Zuogui pills for myelinolysis in a rat model of experimental autoimmune encephalomyelitis

Zuogui pills have been shown to attenuate the inflammatory reaction in a rat model of experimental autoimmune encephalomyelitis （EAE）. The present study attempted to investigate the pathology underlying the influence of Zuogui pills on myelinolysis in EAE rats. Hematoxylin-eosin and Luxol fast blue staining showed that the myelinolysis foci in the cerebrum, cerebellum, brain stem, and the spinal cord of EAE rats were significantly decreased, along with serum myelin basic protein content following treatment with Zuogui pills.

Fourier transform infrared spectroscopy analysis of the active components in serum of rats treated with Zuogui Pill

Objective:To investigate the active components Zuogui Pill,a typical recipe for nourishing kidney essence in the traditional Chinese medicine.Methods:Adult male Sprague Dawley rats were treated with the traditional Chinese herbal medicine Zuogui Pill and the active components found in the serum of the animals were analyzed by Fourier transform infrared(FTIR)spectroscopy.FTIR spectra of serum samples of treated and untreated rats were analyzed and the A2960/A2931 and A1540/A1080 ratios were calculated.Results:A2960/A2931 ratios of the serum samples collected following the administration of Zuogui Pill were significantly higher than those of the normal serum samples.FTIR data were then fitted using a Gaussian equation for wave numbers in the range of 1140e1000 cm
1.ARNA/ADNA ratios in the serum of rats treated with Zuogui Pill were higher than those found in normal rat serum.Conclusion:FTIR spectroscopy could be used as an analytical tool to detect the activecomponents in serum of animals treated with Zuogui Pill.

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

Network pharmacological study of Zuogui pill and Yougui pill for the treatment of postmenopausal osteoporosis

Background:Postmenopausal osteoporosis(PMO)is the most common primary osteoporosis in older women.This condition imposes a huge economic and medical burden on society.Aside from western medicine,traditional Chinese medicine is also widely used for the treatment of PMO,especially in Asian countries.Zuogui pill(ZGP)and Yougui pill(YGP)are classical formulas for the treatment of PMO with potent clinical effects.This study aimed to explore the potential active compounds,potential targets,and potential mechanisms of ZGP and YGP for the treatment of PMO.Methods:Compounds from ZGP and YGP were collected from three online databases,and these compounds were screened by oral bioavailability and drug-likeness.Their corresponding targets were determined from the Medicine Systems Pharmacology Database and Analysis platform.The corresponding targets for PMO were obtained from two disease databases.The target protein-protein interaction was identified through the STRING platforms and the core targets were ascertained by analyzing the protein-protein interaction network by using Cytoscape software.PMO-related genes were identified via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses to identify specific biological processes,cellular components,molecular functions and key signalling pathways of ZGP and YGP for PMO treatment.Results:A total of 68 compounds were screened from ZGP with 168 putative potential target genes,whereas 99 compounds were screened from YGP with 214 potential target genes associated with PMO.Most of the core components included quercetin and kaempferol,and most of the core targets were TP53,AKT1,MAPK1,JUN,TNF,HSP90AA1,APP,IL6,VEGFA,RELA,MAPK8,NR3C1,EGFR,CXCL8,ESR1,FOS and MAPK14.Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that ZGP and YGP treat PMO primarily via regulation of bone formation and resorption,anti-inflammatory immunity and hormonal regulation.Conclusion:Our data provided insights into the mechanisms,by which ZGP and YGP treat PMO.This study points out a direction for further research into ZGP and YGP monomers and pathways and offers a new clinical treatment option for non-traditional Chinese medicine clinicians in the treatment of PMO.

Zuogui(左归丸)and Yougui pills(右归丸)improve perimenopausal syndrome via regulation of apoptosis in mice

OBJECTIVE:To examine,dissect,and understand the molecular mechanisms and combinatorial effects of Zuogui(左归丸,ZGP)and Yougui pills(右归丸,YGP)in 4-vinyl cyclohexene diepoxide(4-VCD)-induced Perimenopausal syndrome(PMS).METHODS:Using the 4-VCD-induced PMS mouse model,uterine and ovary index were measured,and serum sex steroidal hormone levels were evaluated after treatment with ZGP,YGP,ZGP+YGP,estradiol valerate(EV),and Gengnian An(更年安,GNA).Histopathological examinations,ingredient-target network predictions,western blotting,and real-time quantitative polymerase chain reaction(RT-q PCR)analyses were performed to ascertain the possible pharmacological effects and molecular mechanisms of ZYP and YGP.RESULTS:Treatment with ZGP and YGP remarkably improves estrous cyclicity and prevents pathological damage to the uterus.Also,altered sex hormones,including AMH,E2,FSH,LH,P,and T,were restored to normal levels after ZGP and YGP administration.Ingredient-target network analysis showed that the 5 ingredients common to the ZGP and YGP formula modulate 53 targets shared with PMS.Pathwayenrichment analysis further predicted that ZGY and YGP likely regulate of apoptosis and other essential pathways during PMS.In-vivo studies showed that ZGP and YGP suppress PMS via modulating apoptosis through decreasing Caspase-3 and Bcl-2-associated X(Bax)levels and increasing B-cell lymphoma-2(Bcl-2)/Bax and Bcl-2 levels.Importantly,ZGP+YGP treatment modulation effects were somewhat or significantly better compared to ZGP or YGP alone treatment.CONCLUSION:ZGP and YGP represent novel anti-PMS agents whose effects involve restoring altered hormonal levels,protecting the uterus,and regulating apoptosis.

Effects of Zuogui Pill (左归丸) on Wnt Singal Transduction in Rats with Glucocorticoid-induced Osteoporosis

Objective: To reveal the mechanism of Zuogui Pill (左归丸) in treatment of glucocorticoid-induced osteoporosis from the angle of the Wnt signal transduction pathway and to provide further experimental evidence for expounding the scientific connotation of "the kidney dominating the bones" in TCM. Methods: Forty-two male Wistar rats were selected and randomly divided into three groups, control group (n=12), model group (n=15) and Zuogui Pill group (n=15). Form the beginning, The rats were injected dexamethasone for eight weeks to make the model of osteoporosis, and the Zuogui Pill were administered intragastrically to the rats of Zuogui Pill group for eight weeks. The relative morphological parameters were measured in the undecalcified tibial slices. And the protein expression levels of Wnt1, LRP-5 and β-catenin in rat tibial osteoblasts (OB) and bone marrow stromal cells (BMC) were detected by immunohistochemistry. Results: Compared with the control group, TBV% and TFS% decreased significantly, while TRS% increased significantly, and the protein expression of Wnt1, LRP-5 and β-catenin in OB and BMC decreased significantly in the model group. And compared with the model group, TBV% and TFS% increased significantly, and expression levels of Wnt1, LRP-5 and β-catenin proteins increased significantly in the Zuogui pill group. Conclusion: Zuogui Pill can prevent and treat glucocorticoid-induced osteoporosis in rats by up-regulating the expression of the key signal molecules Wnt1, LRP-5 and β-catenin in Wnt signal transduction pathway.

Effects of Zuogui Pill(左归丸) and Yougui Pill(右归丸) on the Expression of Brain-Derived Neurotrophic Factor and Cyclic Adenosine Monophosphate/Protein Kinase A Signaling Transduction Pathways of Axonal Regeneration in Model Rats with Experimental Autoimm

Objective：To study the effects of Zuogui Pill（左归丸,ZGP）and Yougui Pill（右归丸,YGP）on the expressions of brain-derived neurotrophic factor（BDNF）and cyclic adenosine monophosphate（cAMP）/protein kinase A（PKA）signaling of axonal regeneration in the Lewis rats with experimental autoimmune encephalomyelitis（EAE）,in order to explore the possible mechanism of ZGP and YGP on promoting axonal regeneration.Methods：The rats were randomly divided into normal control（NC）,model（MO）,prednisone acetate（PA）,ZGP and YGP groups.The EAE model of rat was established by injecting antigen containing myelin basic protein（MBP）68-86.The brain and spinal cord were harvested on the 14th and 28th day postimmunization（PI）,the protein and mRNA expression of BDNF and PKA in the brain and spinal cord of rats were detected by Western blot analysis and real-time quantitative polymerase chain reaction（PCR）,and the cAMP levels were detected by using enzyme-immunoassay method.Results：（1）On the 28th day PI,the mRNA expression of BDNF in brain white matter and spinal cord of rats in ZGP and YGP groups were up-regulated,especially in YGP group（P〈0.05 or P〈0.01）.（2）On the 14th day PI,the cAMP levels in brain white matters significantly increased in PA and YGP groups compared with MO group（P〈0.05 or P〈0.01）,and the cAMP level in YGP group was higher than that in ZGP group（P〈0.05）.The cAMP level in spinal cord also significantly increased in YGP group compared with MO,PA and ZGP groups,respectively（P〈0.01）.（3）On the 14th day PI,the PKA expression in spinal cord of rats in ZGP group was significantly decreased compared with MO and YGP groups,respectively（P〈0.05）.（4）On the 28th day PI,there was a positive correlation between cAMP and PKA expression in the brain white matter of YGP rats.Conclusions：The results suggest that ZGP and YGP may promote axonal regeneration by modulating cAMP/PKA signal transduction pathway,but the targets of molecular mechanism of ZGP may be different from those of YGP.

Research on the Mechanism of Zuogui Pill(左归丸)and Yougui Pill(右归丸)in Promoting Axonal Regeneration in Model Rats of Autoimmune Encephalomyelitis

Objective：To study the molecular mechanism of Zuogui Pill（左归丸,ZGP） and Yougui Pill（右归丸,YGP） on axonal regeneration in rats with experimental autoimmune encephalomyelitis（EAE）.Methods： EAE rat model was established by bilateral rear pedes subcutaneous injection of antigen made by mixing myelin basic protein（MBP） and complete Freud＇s adjuvant（CFA） in the volume ratio of 1：1.The pathological changes of axonal injury and regeneration in the brain and the spinal cord were observed on the 14th（the acute stage） and the 28th day（the remission stage） after modeling,with hematoxylin-eosin（HE） staining,silver stain, and immunohistochemical staining.The rats treated with prednisone acetate were taken as controls.Results： Observation under the light microscope with HE staining showed a sleeve-like change in rats＇ cerebrospinal parenchyma with inflammatory cell infiltration around the small vessels and neuronic denaturation,while silver staining showed excessive tumefaction and abscission of axon,and immunohistochemical analysis showed decreasing of nerve growth factor（NGF） expression at the acute stage of EAE,which was even more remarkable at the remission stage,showing significant difference as compared with the normal control（P〈0.05）. And the expressions of Nogo A,an axon growth inhibitor,and its receptor（Nogo-66 receptor,Ng R） were significantly higher than those in the normal control at the acute stage（P〈0.01）.However,after the intervention of ZGP and YGP,the pathological changes and axon damage in rats＇ brain and spinal cord were much more alleviated,and the NGF expression was significantly higher than that in the model group at the acute stage （P〈0.05）.The expression of NGF was even stronger during the remission stage,and a better effect was shown by YGP.As for Nogo A and Ng R expressions,they were significantly lower than those in the model group at the acute stage（P〈0.05）,but a better effect was shown by ZGP.Conclusions：ZGP and YGP can prevent axonal injury and promote the axonal regeneration in rats of EAE,and the possible mechanism is to increase the expression of NGF and reduce the expression of Nogo A and its receptor.However,some differences are observed between the two Chinese preparations in their acting times and points,which provides a certain basis for revealing the modern connotation of the Chinese medicine theory on tonifying Shen（肾）-yin and Shen-yang.

Effect of Zuogui Pill(左归丸) on Monoamine Neurotransmitters and Sex Hormones in Climacteric Rats with Panic Attack

Objective： To explore the effects of Chinese medicine prescription Zuogui Pill（左归丸, ZGP） on monoamine neurotransmitters and sex hormones in climacteric rats with induced panic attacks. Methods： Forty-eight climacteric female rats were randomized into 6 groups with 8 rats in each group： the control group, the model group, the low-, medium- and high-dose ZGP groups and the alprazolam group. Rats in the low-, medium- and high-dose ZGP groups were administered 4.725, 9.45, or 18.9 g/kg ZGP by gastric perfusion, respectively. The alprazolam group was treated by gastric perfusion with 0.036 mg/kg alprazolam. The control and model groups were treated with distilled water. The animals were pretreated once daily for 8 consecutive weeks. The behaviors of rats in the open field test and the elevated T-maze（ETM） were observed after induced panic attack, and the levels of brain monoamine neurotransmitters and the plasma levels of sex hormones were measured. Results： Compared with the control group, the mean ETM escape time and the levels of 5-hydroxytryptamine（5-HT） and noradrenalin（NE） of the model group were significantly reduced（P〈0.05）, Compared with the model group, the mean ETM escape time and the 5-HT and NE levels of all the ZGP groups increased significantly（P〈0.05 or P〈0.01）. However, no significant difference was observed in the levels of sex hormones between the groups. Conclusion： Pretreatment with ZGP in climacteric rats may improve the behavior of panic attack, which may be related to increased 5-HT and NE in the brain.