Expression of P450 and nuclear receptors in normal and end-stage Chinese livers

AIM:To investigate the expression of P450 enzyme genes by using end-stage liver disease samples and trimmed normal Chinese donor livers.METHODS:The end-stage liver disease samples[n=93,including hepatocellular carcinoma(HCC),peri-HCC tissue,hepatitis B virus cirrhosis,alcoholic cirrhosis,and severe cirrhosis]and trimmed normal Chinese donor livers(n=35)from The Institute of Organ Transplantation in Beijing,China.Total RNA was extracted,purified,and subjected to real-time RT-PCR analysis.RESULTS:For cytochrome P450 enzymes 1(CYP1)family,the expression of CYP1A2 was decreased 90%in HCC,80%in alcoholic cirrhosis,and 65%in severe cirrhosis.For CYP2 family,the expression of CAR was decreased 50%in HCC,but increased 50%in peri-HCC tissues.Similar decreases(about 50%)of CYP2B6,CYP2C9,CYP2C19,CYP2D6 and CYP2E1 were observed in HCC,as compared to peri-HCC tissues and normal livers.CYP2C19 were decreased in all end-stage liver diseases and CYP2E1 also decreased in alcoholic cirrhosis and severe cirrhosis.For CYP3 family,the expression of PXR was decreased 60%in HCC,together with decreases in CYP3A4,CYP3A5,and CYP3A7.In contrast,the expression of CYP3A7 was slightly increased in HBV cirrhosis.The expression of CYP4A11 was decreased85%in HCC,7%in alcoholic cirrhosis and severe liver cirrhosis,along with decreases in PPARα.The 93 endstage livers had much higher inter-individual variations in gene expression than 35 normal livers.CONCLUSION:The expression of CYP enzyme genes and corresponding nuclear receptors was generally decreased in end-stage liver diseases,and significant differences in gene expression were evident between peri-HCC and HCC.

Nuclear receptors modulate inflammasomes in the pathophysiology and treatment of major depressive disorder

Major depressive disorder(MDD)is highly prevalent and is a significant cause of mortality and morbidity worldwide.Currently,conventional pharmacological treatments for MDD produce temporary remission in<50%of patients;therefore,there is an urgent need for a wider spectrum of novel antidepressants to target newly discovered underlying disease mechanisms.Accumulated evidence has shown that immune inflammation,particularly inflammasome activity,plays an important role in the pathophysiology of MDD.In this review,we summarize the evidence on nuclear receptors(NRs),such as glucocorticoid receptor,mineralocorticoid receptor,estrogen receptor,aryl hydrocarbon receptor,and peroxisome proliferator-activated receptor,in modulating the inflammasome activity and depression-associated behaviors.This review provides evidence from an endocrine perspective to understand the role of activated NRs in the pathophysiology of MDD,and to provide insight for the discovery of antidepressants with novel mechanisms for this devastating disorder.

Nuclear receptors and pathogenesis of pancreatic cancer

Pancreatic ductal adenocarcinoma(PDAC)is a devastating disease with a median overall survival time of5 mo and the five years survival less than 5%,a rate essentially unchanged over the course of the years.A well defined progression model of accumulation of genetic alterations ranging from single point mutations to gross chromosomal abnormalities has been introduced to describe the origin of this disease.However,due to the its subtle nature and concurring events PDAC cure remains elusive.Nuclear receptors(NR)are members of a large superfamily of evolutionarily conserved ligand-regulated DNA-binding transcription factors functionally involved in important cellular functions ranging from regulation of metabolism,to growth and development.Given the nature of their ligands,NR are very tempting drug targets and their pharmacological modulation has been widely exploited for the treatment of metabolic and inflammatory diseases.There are now clear evidences that both classical ligand-activated and orphan NR are involved in the pathogenesis of PDAC from its very early stages;nonetheless many aspects of their role are not fully understood.The purpose of this review is to highlight the striking connections that link peroxisome proliferator activated receptors,retinoic acid receptors,retinoid X receptor,androgen receptor,estrogen receptors and the orphan NR Nur,chicken ovalbumin upstream promoter transcription factorⅡand the liver receptor homologue-1 receptor to PDAC development,connections that could lead to the identification of novel therapies for this disease.

Hepatic drug transporters and nuclear receptors:Regulation by therapeutic agents

The canalicular membrane represents the excretory pole of hepatocytes.Bile is an important route of elimination of potentially toxic endo-and xenobiotics(including drugs and toxins),mediated by the major canalicular transporters:multidrug resistance protein 1(MDR1, ABCB1),also known as P-glycoprotein,multidrug resistance-associated protein 2(MRP2,ABCC2),and the breast cancer resistance protein(BCRP,ABCG2).Their activities depend on regulation of expression and proper localization at the canalicular membrane,as regulated by transcriptional and post-transcriptional events,respectively.At transcriptional level,specific nuclear receptors(NR)s modulated by ligands,co-activators and co-repressors,mediate the physiological requirements of these transporters.This complex system is also responsible for alterations occurring in specific liver pathologies.We briefly describe the major ClassⅡNRs, pregnane X receptor(PXR)and constitutive androstane receptor(CAR),and their role in regulating expression of multidrug resistance proteins.Several therapeutic agents regulate the expression of relevant drug transporters through activation/inactivation of these NRs.We provide some representative examples of the action of therapeutic agents modulating liver drug transporters, which in addition,involve CAR or PXR as mediators.

Chromatin remodeling regulated by steroid and nuclearreceptors

Coactivators and corepressors regulate transcriptionby controlling interactions between sequence-specific transcription factors, the basal transcriptional machinery andthe chromatin environment. This review consider the access of nuclear and steroid receptors to chromatin, theiruse of corepressors and coactivators to modify chromatinstructure and the implications for transcriptional control.The assembly of specific nucleoprotein architectures andtargeted histone modification emerge as central controlling elements for gene expression.

Liver X receptors and epididymal epithelium physiology

Aim： To investigate the roles of liver X receptors （LXR） in the lipid composition and gene expression regulation in the murine caput epididymidis. LXR are nuclear receptors for oxysterols, molecules derived from cholesterol metabolism that are present in mammals as two isoforms： LXRα, which is more specifically expressed in lipid-metabolising tissues, such as liver, adipose and steroidogenic tissues, and macrophages, whereas LXRβ is ubiquitous. Their importance in reproductive physiology has been sustained by the fact that male mice in which the function of both LXR has been disrupted have fertility disturbances starting at the age of 5 months, leading to complete sterility by the age of 9 months. These defects are associated with epididymal epithelial degeneration in caput segments one and two, and with a sperm midpiece fragility, leading to the presence of isolated sperm heads and flagella when luminal contents are recovered from the cauda epididymidis. Methods： The lipid composition of the caput epididymidis of wild-type and LXR-deficient mice was assessed using oil red O staining on tissue cryosections and lipid extraction followed by high performance liquid chromatography or gas chromatography. Gene expression was checked by quantitative real time polymerase chain reaction. Results： Using LXR-deficient mice, we showed an alteration of the lipid composition of the caput epididymidis as well as a significantly decreased expression of the genes encoding SREBPlc, SCD1 and SCD2, involved in fatty acid metabolism. Conclusion： Altogether, these results show that LXR are important regulators of epididymal function, and play a critical role in the lipid maturation processes occurring during sperm epididymal maturation. （Asian J Androl 2007 July; 9： 574-582）

Role of nuclear receptors in breast cancer stem cells

The recapitulation of primary tumour heterogenity and the existence of a minor sub-population of cancer cells,capable of initiating tumour growth in xenografts on serial passages, led to the hypothesis that cancer stem cells(CSCs) exist. CSCs are present in many tumours, among which is breast cancer. Breast CSCs(BCSCs) are likely to sustain the growth of the primary tumour mass, as wellas to be responsible for disease relapse and metastatic spreading. Consequently, BCSCs represent the most significant target for new drugs in breast cancer therapy. Both the hypoxic condition in BCSCs biology and proinflammatory cytokine network has gained increasing importance in the recent past. Breast stromal cells are crucial components of the tumours milieu and are a major source of inflammatory mediators. Recently, the antiinflammatory role of some nuclear receptors ligands has emerged in several diseases, including breast cancer. Therefore, the use of nuclear receptors ligands may be a valid strategy to inhibit BCSCs viability and consequently breast cancer growth and disease relapse.

Effect of Marine Collagen Peptides on Markers of Metabolic Nuclear Receptors in Type 2 Diabetic Patients with/without Hypertension

Objective To explore Effects of marine collagen peptides （MCPs） on markers of metablic nuclear receptors, i.e peroxisome proliferator-activated receptor （PPARs）, liver X receptor （LXRs） and farnesoid X receptor （FXRs） in type 2 diabetic patients with/without hypertension. Method Study population consisted of 200 type 2 diabetic patients with/without hypertension and 50 healthy subjects, all of whom were randomly assigned to MCPs-treated diabetics （n=50）, placebo-treated diabetics （n=50）, MCPs-treated diabetics with hypertension （n=50）, placebo-treated diabetics with hypertension （n=50）, and healthy controls （n=50）. MCPs or placebo （water-soluble starch） were given daily before breakfast and bedtime over three months. Levels of free fatty acid, cytochrome P450, leptin, resistin, adiponectin, bradykinin, NO, and Prostacyclin were determined before intervention, and 1.5 months, and 3 months after intervention. Hypoglycemia and the endpoint events during the study were recorded and compared among the study groups. Result At the end of the study period, MCPs-treated patients showed marked improvement compared with patients receiving placebo. The protection exerted by MCPs seemed more profound in diabetics than in diabetics with hypertension. In particular, after MCPs intervention, levels of free fatty acid, hs-CRP, resistin, Prostacyclin decreased significantly in diabetics and tended to decrease in diabetic and hypertensive patients whereas levels of cytochrome P450, leptin, NO tended to decrease in diabetics with/without hypertension. Meanwhile, levels of adiponectin and bradykinin rose markedly in diabetics following MCPs administration. Conclusion MCPs could offer protection against diabetes and hypertension by affecting levels of molecules involved in diabetic and hypertensive pathogenesis. Regulation on metabolic nuclear receptors by MCPs may be the possible underlying mechanism for its observed effects in the study. Further study into its action may shed light on development of new drugs based on bioactive peptides from marine sources.

Peroxisome proliferator activated receptors at the crossroad of obesity, diabetes, and pancreatic cancer

Pancreatic ductal adenocarcinoma(PDAC) is the fourth cause of cancer death with an overall survival of 5% at five years. The development of PDAC is characteristically associated to the accumulation of distinctive genetic mutations and is preceded by the exposure to several risk factors. Epidemiology has demonstrated that PDAC risk factors may be non-modifiable risks(sex, age, presence of genetic mutations, ethnicity) and modifiable and co-morbidity factors related to the specific habits and lifestyle. Recently it has become evident that obesity and diabetes are two important modifiable risk factors for PDAC. Obesity and diabetes are complex systemic and intertwined diseases and, over the years, experimental evidence indicate that insulin-resistance, alteration of adipokines, especially leptin and adiponectin, oxidative stress and inflammation may play a role in PDAC. Peroxisome proliferator activated receptor-γ(PPARγ) is a nuclear receptor transcription factor that is implicated in the regulation of metabolism, differentiation and inflammation. PPARγ is a key regulator of adipocytes differentiation, regulates insulin and adipokines production and secretion, may modulate inflammation, and it is implicated in PDAC. PPARγ agonists are used in the treatment of diabetes and oxidative stressassociated diseases and have been evaluated for the treatment of PDAC. PPARγ is at the cross-road of diabetes, obesity, and PDAC and it is an interesting target to pharmacologically prevent PDAC in obese and diabetic patients.

Diurnal Variation of Nuclear Receptors in Mice with or without Fasting

Nuclear receptors such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR) regulate the transcription of transporter and cytochrome P450 (CYP). The diurnal variation was observed in some transporters regulated by nuclear receptors. We investigated whether diurnal variation in PXR and CAR exists in mice. We also examined the effect of food intake on the diurnal rhythm of hepatic PXR and CAR using fed and fasted mice. In liver and small intestine of fed mice, the mRNA levels of PXR and CAR were unchanged between 7:00 AM and 7:00 PM. In contrast to fed mice, fasting mice partly exhibited the diurnal variation in PXR, not in CAR. The mRNA levels of PXR at 7:00 AM were significantly higher than that those at 7:00 PM in liver of fasting mice. These results indicated the different effects of fasting in mice on diurnal variation of PXR in each tissue.

Peroxisome proliferator-activated receptors for hypertension

Peroxisome proliferator-activated receptors(PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, which is composed of four members encoded by distinct genes(α, β, γ, and δ). The genes undergo transactivation or transrepression under specific mechanisms that lead to the induction or repression of target gene expression. As is the case with other nuclear receptors, all four PPAR isoforms contain five or six structural regions in four functional domains; namely, A/B, C, D, and E/F. PPARs have many functions, particularly functions involving control of vascular tone, inflammation, and energy homeostasis, and are, therefore, important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. Hence, PPARs also represent drug targets, and PPARα and PPARγ agonists are used clinically in the treatment of dyslipidemia and type 2 diabetes mellitus, respectively. Because of their pleiotropic effects, they have been identified as active in a number of diseases and are targets for the development of a broad range of therapies for a variety of diseases. It is likely that the range of PPARγ agonist therapeutic actions will result in novel approaches to lifestyle and other diseases. The combination of PPARs with reagents or with other cardiovascular drugs, such as diuretics and angiotensin Ⅱ receptor blockers, should be studied.This article provides a review of PPAR isoform characteristics, a discussion of progress in our understanding of the biological actions of PPARs, and a summary of PPAR agonist development for patient management. We also include a summary of the experimental and clinical evidence obtained from animal studies and clinical trials conducted to evaluate the usefulness and effectiveness of PPAR agonists in the treatment of lifestyle-related diseases.

Targeting nuclear receptors for NASH/MASH:From bench to bedside

The onset of metabolic dysfunction-associated steatohepatitis(MASH)or non-alcoholic steatohepatitis(NASH)represents a tipping point leading to liver injury and subsequent hepatic complications in the natural progression of what is now termed metabolic dysfunction-associated steatotic liver diseases(MASLD),formerly known as non-alcoholic fatty liver disease(NAFLD).With no pharmacological treat-ment currently available for MASH/NASH,the race is on to develop drugs targeting multiple facets of hepatic metabolism,inflammation,and pro-fibrotic events,which are major drivers of MASH.Nuclear receptors(NRs)regulate genomic transcription upon binding to lipophilic ligands and govern multiple aspects of liver metabolism and inflammation.Ligands of NRs may include hormones,lipids,bile acids,and synthetic ligands,which upon binding to NRs regulate the transcriptional activities of target genes.NR ligands are presently the most promising drug candidates expected to receive approval from the United States Food and Drug Administration as a pharmacological treatment for MASH.This review aims to cover the current understanding of NRs,including nuclear hormone receptors,non-steroid hormone receptors,circadian NRs,and orphan NRs,which are currently undergoing clinical trials for MASH treatment,along with NRs that have shown promising results in preclinical studies.

Impact of obese levels on the hepatic expression of nuclear receptors and drug-metabolizing enzymes in adult and offspring mice

The prevalence of obesity-associated conditions raises new challenges in clinical medication.Although altered expression of drug-metabolizing enzymes(DMEs)has been shown in obesity,the impacts of obese levels(overweight,obesity,and severe obesity)on the expression of DMEs have not been elucidated.Especially,limited information is available on whether parental obese levels affect ontogenic expression of DMEs in children.Here,a high-fat diet(HFD)and three feeding durations were used to mimic different obese levels in C57BL/6 mice.The hepatic expression of five nuclear receptors(NRs)and nine DMEs was examined.In general,a trend of induced expression of NRs and DMEs(except for Cyp2c29 and 3a11)was observed in HFD groups compared to low-fat diet(LFD)groups.Differentialeffects of HFD on the hepatic expression of DMEs were found in adult mice at different obese levels.Family-based dietary style of an HFD altered the ontogenic expression of DMEs in the offspring older than 15 days.Furthermore,obese levels of parental mice affected the hepatic expression of DMEs in offspring.Overall,the results indicate that obese levels affected expression of the DMEs in adult individuals and that of their children.Drug dosage might need to be optimized based on the obese levels.

Editor profile:Guest editor of special issue “Bile Acids and Nuclear Receptors in Digestive System and Therapy”

Associate Prof.Grace Liejun Guo is a tenured faculty in the Department of Pharmacology and Toxicology in the School of Pharmacy at the Rutgers University in New Jersey,USA.Dr.Guo graduated from the West China University of Medical Sciences in 1993.In 1997,Dr.Guo obtained a MS degree

Nuclear receptors and non-alcoholic fatty liver disease:An update

Non-alcoholic fatty liver disease(NAFLD)has become the leading cause of chronic liver disease in adults and children worldwide.The symptoms of NAFLD range from simple steatosis and non-alcoholic stea-tohepatitis(NASH)to hepatic fibrosis or cirrhosis,even ultimately develops to hepatocellular carcinoma.Nuclear receptors(NRs)are a superfamily of ligand-activated transcription factors,most of which are ligand-activated that control cellular homeostasis in the liver and other tissues.A growing number of studies demonstrated the important role of NRs in NAFLD.In this review,the current findings on the role of NRs in NAFLD are summarized and future perspectives to target NRs for NAFLD are discussed.

Understanding the physiological functions of the host xenobiotic-sensing nuclear receptors PXR and CAR on the gut microbiome using genetically modified mice

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor(PXR) and constitutive androstane receptor(CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids(BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. h PXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.

Aryl hydrocarbon receptor nuclear translocator 2 as a prognostic biomarker and immunotherapeutic indicator for clear cell renal cell carcinoma

Background:In many cancer types,aryl hydrocarbon receptor nuclear translocator 2(ARNT2)has been found to be associated with tumor cell proliferation and prognosis.However,the role of ARNT2 in clear cell renal cell carcinoma(ccRCC)has not been completely elucidated.In this study,the potential role of ARNT2 in ccRCC development was characterized.Methods:A pan-cancer dataset(TCGA-TARGET-GTEx)was accessed from UCSC Xena Data Browser.ARNT2 expression in normal and tumor samples was compared.Univariate Cox regression was performed to evaluate the prognostic value of ARNT2.Single sample gene set enrichment analysis(ssGSEA)was used to estimate the enrichment of functional pathways and gene signatures.CIBERSORT and ESTIMATE methods evaluated the immune infiltration.The ARNT2 expression was determined in ccRCC tissue and cell lines using RT-qPCR and Western blot.Results:ARNT2 expression was significantly dysregulated in 23 out of 30 cancer types.Pan-cancer data revealed a strong correlation between ARNT2 expression and immune modulators,immune cell infiltration,and genomic alternations.In ccRCC patients,the low-ARNT2 expression group had higher immune infiltration,CD8 T cells,and programmed cell death ligand 1 expression,as well as higher enrichment score of immunotherapeutic predictors than those in the high-ARNT2 expression group.Low-ARNT2 expression group was more responsive to immunotherapy.Moreover,low ARNT2 expression was observed in ccRCC tissue and cell lines.Conclusions:Dysregulated ARNT2 expression is involved in cancer development and the modulation of the immune microenvironment.ARNT2 can be potentially used as a prognostic indicator and an immunotherapeutic indicator for ccRCC.

Changes in Physiological and Biochemical Characteristics of Floral Organ Development in a Soybean Cytoplasmic-nuclear Male Sterile Line

[Objectives]This study was conducted to explore the mechanism of soybean cytoplasmic-nuclear male sterility.[Methods]With soybean cytoplasmic-nuclear male sterile line JLCMS9 A and its homotype maintainer line JLCMS9 B as experimental materials,the activity of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT),malondialdehyde(MDA)content,starch content,soluble protein content,soluble sugar content and free proline content in flower buds,alabastrums and mature flowers were determined,and the contents and changes of auxin(IAA),gibberellin(GA3),isopentenyl adenosine(iPA)and abscisic acid(ABA)at the three stages were analyzed.[Results]The activity of SOD and CAT and the contents of MDA and free proline in the sterile line at the flower bud stage were lower than those of the maintainer line,but the opposite was true at the alabastrum stage and the flowering stage,and their values were higher than those of the maintainer line;the POD activity of the sterile line was significantly lower than that of the maintainer line at the flower bud stage,and the opposite was true at the alabastrum stage and the flowering stage,and its values were higher than those of the maintainer line;and the starch content and soluble sugar content of sterile line 9 A showed an overall upward trend,and were significantly lower than those of the maintainer line 9 B at the alabastrum stage and the flowering stage.During the whole development process of floral organs,the content of IAA in sterile line 9 A showed a trend of first increasing and then decreasing,and the content of iPA increased gradually.The contents of hormones in the sterile line were lower than those in the maintainer line.The ratios of IAA/ABA,IAA/GA3,IAA/iPA and ABA/GA3 in the sterile line were significantly different from those in the maintainer line.It is inferred that the abnormal physiological characteristics of floral organ development are related to the cytoplasmic-nuclear male sterility of soybean.The alabastrum stage may be a critical period for the occurrence of abnormal physiological and biochemical indexes in the floral organs of soybean cytoplasmic-nuclear male sterile lines.[Conclusions]This study provides a theoretical basis for the breeding of fine sterile lines of soybean and the research on the mechanism of sterility.

Transcriptional Crosstalk between Nuclear Receptors and Cytokine Signal Transduction Pathways in Immunity

The nuclear receptor superfamily and the transcriptional factors associated with cytokines are inherently different families of signaling molecules and activate gene transcription by binding to their respective responsive element.However,it has become increasingly clear from our works and others that nuclear receptors are important regulators of cytokine production and function through complex and varied interactions between these distinct transcriptional factors.This review provides a general overview of the mechanism of action of nuclear receptors and their transcriptional crosstalk with transcriptional factors associated with cytokine transduction pathways.One of the most important mechanistic aspects is protein to protein interaction through a direct or co-regulator-mediated indirect manner.Such crosstalk is crucially involved in physiological and therapeutic roles of nuclear receptors and their iigands in immunity, inflammation and cytokine-related tumors.Cellular & Molecular Immunology.2004;1(6):416-424.

Biochanin A attenuates spinal cord injury in rats during early stages by inhibiting oxidative stress and inflammasome activation

Previous studies have shown that Biochanin A,a flavonoid compound with estrogenic effects,can serve as a neuroprotective agent in the context of cerebral ischemia/reperfusion injury;howeve r,its effect on spinal cord injury is still unclea r. In this study,a rat model of spinal cord injury was established using the heavy o bject impact method,and the rats were then treated with Biochanin A(40 mg/kg) via intrape ritoneal injection for 14 consecutive days.The res ults showed that Biochanin A effectively alleviated spinal cord neuronal injury and spinal co rd tissue injury,reduced inflammation and oxidative stress in spinal cord neuro ns,and reduced apoptosis and pyroptosis.In addition,Biochanin A inhibited the expression of inflammasome-related proteins(ASC,NLRP3,and GSDMD)and the Toll-like receptor 4/nuclear factor-κB pathway,activated the Nrf2/heme oxygenase 1 signaling pathway,and increased the expression of the autophagy markers LC3 Ⅱ,Beclin-1,and P62.Moreove r,the therapeutic effects of Biochanin A on early post-s pinal cord injury were similar to those of methylprednisolone.These findings suggest that Biochanin A protected neurons in the injured spinal cord through the Toll-like receptor 4/nuclear factor κB and Nrf2/heme oxygenase 1 signaling pathways.These findings suggest that Biochanin A can alleviate post-spinal cord injury at an early stage.