Salidroside Ameliorates Lung Injury Induced by PM_(2.5) by Regulating SIRT1-PGC-1αin Mice

Objective This study aimed to clarify the intervention effect of salidroside(SAL)on lung injury caused by PM_(2.5) in mice and illuminate the function of SIRT1-PGC-1ɑaxis.Methods Specific pathogen-free(SPF)grade male C57BL/6 mice were randomly assigned to the following groups:control group,SAL group,PM_(2.5) group,SAL+PM_(2.5) group.On the first day,SAL was given by gavage,and on the second day,PM_(2.5) suspension was given by intratracheal instillation.The whole experiment consist of a total of 10 cycles,lasting 20 days.At the end of treatment,blood samples and lung tissues were collected and analyzed.Observation of pathological changes in lung tissue using inverted microscopy and transmission electron microscopy.The expression of inflammatory,antioxidants,apoptosis,and SIRT1-PGC-1ɑproteins were detected by Western blotting.Results Exposure to PM_(2.5) leads to obvious morphological and pathologica changes in the lung of mice.PM_(2.5) caused a decline in levels of antioxidant-related enzymes and protein expressions of HO-1,Nrf2,SOD2,SIRT1 and PGC-1ɑ,and an increase in the protein expressions of IL-6,IL-1β,Bax,caspase-9 and cleaved caspase-3.However,SAL reversed the aforementioned changes caused by PM_(2.5) by activating the SIRT1-PGC-1α pathway.Conclusion SAL can activate SIRT1-PGC-1ɑ to ameliorate PM2.5-induced lung injury.

Salidroside attenuates oxygen and glucose deprivation-induced neuronal injury by inhibiting ferroptosis

Objective: To evaluate the effect of salidroside on oxygen and glucose deprivation(OGD)-treated NT2 cells and its underlying mechanisms of action.Methods: Retinoic acid was used to induce the differentiation of NT2 cells into neurons. The effects of salidroside on survival, apoptosis, inflammatory response, and oxidative stress of neurons undergoing OGD were evaluated. Using precursor cells as controls, the effect of salidroside on the differentiation progression of OGDtreated cells was evaluated. In addition, the effect of erastin, a ferroptosis inducer, on NT2 cells was examined to investigate the underlying mechanisms of neuroprotective action of salidroside.Results: Salidroside alleviated the effects of OGD on neuronal survival, apoptosis, inflammation, and oxidative stress, and promoted NT2 cell differentiation. Moreover, salidroside prevented ferroptosis of OGD-treated cells, which was abolished following erastin treatment, indicating that ferroptosis mediated the regulatory pathway of salidroside.Conclusions: Salidroside attenuates OGD-induced neuronal injury by inhibiting ferroptosis and promotes neuronal differentiation.

Neuroprotective effects of salidroside on focal cerebral ischemia/reperfusion injury involve the nuclear erythroid 2-related factor 2 pathway

Salidroside,the main active ingredient extracted from Rhodiola crenulata,has been shown to be neuroprotective in ischemic cerebral injury,but the underlying mechanism for this neuroprotection is poorly understood.In the current study,the neuroprotective effect of salidroside on cerebral ischemia-induced oxidative stress and the role of the nuclear factor erythroid 2-related factor 2（Nrf2）pathway was investigated in a rat model of middle cerebral artery occlusion.Salidroside（30 mg/kg）reduced infarct size,improved neurological function and histological changes,increased activity of superoxide dismutase and glutathione-S-transferase,and reduced malon-dialdehyde levels after cerebral ischemia and reperfusion.Furthermore,salidroside apparently increased Nrf2 and heme oxygenase-1 expression.These results suggest that salidroside exerts its neuroprotective effect against cerebral ischemia through anti-oxidant mechanisms and that activation of the Nrf2 pathway is involved.The Nrf2/antioxidant response element pathway may become a new therapeutic target for the treatment of ischemic stroke.

Protective Role of Salidroside against Aging in A Mouse Model Induced by D-galactose

Objective To investigate the protective effects of putative AGEs （advanced glycation endproducts） inhibitor salidroside against aging in an accelerated mouse aging model induced by D-galactose. Methods A group of 5-month-old C57BL/6J mice were treated daily with D-galactose, D-galactose combined with salidroside, salidroside alone, and control buffer for 8 weeks. At the end of the treatment, serum AGEs levels, neurological activities, expression of glial fibrillary acidic protein （GFAP） and neurotrophin-3 （NT-3） in the cerebral cortex, as well as lymphocyte proliferation and IL-2 production were determined. Results D-galactose induced mouse aging model was developed as described before. As expected, salidroside blocked D-galactose induced increase of serum AGEs levels. It also reversed D-galactose induced aging effects in neural and immune system, as evidenced by improving motor activity, increasing memory latency time, and enhancing lymphocyte mitogenesis and interleukin-2 （IL-2） production. Furthermore, elevated expression of GFAP and NT-3 in the aged model mice was also reduced upon salidroside treatment. Conclusion Salidroside inhibits AGEs formation in vivo, which at least partially contributes to its anti-aging effect in D-galactose induced aging model.

Effects of salidroside on glioma formation and growth inhibition together with improvement of tumor microenvironment

Objective：To test the effects of salidroside on formation and growth of glioma together with tumor microenvironment.Methods：Salidroside extracted from Rhodiola rosea was purified and treated on human glioma cells U251 at the concentration of 20 μg/mL.3-（4,5-dimethylthiazol-2-yl）-2,5-dephenyltetrazolium bromide （MTT） assay for cytotoxicity and flow cytometry （FCM） for cell cycle analysis were performed.Then for in vivo study,xenotransplantation tumor model in nude mice was generated and treated with salidroside at the concentration of 50 mg/kg.d for totally 20 d.Body weight and tumor size were detected every 2 d after the treatment.The levels of 8-isoprostane,superoxide dismutase （SOD） and malondialdehyde （MDA）,special markers for oxidative stress,were detected while immunofluoresence staining was performed for astrocyte detection.Results：For in vitro study,salidroside could decrease the viability of human glioma cells U251 and the growth of U251 cells at G0/G1 checkpoint during the cell cycle.For in vivo study,salidroside could also inhibit the growth of human glioma tissue in nude mice.The body weight of these nude mice treated with salidroside did not decrease as quickly as control group.In the tumor xenotransplantation nude mice model,mice were found of inhibition of oxidative stress by detection of biomarkers.Furthermore,overgrowth of astrocytes due to the stimulation of oxidative stress in the cortex of brain was inhibited after the treatment of salidroside.Conclusions：Salidroside could inhibit the formation and growth of glioma both in vivo and in vitro and improve the tumor microenvironment via inhibition of oxidative stress and astrocytes.

Salidroside Inhibits Lipopolysaccharide-ethanol-induced Activation of Proinflammatory Macrophages via Notch Signaling Pathway

Activation of macrophages is a key event for the pathogenesis of various inflammatory diseases.Notch signaling pathway recently has been found to be a critical pathway in the activation of proinflammatory macrophages.Salidroside (Sal),one of main bioactive components in Rhodiola crenulata (Hook.F.et Thoms) H.ohba,reportedly possesses anti-inflammatory activity and ameliorates inflammation in alcohol-induced hepatic injury.However,whether Sal regulates the activation of proinflammatory macrophages through Notch signaling pathway remains unknown.The present study investigated the effects of Sal on macrophage activation and its possible mechanisms by using both alcohol and lipopolysaccharide (LPS) to mimic the microenvironment of alcoholic liver.Detection of THP-1-derived macrophages exhibited that Sal could significantly decrease the expression of tumor necrosis factor-α(TNF-α),interleukinbeta (IL-1β)and IL-6 in the macrophages at both mRNA and protein levels.Furthermore,Sal significantly suppressed NF-kB activation via Notch-Hes signaling pathway in a dose-dependent manner.Moreover,in the microenvironment of alcoholic liver,the expression of Notch-dependent pyruvate dehydrogenase phosphatase 1 (PDP1) was elevated,and that of Ml gene expression [inducible NO synthase (NOS2)] was up-regulated.These changes could all be effectively ameliorated by Sal.The aforementioned findings demonstrated that Sal could inhibit LPS-ethanol-induced activation of proinflammatory macrophages via Notch signaling pathway.

LC-MS determination and pharmacokinetic study of salidroside in rat plasma after oral administration of suspensions of traditional Chinese medicine Erzhi Wan and Fructus Ligustri lucidi

A simple,rapid and sensitive liquid chromatography-mass spectrometry（LC-MS）method was developed for the determination of salidroside in rat plasma and study of its pharmacokinetics after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi into Wistar rats.Plasma sample of 200 μL was extracted with acetic ether-isopropanol（2∶1）and the extraction was performed on a Kromasil C18 column（150 mm×4.6 mm,5 μm）with the mobile phase of methanol-water（41∶59,v/v）within a run time of 6.0 min.The analyte was monitored with positive electrospray ionization（ESI）by selected ion monitoring（SIM）mode.The target ions were m/z 323.05 for salidroside and m/z 411.05 for internal standard（IS）geniposide.A good linear relationship was obtained over the range of 5.0-500.0 ng/mL and the lower limit of quantification was 5.0 ng/mL.The validated method was successfully applied to the pharmacokinetic study of salidroside in rat plasma after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi.

Salidroside Attenuates LPS-stimulated Activation of THP-1 Cell-derived Macrophages through Down-regulation of MAPK/NF-kB Signaling Pathways

Summary： Excessive activation of macrophages is implicated in various inflammatory injuries. Salidroside （Sal）, one of the main bioactive components ofRhodiola Sachalinensis, has been reported to possess anti-inflammatory activities. This study aimed to examine the effect of Sal on the activa- tion of macrophages and the possible mechanism. The lipopolysaccharide （LPS）-stimulated phrobol 12-myristate 13-acetate （PMA）-differentiated THP-1 macrophage models were established. The changes in the inflammatory profiles of THP-l-derived macrophages were determined. The results showed that Sal significantly decreased the expression of inducible nitric oxide synthase （iNOS）, cyclooxygenase-2 （COX2）, interleukin-lbeta （IL-1β）, interleukin-6 （IL-6） and tumor necrosis fac- tor-or （TNF-a） at both mRNA and protein levels in THP-l-derived macrophages, and the effect was dose-depedent. Moreover, NF-B activation was significantly suppressed and the phosphorylation of ERK, p38 and JNK was substantially down-regulated after Sal treatment. The findings suggested that Sal can suppress the activation of LPS-stimulated PMA-differetiated THP-1 cells, as evidenced by the decreased expression of iNOS, COX2, IL-1β, IL-6 and TNF-a, and the mechanism involves the inhibition of NF-r,B activation and the phosphorylation of the MAPK signal pathway.

Anti-fatigue effects of salidroside in mice

Objective： To study the anti-fatigue effects of salidroside in mice. Methods： Totally 120 normal male Kunming mice were randomized into 5 groups （4 salidroside intervention groups and the control group） based on body weight. The control group was given distilled water and the 4 intervention groups were given various doses of salidroside （60, 180, 360, 720 mg/kg） for 15 consecutive days, respectively. The levels of lactate, serum urea nitrogen, muscle and liver glycogen, the longest swimming time and hemoglobin were determined before and after swimming test. Results： Different doses of salidroside significantly lengthened the swimming time and increased the contents of hemoglobin and muscle and liver glycogen, while reducing that of lactate in blood significantly compared with control group, especially in the 180 mg/kg salidroside group. Conclusion： Salidroside has noticeable anti-fatigue effect on mice. These effects were dose-dependent, and the strongest effect on most biomarkers was seen with an intermediate dose.

Salidroside improves endothelial function and alleviates atherosclerosis by activating a mitochondria-related AMPK/PI3K/Akt/eNOS pathway

Aim Salidroside （SAL） is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea. A recent study has reported that SAL can efficiently decrease atherosclerotic plaque formation in low-density lipoprotein receptor - deficient mice. This study was to investigate the molecular mechanism of antiatherogenic effects of SAL. Method Six-week old apoE-/- male mice were fed a high-fat diet for 8 weeks and then were ad- ministered with SAL for another 8 weeks. Atherosclerotic lesion and vascular function were analyzed. Primary cul- tured human umbilical vein endothelial cells （HUVECs） were prepared. Superoxide anion （O2^-）, NO produc- tion, mitochondrial membrane potential （△ψm） and intracellular ATP and AMP levels were measured. Expression of eNOS and AMPK were analyzed by Western blot. Result SAL significantly improved endothelial function asso- ciated with increasing eNOS activation thus reduced the atherosclerotic lesion area. SAL increased eNOS-Serl177 phosphorylation and decreased eNOS-Thr495 phosphorylation. SAL significantly activated AMP-activated protein ki- nase （AMPK）. Both AMPK inhibitor and AMPK small interfering RNA （siRNA） abolished SAL-induced Akt- Ser473 and eNOS-Serl177 phosphorylation. In contrast, LY294002, the PI3k/Akt pathway inhibitor, abolished SAL-induced phosphorylation and expression of eNOS. SAL decreased cellular ATP content and increased the cel- lular AMP/ATP ratio, which was associated with the activation of AMPK. SAL was found to decrease A^m, which is likely consequence of reduced ATP production. Conclusion The action of SAL to reduce atherosclerotic lesion formation may at least be attributed to its effect on improving endothelial function by promoting nitric oxide （NO） production, which was associated with mitochondria depolarization and subsequent activation of the AMPK/PI3 IC/ Akt/eNOS pathway. Taken together, our data described the effects of SAL on mitochondria, which played critical roles in improving endothelial function in atherosclerosis.

Protective Effect of Salidroside on Mitochondrial Disturbances via Reducing Mitophagy and Preserving Mitochondrial Morphology in OGD-induced Neuronal Injury

Salidroside is the active ingredient extracted from Rhodiola rosea,and has been reported to show protective effects in cerebral ischemia,but the exact mechanisms of neuronal protective effects are still unrevealed.In this study,the protective effects of salidroside(1 jimol/L)in ameliorating neuronal injuries induced by oxygen-glucose deprivation(OGD),which is a classical model of cerebral ischemia,were clarified.The results showed that after 8 h of OGD,the mouse hippocampal neuronal cell line HT22 cells showed increased cell death,accompanied with mitochondrial fragmentation and augmented mitophagy.However,the cell viability of HT22 cells showed significant restoration after salidroside treatment.Mitochondrial morphology and mitochondrial function were effectively preserved by salidroside treatment.The protective effects of salidroside were further related to the prevention of mitochondrial over-fission.The results showed that mTOR could be recruited to the mitochondria after salidroside treatment,which might be responsible for inhibiting excessive mitophagy caused by OGD.Thus,salidroside was shown to play a protective role in reducing neuronal death under OGD by safeguarding mitochondrial function,which may provide evidence for further translational studies of salidroside in ischemic diseases.

Approaches to biosynthesis of salidroside and its key metabolic enzymes

As a main component of efficiency in Rhodiola plants, salidroside is a promising environmental acclamation medicine and possesses specific medical properties against symptoms of fatigue, old age, microwave radiation, viral infections and tumors. Salidroside plays important roles, especially in military, aerospace, sport and healthcare medicine and has, therefore, recently, drawn more and closer attention. This article probes mainly into the probable biosynthetic pathway of salidroside following a brief introduction of the exploitation and utilization values of Rhodiola plants and the current condition of its natural resources. We have come to the conclusion that tyrosol, the aglycon of salidroside, is biosynthesized through the well-characterized shikimic acid pathway. A molecule of glucose is transferred by the UDP-glucosyltransferase （or possibly by the β-D-glucosidase too） to the tyrosol to form salidroside. On the other hand, salidroside may be degraded into tyrosol and glucose by β-D-glucosidase. Progress in research of these two key-enzymes, involved in the metabolism of salidroside, is finally elaborated.

Separation of salidroside from the fermentation broth of engineered Escherichia coli using macroporous adsorbent resins

Salidroside(8-O-β-D-glucoside of tyrosol),a plant-derived natural product,is used for treatment of hypoxia,fatigue and aging diseases.The availability of salidroside is restricted since it is extracted from3-5 years old Rhodiola roots,which grow very slowly in the cold region of northern hemisphere of Earth.Our laboratory has constructed an engineered Escherichia coli and established a fermentation process to produce salidroside from glucose.In this article,nine macroporous resins from polarity to non-polarity,including NKA-9,S-8,AB-8,SP825,D101,LSA-8,LX-12,LX-18 and LX-68 resins,were tested to separate salidroside from fermentation broth.After static and dynamic experiments,the weakly polar SP825 resin had a better separation efficiency among nine resins.The adsorption kinetic and isotherm of salidroside on the SP825 resin were determined,and the pseudo-second-order kinetic model and Langmuir model could be fitted well.The effects of the pH on adsorption and ethanol concentration on desorption were investigated,and an optimal separation process was established.The adsorption for salidroside in the SP825 resin column was conducted with loading 150 ml at pH 7,and desorpted by washing 50 ml of80%ethanol solution.Under the best process conditions,the purity and yield of salidroside in the final product were 91.6%and 74.0%,respectively.The results showed that the macroporous SP825 resin would be feasible and effective to prepare salidroside and has promising application in the downstream process of microbial fermentation.

Salidroside Pretreatment to Mesenchymal Stem Cells Improves Cell Survival and Migration to Promote Diabetic Wound Healing

Objective Diabetic patients pose a greater challenge in managing chronic wound healing,leading to a higher amputation risk compared to non-diabetic patients.Due to their paracrine function by secreting various cytokines and angiogenic factors,mesenchymal stem cells(MSCs)have been acknowledged to be a potential agent in modulating wound healing process.However,post-transplanted MSCs are vulnerable to death,indicating poor survival and migration ability in the wound site of the host,especially under hyperglycemia.As hyperglycemia induces reactive oxygen species(ROS)generation and cellular apoptosis,improvement of MSCs survival and migration potentials under hyperglycemia could contribute to a more efficient MSCs-based wound healing therapy.Salidroside(Sa),a small-molecule drug derived from Rhodiola plant,has been proved to enhance the paracrine function of skeletal muscle cells,as well as their migration even under hypoxichyperglycemia.Herein,we investigated whether Sa could improve the survival and migration potentials of MSCs,subsequently enhance the wound healing process under hyperglycemia.Methods MSCs were cultured under three conditions:low glucose,high glucose,and high glucose+Sa.qPCR analysis and western blotting were done to examine the mRNA and protein expression level of several factors which are important in upregulating the wound healing process.MTT colorimetric assay,intracellular ROS detection,and flow cytometry assay were employed to examine the effect of Sa in MSCs survival.Transwell chamber assay,scratch assay,and phalloidin staining were done to elucidate the role of Sa in regulating MSCs migration potential.For in vivo experiment,diabetic wound healing mice model was generated to elucidate the effect of Sa-pretreated MSCs transplantation in wound closure rate,as well as re-epithelization status,observed with hematoxylin and eosin staining.The diabetic wound healing mice model were divided into three groups:1)mice injected with PBS,2)mice transplanted with PBS-pretreated MSCs,and 3)mice transplanted with Sa-pretreated MSCs.Results(1)Hyperglycemic condition induced the generation of ROS and suppressed total cell number of MSCs,while Sa treatment into MSCs restored these hyperglycemia-induced alterations.In line with this,total apoptotic cells were also suppressed by treating MSCs with Sa.The expression level of cell survival factor,heme-oxygenase 1(HO-1),was enhanced in Sa-pretreated MSCs.Further treatment of HO-1 inhibitor into Sa-pretreated MSCs nullified the ROS level and total apoptotic cells,indica-ting the importance of HO-1 in mediating the Sa-induced survival of MSCs under hyperglycemia.(2)Transwell chamber and scratch assay results showed that Sa-pretreated MSCs have a higher migration potential under hyperglycemia,supported by higher F-actin polymerization fractal dimension.Fibroblast growth factor 2(FGF2)and hepatocyte growth factor(HGF)expression level,which are essential factors for cell migration,were also improved in Sa-pretreated MSCs under hyperglycemia.(3)In diabetic wound healing mice model,transplantation of Sa-pretreated MSCs resulted in significantly improved wound closure rate and re-epithelization.The protein levels of HO-1,FGF2,and HGF were also enhanced in the tissues obtained from the wound site of diabetic wound healing mice model which were transplanted with Sa-pretreated MSCs.Conclusions Salidroside pretreatment on MSCs could improve their survival and migration potentials,subsequently promoting wound healing process under hyperglycemia.This prospective MSC-based therapy could serve as a novel strategy to improve diabetic wound healing.

Mechanistic study on the pharmacokinetic process of salidroside in hypoxic rats

OBJECTIVE To investigate the effect of hypoxia on the pharmacokinetic process of salidrosidein rats and to explore its underlying mechanisms.METHODS The Caco-2 cell monolayer was exposed to 1% oxygen(O_2) concentration for 24 h to build the hypoxiccell model.The transportation mode of salidroside was investigated with the aid of this hypoxia model by detecting the apparent permeability coefficient(P app).Healthy Sprague Dawley(SD) rats were exposed to 9% O_2 for 72 h for the construction of hypoxic rat model.Liver sample was subsequently collected from the hypoxic rats with an aim to identify enzymes responsible for salidroside metabolism.The expression levels of salidroside-transporting and salidroside-metabolizing enzymes,including Sodium-dependent glucose cotransporters(SGLT1),β-glucosidase(GBA3)and sulfotransferase(SULT2A1),were thereafter detected by RT-PCR and Western blot.The metabolic activity of GBA3 and SULT2A1 was monitored by rat liver microsome incubation.In addition,the renal function of rats under hypoxia was assessed by detecting concentrations of blood urea nitrogen and creatinine.RESULTS The AUC and t1/2 values of salidroside in hypoxic rats were more than doubled,while the in vivo clearance was significantly reduced.Mechanistic study demonstrated that the Papp A-B/Papp B-A eualsto 10.3,indicating the potential active transport of salidrosile.The expression of SGLT1 and GBA3 was significantly decreased,which indicated a reduced metabolism of salidroside under hypoxia.Moreover,rat under hypoxia was found to suffer from renal dysfunction,with an abnormal value of blood urea nitrogen.CONCLUSION Due to the reduced metabolism and the abnormal renal function under hypoxia,the systemic exposure of salidroside in rats was significantly enhanced.

Inhibition of Salidroside on Salivary Gland Adenoid Cystic Carcinoma

To evaluate the role of salidroside on proliferation,apoptosis and invasiveness of salivary gland adenoid cystic carcinoma cells（SACC）,immunocytochemical staining was employed to detect proliferating cell nuclear antigen（PCNA）,caspase 3 and caspase 8 expression in SACC-2 cells.Modified Boyden chamber assay combined with laser confocal microscopy（LSCM） was used to evaluate the invasion and migration abilities of SACC-2 cells at different time point.Immunohistochemistry staining revealed that the expression of PCNA was significantly decreased（P0.01） after salidroside treatment.In contrast,salidroside treatment led to increased caspase 3 and caspase 8 in SACC-2 cells.Cell migration depth and number of cells that penetrated Boyden chamber were also decreased by salidroside.Salidroside potently inhibits the proliferation and simultaneously induces the apoptosis of SACC-2 cells.Migration and invasion of SACC-2 cells are also inhibited.Our data throw light on potential clinical application of salidroside to the patients with SACC.

Preparation and in vitro Evaluation of Salidroside Nanoparticle

[Objectives]To use poly(lactic-co-glycolic acid)(PLGA)nanoparticles to improve the bioavailability and brain entrance capability of Salidroside(Sal).[Methods]An emulsion solvent evaporation approach was used to create PLGA nanoparticles(Sal-NP).The preparation parameters were optimized using a single factor experiment.The particle size and zeta potential were determined using the laser particle analyzer,and the morphology of the nanoparticles was observed using transmission electron microscopy.The encapsulation efficiency and drug loading were determined using HPLC.Subsequently,the in vitro drug release was determined using a dynamic dialysis method,and the cellular uptake and cytotoxicity were determined using the bEnd3 cell model.[Results]The ultrasonic time and power for preparing Sal-NP were 6 min and 100 W,respectively.The size of the nanoparticles was 162.0±74.86 nm,and the morphology of Sal-NP was spherical like.After 48 h,the cumulative release of Sal-NP was 62%,indicating that Sal showed a controlled release property in Sal-NP.Cellular uptake study showed that the PLGA nanoparticles remarkably increased the internalization than control group(P<0.001).In addition,Sal-NPs were non-toxic to cells at concentrations ranging from 12.5 to 100μM.[Conclusions]PLGA nanoparticles is promising to be exploited in Alzheimer's disease research due to the increasing absorption and controlled release advantages for Sal.

Salidroside Ameliorates Vascular Endothelial Cell Senescence through Downregulation of KLF4

Salidroside is extensively used as a herbal medicine worldwide, and it has been shown to protect against disruption of endothelial homeostasis and act as an anti-aging agent. The present study aimed to investigate the ameliorative effects of salidroside on homocysteine (Hcy)-induced cell senescence in human umbilical vein endothelial cells (HUVECs) that were mediated via inhibition of Krüppel-like factor 4 (KLF4). An endothelial cell senescence model was induced by Hcy. The cell viability, activities of telomerase and lactate dehydrogenase (LDH), and the level of reactive oxygen species were determined using commercial kits. The expression levels of KLF4, p53 and p21 were determined via western blot analysis, whereas the mRNA expression levels of KLF4 were detected by reverse transcription-quantitative PCR. Small interfering RNA-mediated knockdown of KLF4 was found to reverse Hcy-induced cell senescence. Hcy treatment led to an accelerated cell senescence, as evidenced by decreases in both cell viability and telomerase activity, whereas increases were noted in the leakage of LDH and the level of reactive oxygen species, in addition to an up-regulation of the protein levels of p53 and p21, and up-regulation of KLF4 at both the mRNA and protein level. Treatment with salidroside ameliorated Hcy-induced cell senescence in a dose-dependent manner. Taken together, these results suggested that Hcy may induce cell senescence through upregulation of KLF4, and this may be reversed by treatment with salidroside. Therefore, salidroside was shown to inhibit Hcy-induced cell senescence through KLF4 inhibition.

Scalable synthesis of salidroside

By analyzing the key steps that restricted the industrial synthesis of salidroside, selective protection of tyrosol with different acylation reagents was adopted. The strategy facilitated the crystallization of intermediates, which allowed the scalable synthesis of salidroside. It included a reaction of penta-O-acetyl-β-D-glucose with acyl protected tyrosol in the presence of Lewis acid catalyst（ZnCl2）, followed by deacylation under basic condition（NaOMe/MeOH） to give the salidroside. The total yield of this three-step reaction was 47%. Final product and intermediates were purified by recrystallization, which significantly reduced the cost and made the large scale synthesis feasible.