the brain iron content was significantly decreased(P＜0.01),the mitochondrial damage was alleviated,the learning and memory ability was significantly increased(P＜0.05),the brain neuron damage was alleviated(P＜0.01),and the expression levels of GSH,GCLC and GLS2 were significantly increased(P＜0.05).CONCLUSION:The YHG can improve the cognitive function of APP/PS1 HAMP-/-mice,and its mechanism may be related to the regulation of ferroptosis amino acid metabolism and the enhancement of antioxidant capacity.

Icariin ameliorates viral myocarditis by inhibiting TLR4-mediated ferroptosis

@#Objective: To explore the mechanism by which icariin alleviates viral myocarditis. Methods: CVB3-induced cardiomyocytes were used as an in vitro model of viral myocarditis to assess the effects of icariin treatment on cell viability, inflammation, and apoptosis. Moreover, the effects of icariin on ferroptosis and TLR4 signaling were assessed. After AC16 cells were transfected with TLR4 overexpression plasmids, the role of TLR4 in mediating the regulatory effect of icariin in viral myocarditis was investigated. Results: Icariin significantly elevated cell viability and reduced inflammatory factors TNF-α, IL-1β, IL-6, and IL-18. Flow cytometry revealed that icariin decreased apoptosis rate, and the protein expression of Bax and cleaved caspase 3 and 9 in CVB3-induced cardiomyocytes. Additionally, it suppressed ferroptosis including lipid peroxidation and ferrous ion, as well as the TLR4 signaling. However, TLR4 overexpression abrogated the modulatory effects of icariin. Conclusions: Icariin mitigates CVB3-induced myocardial injury by inhibiting TLR4-mediated ferroptosis. Further animal study is needed to verify its efficacy.

Icariin plus curcumol enhances autophagy through the mTOR pathway and promotes cathepsin B-mediated pyroptosis of prostate cancer cells

@#Objective: To examine the effect of icariin plus curcumol on prostate cancer cells PC3 and elucidate the underlying mechanisms. Methods: We employed the Cell Counting Kit 8 assay and colony formation assay to assess cell viability and proliferation. Autophagy expression was analyzed using monodansylcadaverine staining. Immunofluorescence and Western blot analyses were used to evaluate protein expressions related to autophagy, pyroptosis, and the mTOR pathway. Cellular damage was examined using the lactate dehydrogenase assay. Moreover, cathepsin B and NLRP3 were detected by co-immunoprecipitation. Results: Icariin plus curcumol led to a decrease in PC3 cell proliferation and an enhancement of autophagy. The levels of LC3- Ⅱ/LC3-Ⅰ and beclin-1 were increased, while the levels of p62 and mTOR were decreased after treatment with icariin plus curcumol. These changes were reversed upon overexpression of mTOR. Furthermore, 3-methyladenine resulted in a decrease in inflammatory cytokines, pyroptosis-related protein levels, and lactate dehydrogenase concentration, compared to the icariin plus curcumol group. Inhibiting cathepsin B reversed the regulatory effects of icariin plus curcumol. Conclusions: Icariin plus curcumol demonstrates great potential as a therapeutic agent for castration-resistant prostate cancer by enhancing autophagy via the mTOR pathway and promoting pyroptosis mediated by cathepsin B. These findings provide valuable insights into the molecular mechanisms underlying the therapeutic potential of icariin and curcumol for prostate cancer treatment.

Effect of Icariin on Peripheral Blood Dendritic Cells and Th17/Treg Balance in Myocardial Remodeling Model of Dahl Salt-sensitive Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of icariin （ICA）-mediated vitamin D system on peripheral blood dendritic cells （DCs） and helper T cells 17 （Th17）/regulatory T cells （Treg） balance in myocardial remodeling model of Dahl salt-sensitive rats. MethodFifty SPF Dahl salt-sensitive rats were divided into model group， vitamin D group （3×10-5 mg·kg-1·d-1）， and high-， medium-， and low-dose ICA groups （120， 60， 30 mg·kg-1·d-1）， and 10 Dahl salt-resistant rats were used as normal group. The myocardial remodeling model was established by feeding rats with a high-salt diet containing 8% NaCl. After six weeks of modeling， the normal group and the model group were given an equal volume of ultrapure water by gavage， and other groups were continuously administrated for six weeks. Cardiac echocardiography， hematoxylin-eosin （HE） staining， and Masson staining were used to observe the pathological changes in cardiac structure and fibrosis. The levels of serum 25（OH）D3， B-type N-terminal pro-brain natriuretic peptide （NT-ProBNP）， interleukin （IL）-17， transforming growth factor （TGF）-β1， IL-12， and IL-10 were detected by enzyme-linked immunosorbent assay （ELISA）. The phenotype of peripheral blood DCs and the ratio of Th17/Treg cells of rats were detected by flow cytometry. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the mRNA and protein expressions of vitamin D receptor （VDR），1α-hydroxylase （CYP27B1）， and 24-hydroxylase （CYP24A1） in peripheral blood DCs of rats. ResultCompared with the control group， the rats in the model group had pathological changes such as disordered arrangement of myocardial cells and cytoplasmic hypertrophy and swelling. Myocardial collagen fibers proliferated significantly， and the arrangement of myocardial fibers was disordered. The levels of serum 25（OH）D3 and IL-10 were significantly decreased， and the levels of serum IL-17， TGF-β1， IL-6， IL-12， and NT-ProBNP were significantly increased （P<0.05）. The costimulatory molecules CD40， CD80， CD86， and MHC-Ⅱ were highly expressed in the peripheral blood DCs， and the expression of CD11 and CD11b was lower （P<0.05）. The proportion of Th17 cells in the peripheral blood was significantly increased， and the proportion of Treg cells was decreased. The ratio of Th17/Treg was increased （P<0.05）. The mRNA and protein expressions of CYP24A1 in peripheral blood DCs increased， and the mRNA and protein expressions of CYP27B1 and VDR decreased （P<0.05）. Compared with the model group， the arrangement of myocardial fibers in each drug administration group was relatively regular， and the swelling of myocardial cells was significantly reduced. The pathological morphology of myocardial tissue was improved to varying degrees. The pathological changes in myocardial tissue were improved and alleviated to varying degrees. The drug could reduce the serum levels of NT-ProBNP， IL-17， TGF-β1， IL-6， and IL-12 and increase the level of serum 25（OH）D3 and IL-10 （P<0.05）. The expression of costimulatory molecules CD40， CD80， CD86， and MHC-Ⅱ in the peripheral blood DCs of rats was decreased， and the expression of CD11 and CD11b molecules was increased （P<0.05）. The drug could reduce the proportion of Th17 cells in peripheral blood and the ratio of Th17/Treg cells and increase the proportion of Treg cells （P<0.05）. It could decrease the mRNA and protein expressions of CYP24A1 in peripheral blood DCs of rats and elevate the mRNA and protein expression of VDR and CYP27B1 （P<0.05）. ConclusionICA can regulate the phenotype of peripheral blood DCs and the ratio of Th17/Treg cells by regulating the vitamin D system and play a role in improving myocardial remodeling from the perspective of immune balance.

Icariin Regulates Glucocorticoid-induced Autophagy of Bone Microvascular Endothelial Cells Through PI3K/Akt/mTOR Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the impact of icariin （ICA） on autophagy in glucocorticoid-induced bone microvascular endothelial cells （BMECs） mediated by the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. MethodBMECs were isolated and cultured from femoral heads obtained during total hip arthroplasty and identified using immunofluorescence staining. The experimental cells were divided into four groups： A control group， a glucocorticoid group （100 mg·L-1 hydrocortisone）， an ICA group （100 mg·L-1 hydrocortisone+6.7×10-3 mg·L-1 ICA）， and a Rapamycin group （100 mg·L-1 hydrocortisone+6.7×10-3 mg·L-1 ICA+1 mg·L-1 rapamycin）. Autophagy in BMECs was induced using 100 mg·L-1 hydrocortisone. LC3 fluorescence staining was used to observe the peak of autophagy at different time points. Western blot analysis was employed to analyze the expression of autophagy-related proteins and PI3K/Akt/mTOR pathway proteins in each group. Electron microscopy was used to observe autophagosomes and autolysosomes in the cells. ResultHydrocortisone at 100 mg·L-1 induced autophagy in BMECs， reaching a peak at around 5 hours， which then declined with further intervention. Compared to the control group， the glucocorticoid group showed cell membrane damage， disordered organelle arrangement， and a large number of autophagosomes and autolysosomes. Compared to the glucocorticoid group， the ICA group had more intact cell membranes， sparser organelle arrangement， and fewer autophagosomes and autolysosomes. Compared to the ICA group， the Rapamycin group showed cell membrane damage， disordered organelle arrangement， and more autophagosomes and autolysosomes. Compared to the control group， the glucocorticoid group had significantly increased expression of light chain 3B （LC3B）， Atg4B， and p62 （P<0.01）. Compared to the glucocorticoid group， the ICA group showed significantly decreased expression of LC3B， Atg4B， p62， and Beclin-1 （P<0.01）. Compared to the ICA group， the Rapamycin group had significantly increased expression of Atg4B and p62 （P<0.01）. Compared to the control group， the glucocorticoid group had significantly decreased expression of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR （P<0.01）. Compared to the glucocorticoid group， the ICA group showed significantly increased expression of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR （P<0.01）. Compared to the ICA group， the Rapamycin group had significantly decreased expression of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR （P<0.01）. Ubiquitination levels were significantly decreased in the glucocorticoid group compared to the control group （P<0.01）. Compared to the glucocorticoid group， ubiquitination levels were significantly increased in the ICA group （P<0.01）， and significantly decreased in the Rapamycin group compared to the ICA group （P<0.01）. ConclusionThe glucocorticoid-induced autophagy in BMECs is time-dependent. ICA inhibits glucocorticoid-induced autophagy in BMECs， and this effect may be related to the regulation of the PI3K/Akt/mTOR pathway.

Mechanism of icariin regulating the NLRP3 inflammasome against cerebral ischemia reperfusion / 中国实验动物学报

Objective To investigate the mechanism of icariin regulating the NLRP3 inflammasome in the treatment of cerebral ischemia-reperfusion injury in rats.Methods A rat model of focal cerebral ischemia-reperfusion was induced using the thread embolism method.At 24 hours post-operation,the rats were randomly allocated into a sham operation group,model group,butylphthalide group(70 mg/kg),ICA-low dose(20 mg/kg),ICA-middle dose(40 mg/kg),and ICA-high dose(80 mg/kg)groups.The corresponding drugs were administered by gavage at 10 mL/kg once a day for 13 consecutive days.One hour after the last administration,neurological function was scored.The cerebral cortex was observed by hematoxylin-eosin(HE)staining.Expression of interleukin(IL)-1β and IL-18 in the cerebral cortex was determined by immunohistochemistry.Expression of NLRP3,ASC,and Caspase-1 in the cerebral cortex was determined by Western Blot.Results In contrast to the sham operation group,there was a notable increase in neural function scores within the model group.The ischemic area around the visible cerebral cortex showed neuron necrosis at various level or glial cell proliferation,and the number of intact neurons was significantly reduced.IL-1β and IL-18 positive cells were significantly increased.Expression of NLRP3,ASC,and Caspase-1 was significantly increased(P＜0.01,P＜0.05).After treatment with icariin,the neural function score was decreased significantly.The degree of neuronal necrosis in the peri-ischemic area was significantly reduced,and the number of intact neurons was significantly increased.IL-1 β and IL-18-positive cells were decreased significantly.Expressions of NLRP3,ASC,and Caspase-1 were significantly decreased(P＜0.01,P＜0.05).Conclusions Treatment of cerebral ischemia-reperfusion injury by icariin may be related to regulation of the NLRP3 inflammasome.

Effects of icariin on BDNF/TkB signaling pathway and synaptic plasticity regulation on fear memory improvement in rats with post-traumatic stress disorder / 中国医科大学学报

Objective To investigate the effects and mechanisms of icariin on changes in fear memory in post-traumatic stress disorder(PTSD)rats.Methods Thirty male SD rats were used to construct a rat model of single prolonged stress(SPS).The model rats were randomly divided into the SPS,icariin,and icariin + K252a(tyrosine kinase receptor B inhibitor)groups(n= 10 each;another 10 normal rats were used as the control group).The icariin and icariin + K252a groups were administered 20 mg/kg icariin by gavage once per day after SPS,while the control and SPS groups were administered the same dose of normal saline.K252a cells were injected into the lateral ventricles.After 2 weeks,anxiety,depression,and fear memory disorder in rats in each group were detected by the mine experiment,ele-vated cross maze experiment,and conditional fear test.The binding activity of icariin to brain-derived neurotrophic factor(BDNF)and the BDNF and TrkB expressions in the rat amygdala were detected by immunohistochemistry.The relative expressions of BDNF and TrkB pro-teins were detected by Western blotting.The expressions of postsynaptic density protein 95(PSD95)and synaptophysin(SYN)in the rat amygdala were detected using immunofluorescence.Results Icariin showed strong binding to BDNF.Compared with the control group,the times of entering the central area and the percentage of movement distance in the central area in the SPS group and the icariin+K252a group were significantly reduced.The open arm entry(OE)and arm opening time(OT)were significantly reduced,the freezing time and defecation times were significantly increased,and the expressions of the BDNF,TrkB,PSD95,and SYN proteins were significantly reduced(P<0.05).Compared with the SPS group,the icariin group rats had significantly increased times of entering the central area and percentages of movement distance in the central area,significantly increased OE and OT,significantly reduced the time of immobilization and defecation,and significantly increased the expressions of BDNF,TrkB,PSD95,and SYN proteins(P<0.05).Conclusion Icariin effectively alleviated the fear memory impairment induced by SPS in rats.This protective effect is related to BDNF/TrkB signaling pathway activation and upregulated PSD95 and SYN expression.

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.

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.

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.

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.

To Explore the Mechanism of Icariin Combined with Prednisone on Steroid-Resistant Nephrotic Syndrome Based on Network Pharmacology and Experimental Verification / 世界科学技术-中医药现代化

Objective Based on network pharmacology,molecular docking and animal experiment,this study explored the mechanism of Icariin combined with Prednisone in the treatment of steroid-resistant nephrotic syndrome(SRNS)in rats.Methods The targets of Icariin active components were determined by TCMSP,GeneCards and CTD databases.The targets of SRNS were screened from GeneCards,OMIM,CTD and DRUGBANK databases,Remove and integrate.Venn graph was drawn to obtain the intersection target.A protein-protein interaction network was constructed using STRING database,and core targets were screened by topological analysis using Cytoscape 3.7.2.DAVID 6.8 was employed for GO term enrichment and KEGG pathway enrichment.And AutoDockTools Vina,PyMOL for molecular docking.SRNS rat model was constructed.The body weight of rats was measured,The 24-hour urinary protein quantity was detected,and the pathological morphology of renal tissue was observed by HE staining.,and the expression of core target proteins was de tected by Western blot.Results Network pharmacologic analysis showed that 139 targets corresponding to Icariin active ingredients were obtained,and 58 common targets were obtained by intersection with 1476 SRNS related targets.In PPI network,the top three degree values were Akt,JUN and IL6.KEGG enrichment analysis showed that Icariin played a therapeutic role in SRNS mainly by PI3K/AKT singnaling pathway.Molecular docking verification showed that Akt,PI3K and autophagy-related protein LC3-II had good binding activity with Icariin.Animal experiment result showed that Icariin combined with Prednisone significantly increased the body weight of SRNS rats(P<0.05),decreased the 24 hour urine protein quantity(P<0.01),and improved the glomerular morphological changes.The results of Western-blotting showed that Icariin combined with Prednisone could significantly improve the abnormal decrease of LC3-II protein and the abnormal increase of Akt and PI3K protein expression in renal tissue of SRNS rats(P<0.01).Conclusion Icariin can play a role in the treatment of SRNS through a multi-target and multi-pathway,regulate the PI3K-Akt signaling pathway,and increase the autophagy activity of kidney tissue in SRNS rats,which may be one of its main mechanisms.

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.

Exploring the mechanism of icariin in regulat-ing cardiac microvascular endothelial cells based on network pharmacology,molecular docking and in vitro experiments / 中国药理学与毒理学杂志

OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro-vascular endothelial cells(CMEC)after oxygen-glucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment to construct a myocardial ischemia-reperfusion model,and were divided into normal,model,low(10 μmol·L-1),medium(20 μmol·L-1)and high(40 μmol·L-1)ICA group,and high ICA+ inhibitor group(40 μmol·L-1+20 nmol·L-1).CCK-8 assay was used to assess the protective ability of ICA against CMEC,and cell migration assay and tube-formation assay were used to detect the migration and generation ability of CMEC.The TCMSP database,Swiss-Target database and literature mining methods were used to col-lect ICA-related targets,the GeneCards data-base was used to collect target genes related to myocardial ischemia/reperfusion,and Cytoscape 3.8.0 software was used to construct a"drug-tar-get-disease"network.The potential targets were imported into STRING 11.5 database to obtain the PPI network.GO and KEGG enrichment analyses were performed on the potential targets using the DAVID database.Molecular docking was performed using AutoDock-vina 1.1.2 soft-ware.Western blot detected the expression of related proteins.RESULTS After CMEC was subjected to OGD/R treatment,ICA had a protec-tive effect at 10-160 μmol·L-1;the results of the cell migration assay showed that each group of ICA could promote the migratory effect of CMEC(P＜0.01,P＜0.01);and the results of tube-for-mation assay showed that each group of ICA could significantly promote the generation of branches(P＜0.01)and the capillary length exten-sion(P＜0.05).Network pharmacology collected a total of 23 ICA action targets,1500 disease tar-gets and 12 key targets.GO function enrichment analysis found 85 results.KEGG pathway enrich-ment analysis found 53 results,involving AGE-RAGE signaling pathway,sphingolipid signaling pathway and VEGF signaling pathway.Molecu-lar docking results showed that ICA had better binding with core targets PRKCB,PRKCA and PTGS2.Western blot results showed that ICA could regulate the expression of PRKCB,PRKCA and PTGS2 proteins.The results of cell migra-tion assay,tube-formation assay and protein expression were reversed after addition of PKC inhibitor.CONCLUSION The potential mecha-nism of action of ICA against myocardial isch-emia-reperfusion injury may be related to the reg-ulation of processes such as CMEC migration and angiogenesis,and it functions through the key target gene PKC.