Integration of network pharmacology with experimental validation to reveal the mechanism of action of Longdan Xiegan Decoction against HSV2 infection and determine its effective components.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese Medicine (TCM) has made enormous strides recently in the discovery of anti-herpes simplex virus (HSV) drugs under the guidance of TCM theory. Longdan Xiegan Decoction (LXD), a formulation recorded in the Pharmacopoeia of the People's Republic of China, has proved to be effective against HSV infection. However, its effective components and action mechanism remain unclear. AIM OF THE STUDY: To investigate the effective components and mechanisms of LXD in treating HSV infection based on network pharmacology and experimental validation. MATERIALS AND METHODS: The anti-HSV activities of key compounds predicted by network analysis were detected by antiviral tests. High-performance liquid chromatography (HPLC) was applied to identify the main components of the LXD aqueous extract. Time-of-addition assay and infectivity inhibition reversibility assay were conducted to identify the potential antiviral mechanisms of licochalcone B (LCB). Additionally, we assessed the antiviral effect of LCB in vivo by use of body weight, viral load, histological analysis, and scoring of genital lesions in an HSV2-infected mouse model. RESULTS: Our data demonstrated that some components exhibited significant anti-HSV1/2 activity in vitro, including quercetin, kaempferol, wogonin, formononetin, naringenin, baicalein, isorhamnetin, glabridin, licochalcone A, echinatin, oroxylin A, isoliquiritigenin, pinocembrin, LCB and acacetin. HPLC analysis showed that LCB was the main component of LXD aqueous extract. In vitro experiments revealed that LCB not only inactivated HSV2 particles, but also inhibited HSV2 multiplication through the inhibition of the phosphorylation of Akt and its downstream targets. In vivo experiments confirmed that LCB could significantly reduce viral titer, delay weight loss, and alleviate pathological changes in vaginal tissue in vaginal infection mouse models. CONCLUSION: LCB acted as the main component of LXD, with significant anti-HSV2 infection effects both in vivo and in vitro. This study provides additional evidence of the healing efficacy of LXD against HSV infection and presents an efficient analytical method for further investigation of the mechanisms of TCM in prevention and treatment of various diseases.

Clinical Observation on the Acupuncture at the Ghost Points Combined with Longdan Xiegan Decoction in the Treatment of Post-stroke Insomnia of Liver-Fire Disturbing the Heart Type / 广州中医药大学学报

Objective To observe the clinical efficacy of acupuncture on ghost points combined with Longdan Xiegan Decoction in the treatment of post-stroke insomnia of liver-fire disturbing the heart type.Methods Sixty patients with liver-fire disturbing the heart type of post-stroke insomnia were randomly divided into observation group and control group,with 30 patients in each group.The control group was given oral use of modified Longdan Xiegan Decoction,and the observation group was given acupuncture at the ghost points on the basis of the treatment of the control group.Both groups were treated for 4 weeks.After 1 month of treatment,the clinical efficacy of the two groups was evaluated,and the changes in the Pittsburgh Sleep Quality Index(PSQI)score and the Athens Insomnia Scale(AIS)score were observed before and after treatment in the two groups.The changes of Self-Rating Depression Scale(SDS)scores were compared before and after treatment in the two groups of patients.Results(1)After treatment,the PSQI scores of the two groups of patients were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the PSQI scores,with a statistically significant difference(P＜0.05).(2)After treatment,the AIS scores of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving the AIS scores,and the difference was statistically significant(P＜0.05).(3)After treatment,the SDS scores of patients in the two groups were significantly improved(P＜0.05),and the observation group was significantly superior to the control group in improving SDS scores,and the difference was statistically significant(P＜0.05).(4)The total effective rate of the observation group was 86.66%(26/30),while that of the control group was 76.66%(23/30).The efficacy of the observation group was superior to that of the control group,and the difference was statistically significant(P＜0.05).Conclusion Acupuncture at the ghost points combined with Longdan Xiegan Decoction for the treatment of liver-fire disturbing the heart type of post-stroke insomnia can significantly improve the clinical symptoms of the patients,and the therapeutic effect is remarkable.

Longdan Xiegan Decoction alleviates experimental autoimmune uveitis in rats by inhibiting Notch signaling pathway activation and Th17 cell differentiation.

This study aimed to investigate the dynamic effects of the traditional Chinese medicine compound Longdan Xiegan Decoction (LXD) on the inhibition of Notch signaling pathway activation and T helper (Th) cell differentiation in rats with experimental autoimmune uveitis (EAU). Based on a network pharmacology strategy, we conducted protein interaction network analysis to construct an active ingredient-disease treatment network. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were further used to screen out the possible signaling pathways regulated by LXD in the treatment of uveitis. In the subsequent functional studies, we established an EAU rat model and investigated the regulatory role of LXD in the Notch signaling pathway and Th cell differentiation in rats with EAU. Female Lewis rats were randomly divided into a normal control (NC) group, an EAU group, and an LXD group. After the induction of EAU, the ocular inflammation and pathological changes in the rats in each group were observed; for documentation, a scanning laser ophthalmoscope (SLO) was used to observe fundus inflammation on day 12 after immunization. Additionally, quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of Notch1, DLL4, IL-10 and IL-17A in the spleen, lymph nodes and ocular tissues of each group at 0, 6, 9, 12, 15 and 18 days after immunization. In addition, the dynamic frequencies of the CD4+, CD8+, Th17 and Treg cell subsets in the spleen, lymph nodes and ocular tissues were measured by flow cytometry. We found that the Notch signaling pathway was activated and the Th17 frequency was elevated in rats with EAU, leading to disrupted CD4+/CD8+ and Th17/Treg balance. The expression of Notch1, DLL4 and IL-17 mRNA and proteins in the EAU and LXD groups reached a peak on day 12, and then gradually decreased (all P < 0.05), and the ratios of the CD4+/CD8+ and Th17/Treg also peaked on day 12. However, after treatment with LXD, the expression of Notch1, DLL4 and IL-17 mRNA and proteins was significantly decreased (all P < 0.05), and the CD4+/CD8+ and Th17/Treg ratios significantly gradually returns to balance. LXD can efficiently inhibit Th17 cell differentiation, decrease inflammatory cytokine expression, and restore the CD4+/CD8+ and Th17/Treg balance by inhibiting the activation of the Notch signaling pathway in rats with EAU, thus effectively alleviating eye inflammation, protecting eye tissue structures, and positively regulating the immune state of the whole body and the intraocular microenvironment.

Inhibitory effects of Longdan Xiegan Decoction on Notch signaling in rats with experimental autoimmune uveitis / 眼科新进展

Objective To investigate the effects of Longdan Xiegan Decoction on the expression of Notch signaling-related genes in rats with experimental autoimmune uveitis (EAU).Methods Totally 24 Lewis rats were randomly divided into normal control group,EAU model group and Longdan Xiegan Decoction treatment group.A rat model of EAU was established in the EAU model group and Longdan Xiegan Decoction treatment group,followed by intragastrical administration of Longdan Xiegan Decoction in the latter group after successful modeling.On day 12 after different treatments,the spleen and lymph nodes were isolated from rats of all the three groups for the collection of CD4 + T ceils,and the levels of both Th17 and Treg cells were analyzed by flow cytometry to calculate the ratio of Th17 to Treg cells.Meanwhile the levels of Notch1,Notch2,Notch3,Notch4 mRNAs expression were monitored by qRT-PCR.Moreover,the expressions of Notch signaling-related proteins were further detected using ELISA technique.Results Flow cytometry showed increased Th17 cell level but decreased Treg cell level in the EAU model group when compared with the normal control group.However,after treatment with Longdan Xiegan Decoction,the Th17 level was decreased,whereas the Treg level was increased,and the ratio of Th17 to Treg gradually restored to equilibrium when compared to EAU model group.qRT-PCR results showed that the expression levels of Notch1,Notch2 and Notch4 mRNAs in Longdan Xiegan Decoction treatment group were significantly higher than those in the normal control group (all P ＜ 0.01),but lower than those in EAU model group on day 12 after treatment (all P ＜ 0.01),without Notch 3 expression in the spleen and lymph nodes.In addition,Notch 1,Notch 2 and Notch 4 protein levels of the spleen and lymph nodes in Longdan Xiegan Decoction treatment group showed the similar tendency as compared to those of Notch 1,Notch 2 and Notch 4 mRNAs (all P ＜ 0.01),and the expression of Notch 3 protein was still not observed.Conclusion Longdan Xiegan Decoction can effectively relieve the imbalance of Th17/Treg cells in EAU rats,and its mechanism may involve the differentiation of naive CD4+ T cells into Th17 and Treg cells,which is regulated by Notch signaling.

[Effect of chloroform extracts from Longdan Xiegan decoction in inhibiting hydrolytic enzyme activity of Candida albicans isolated from VVC patients].

To investigate the inhibitory effect and mechanism of chloroform extracts from Longdan Xiegan decoction(CELX) against hydrolytic enzymes activity of Candida albicans isolated from vulvovaginal candidiasis(VVC) patients. Secreted aspartyl proteinase(Sap), phospholipase(PL) and lipase(Lip) positive strains were identified from 15 strains of C. albicans with milk culture medium, egg yolk culture medium and tween-80 medium, respectively. Then, the activities of Sap, PL, and Lip were detected in the above media. qRT-PCR was used to detect the changes in gene expressions of aspartic protease(SAP1-7,10), phospholipase B(PLB1-2) and lipase(LIP3-6). Secreted aspartyl proteinase and phospholipase of 15 VVC clinical strains were positive, and lipase of 11 strains were positive. Compared with the blank control group, the drug CELX-containing medium(milk medium, egg yolk culture medium, tween-80 medium) experiment showed that the sedimentation of colonies decreased gradually in each culture medium with the increase of CELX dose. When the concentration of CELX was 256 mg•L⁻¹, the colony almost disappeared, which indicated the enzyme activity was significantly weakened. The results of qRT-PCR showed that SAP1, SAP2, SAP3, SAP4, SAP7, SAP9 and SAP10 were down-regulated by 62%, 55%, 62%, 84%, 61%, 51%, 68%, respectively, except for SAP5 and SAP6; and PLB1, LIP3, LIP4, LIP6 were down-regulated by 67%, 51%, 54%, 55%, respectively. The findings suggested that CELX may inhibit the activities of Sap, PL, and Lip, which are important virulence factors of C. albicans.

Effect of chloroform extracts from Longdan Xiegan decoction in inhibiting hydrolytic enzyme activity of Candida albicans isolated from VVC patients / 中国中药杂志

To investigate the inhibitory effect and mechanism of chloroform extracts from Longdan Xiegan decoction(CELX) against hydrolytic enzymes activity of Candida albicans isolated from vulvovaginal candidiasis(VVC) patients. Secreted aspartyl proteinase(Sap), phospholipase(PL) and lipase(Lip) positive strains were identified from 15 strains of C. albicans with milk culture medium, egg yolk culture medium and tween-80 medium, respectively. Then, the activities of Sap, PL, and Lip were detected in the above media. qRT-PCR was used to detect the changes in gene expressions of aspartic protease(SAP1-7,10), phospholipase B(PLB1-2) and lipase(LIP3-6). Secreted aspartyl proteinase and phospholipase of 15 VVC clinical strains were positive, and lipase of 11 strains were positive. Compared with the blank control group, the drug CELX-containing medium(milk medium, egg yolk culture medium, tween-80 medium) experiment showed that the sedimentation of colonies decreased gradually in each culture medium with the increase of CELX dose. When the concentration of CELX was 256 mg•L⁻¹, the colony almost disappeared, which indicated the enzyme activity was significantly weakened. The results of qRT-PCR showed that SAP1, SAP2, SAP3, SAP4, SAP7, SAP9 and SAP10 were down-regulated by 62%, 55%, 62%, 84%, 61%, 51%, 68%, respectively, except for SAP5 and SAP6; and PLB1, LIP3, LIP4, LIP6 were down-regulated by 67%, 51%, 54%, 55%, respectively. The findings suggested that CELX may inhibit the activities of Sap, PL, and Lip, which are important virulence factors of C. albicans.

Treatment of Senile Post-herpetic Neuralgia with Integrated Traditional Chinese and Western Medicine / 国际中医中药杂志

Objective To observe clinical effect of treating post-herpetic neuralgia of the old with integrated traditional Chinese and western medicine. Methods 98 patients with postherpetic neuraligia were randomly divided into a treatment group and a control group. The treatment group was treated with Longdan Xiegan decoction plus aciclovir, and the control group was treated with aciclovir. A three months' follow-up was performed after the treatment to observe clinical effect of the treatment group and the control group and their difference. Results The therapeutic effect in the treatment group was significantly better than the control group, with P＜0.01.Conclusion Integrated traditional Chinese and western medicine therapy has better effect in treating senile herpes zoster than exclusive western medicine therapy.

Effect of Chinese Herbal Medicine Compatibility on Aristolochic Acid Content in Caulis Aristolochiae Manshuriensis / 广州中医药大学学报

【Objective】To compare aristolochic acid(AA)content in Caulis Aristolochiae Manshuriensis(CAM)and in the separated prescriptions of Longdan Xiegan Decoction(LXD),and to explore the effect of Chinese herbal medicine compatibility on AA content.【Methods】According to the herbs percentage compared to LXD,the herbs samples were named after: CAM group,LXD group,CAM group with heat-clearing herbs,CAM group with yin-nourishing herbs,CAM group with herbs for promoting urination,CAM group with Radix Glycyrrhizae,and LXD group with CAM removed.The AA content was determined by reverse phase HPLC.The chromatographic conditions were as follows: C18 Diamonsiltm analytical column,gradient eluted with a mixture of methyl alcohol,water and acetic acid(volume proportion as 74:24:1),and detection wavelength at 315 nm.【Results】AA content was the lowest in LXD group,and was lower in CAM group with yin-nourishing herbs than that in CAM group.【Conclusion】Subjecting to the theory of Chinese herbal medicine compatibility,it is feasible to reduce the CAM toxicity by composing a prescription with proper proportion of herbs.