Study on analgesic effect of Shentong Zhuyu Decoction in neuropathic pain rats by network pharmacology and RNA-Seq.

ETHNOPHARMACOLOGICAL RELEVANCE: Shentong Zhuyu Decoction (STZYD) is a traditional prescription for promoting the flow of Qi and Blood which is often used in the treatment of low back and leg pain clinicall with unclear mechanism. Neuropathic pain (NP) is caused by disease or injury affecting the somatosensory system. LncRNAs may play a key role in NP by regulating the expression of pain-related genes through binding mRNAs or miRNAs sponge mechanisms. AIM OF THE STUDY: To investigate the effect and potential mechanism of STZYD on neuropathic pain. METHODS: Chronic constriction injury (CCI) rats, a commonly used animal model, were used in this study. The target of STZYD in NP was analyzed by network pharmacology, and the analgesic effect of STZYD in different doses (H-STZYD, M-STZYD, L-STZYD) on CCI rats was evaluated by Mechanical withdrawal thresholds (MWT) and thermal withdrawal latency (TWL). Meanwhile, RNA-seq assay was used to detect the changed mRNAs and lncRNAs in CCI rats after STZYD intervention. GO analysis, KEGG pathway analysis, and IPA analysis were used to find key target genes and pathways, verified by qPCR and Western Blot. The regulatory effect of lncRNAs on target genes was predicted by co-expression analysis and ceRNA network construction. RESULTS: We found that STZYD can improve hyperalgesia in CCI rats, and H-STZYD has the best analgesic effect. The results of network pharmacological analysis showed that STZYD could play an analgesic role in CCI rats through the MAPK/ERK/c-FOS pathway. By mRNA-seq and lncRNA-seq, we found that STZYD could regulate the expression of Cnr1, Cacng5, Gucy1a3, Kitlg, Npy2r, and Grm8, and inhibited the phosphorylation level of ERK in the spinal cord of CCI rats. A total of 27 lncRNAs were associated with the target genes and 30 lncRNAs, 83 miRNAs and 5 mRNAs participated in the ceRNA network. CONCLUSION: STZYD has the effect of improving hyperalgesia in CCI rats through the MAPK/ERK/c-FOS pathway, which is related to the regulation of lncRNAs to Cnr1 and other key targets.

[UPLC fingerprint and determination of five components of substance benchmark of classical prescription Shentong Zhuyu Decoction].

Fingerprints of 18 batches of substance benchmark of Shentong Zhuyu Decoction(SZD) were established by UPLC under the following conditions: Waters Sun Fire C_(18) column(3.0 mm×150 mm, 3.5 µm), column temperature of 35 ℃, gradient elution with mobile phase of acetonitrile(A)-0.1% phosphoric acid aqueous solution(B) at the flow rate of 0.4 mL·min~(-1), and detection by wavelength switching. A total of 16 common peaks were identified. The similarities among the fingerprints were calculated by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 Edition) and the result showed they were in the range of 0.911-0.988. Based on the 16 common peaks, cluster analysis(CA), principal component analysis(PCA), and partial least square discriminant analysis(PLS-DA) all categorized the 18 batches of samples into two groups(S1, S2, S5-S8, S14, and S17 in one group, and S1, S2, S5-S8, S14, and S17 in another), and 11 most influential components were screened. Five known components with great difference among samples(hydroxysafflor yellow A, ferulic acid, benzoic acid, ecdysone, and ammonium glycyrrhizinate) were determined. The combination of multi-component content determination and fingerprints can reflect the overall cha-racteristics of the primary standards of SZD, which is simple, feasible, reproducible, and stable. This study can serve as a reference for the quality control of the primary standards of SZD.

Metabolomics combined with network pharmacology to study the mechanism of Shentong Zhuyu decoction in the treatment of rheumatoid arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Shentong Zhuyu decoction (STZYD) was first recorded in the classic of "Yilin Gaicuo" written by Wang Qingren, and recognized by the Chinese National Administration of Traditional Chinese Medicine as one of the 100 classic formulas. The formula has been widely used in the treatment of rheumatoid arthritis (RA) with significant clinical effects. However, its mechanism of action is not completely clear. AIM OF THE STUDY: This study aimed to explore the mechanism of STZYD in the treatment of RA by network pharmacology and metabolomics. MATERIALS AND METHODS: The effects of STZYD anti-RA were investigated by paw swelling, arthritis score, cytokine level, histopathological and micro-CT analysis in adjuvant-induced arthritis (AIA) rats. The chemical constituents of STZYD and absorbed constituents in AIA rat serum were analyzed by UPLC-Q-Exactive MS/MS. Based on the characterized chemical components, the network pharmacology was used to find potential targets and signaling pathways of STZYD in RA treatment. Meanwhile, the predicted pathway was determined by the Western blot (WB). Subsequently, non-targeted metabolomics of serum was performed to analyze metabolic profiles, potential biomarkers, and metabolic pathways of STZYD in the treatment of RA based on LC-MS technology. RESULTS: STZYD significantly alleviated RA symptoms by improving paw redness and swelling, bone and cartilage damage, synovial hyperplasia, and infiltration of inflammatory cells, and decreased the generation of pro-inflammatory cytokines IL-1ß, IL-6, IL-17A and TNF-α in AIA rats. Totally, 59 chemical components of STZYD and 24 serum migrant ingredients were identified. A total of 655 genes of potential bioactive components in STZYD and 1025 related genes of RA were obtained. TNF signaling pathway was considered to one of the main signaling pathways of STZYD anti-RA by KEGG analysis, including a wide range intracellular signaling pathways. NF-κB signaling pathway regulates inflammation and immunity in the TNF signaling pathway. STZYD markedly inhibited the expression of NF-κB signaling pathway. Ten potential biomarkers were found in metabolomics based on LC-MS technology. Alanine, aspartate and glutamate metabolism, arachidonic acid metabolism are the most related pathways of STZYD anti-RA. CONCLUSION: The study based on serum pharmacochemistry, network pharmacology and metabolomics indicated that STZYD can improve RA through regulating inflammation and immunity related pathways, and provided a new possibility for treatment of RA.

UPLC fingerprint and determination of five components of substance benchmark of classical prescription Shentong Zhuyu Decoction / 中国中药杂志

Fingerprints of 18 batches of substance benchmark of Shentong Zhuyu Decoction(SZD) were established by UPLC under the following conditions: Waters Sun Fire C_(18) column(3.0 mm×150 mm, 3.5 μm), column temperature of 35 ℃, gradient elution with mobile phase of acetonitrile(A)-0.1% phosphoric acid aqueous solution(B) at the flow rate of 0.4 mL·min~(-1), and detection by wavelength switching. A total of 16 common peaks were identified. The similarities among the fingerprints were calculated by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 Edition) and the result showed they were in the range of 0.911-0.988. Based on the 16 common peaks, cluster analysis(CA), principal component analysis(PCA), and partial least square discriminant analysis(PLS-DA) all categorized the 18 batches of samples into two groups(S1, S2, S5-S8, S14, and S17 in one group, and S1, S2, S5-S8, S14, and S17 in another), and 11 most influential components were screened. Five known components with great difference among samples(hydroxysafflor yellow A, ferulic acid, benzoic acid, ecdysone, and ammonium glycyrrhizinate) were determined. The combination of multi-component content determination and fingerprints can reflect the overall cha-racteristics of the primary standards of SZD, which is simple, feasible, reproducible, and stable. This study can serve as a reference for the quality control of the primary standards of SZD.

Integrated serum pharmacochemistry and network pharmacological analysis used to explore possible anti-rheumatoid arthritis mechanisms of the Shentong-Zhuyu decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Shentong-Zhuyu decoction (STZYD) has been recognized by the Chinese National Administration of Traditional Chinese Medicine (TCM) as a classic TCM formula. Use of STZYD has shown a satisfactory clinical therapeutic outcome for rheumatoid arthritis (RA); despite this, its bioactive chemical composition and relevant mechanism(s) of this action have not been clearly elucidated. AIM OF THE STUDY: To explore the bioactive chemical composition of STZYD used for RA treatment and its possible mechanism(s) of action. MATERIALS AND METHODS: Serum pharmacochemistry mediated by the UPLC-Q-Exactive MS/MS method was employed to identify the absorbed phytochemical compounds in serum derived from STZYD, which were commonly considered as the potential bioactive compounds. And then, these components were used to construct a compound-target network for RA using a network pharmacology approach, to predict the possible biological targets of STZYD along with potential signaling pathways. Afterwards, we established a Complete Freund's adjuvant (CFA)-induced RA rat model, and observed the anti-RA effect of STZYD by a series of indexes, including foot swelling, ankle diameter, arthritis score, morphological and radiographic analysis, serum inflammatory factors, and histopathological analysis of synovial tissues. Particularly, the predicted pathway by the combination of serum pharmacochemistry and network pharmacology was further validated using RT-qPCR, Western blot, and immunohistochemical analyses in animal experiment. RESULTS: Totally, 38 compounds derived from STZYD have been identified by serum sample analysis. Based on it, 387 genes related to these identified compounds in STZYD and 3807 genes related to RA were collected by network pharmacology. Critically, KEGG analysis indicated that the PI3K/AKT signaling pathway was recommended as one of the main pathway related to anti-RA effect of STZYD. Experimentally, STZYD significantly alleviated CFA-induced arthritis without any visible side-effects. Compared to the RA model group without any treatment, the treatment of STZYD significantly reduced the expression of both mRNA and protein targets in the PI3K/AKT signaling pathway. Furthermore, this result was also corroborated by immunohistochemistry analysis. All these studies could effectively corroborate the predicted result as above, suggested that the feasibility of this integrated strategy. CONCLUSION: This study provided a useful strategy to identify bioactive compounds and the potential mechanisms for TCM formula by integrating serum pharmacochemistry and network pharmacology.

[Efficacy and safety of Shentong Zhuyu Decoction for lumbar disc herniation: systematic review and Meta-analysis].

To evaluate the clinical efficacy and safety of Shentong Zhuyu Decoction in the treatment of lumbar disc herniation, in order to provide evidence for its clinical application. PubMed, Cochrane Library, EMbase, CNKI, WanFang, VIP, CBM and Clinical Trials.gov databases were sysmatically retrieved. Two researchers independently searched, screened and extracted data. Randomized controlled trials in line with the inclusion criteria were included in the study. The article quality was assessed with the bias risk tool in the Cochrane Handbook. Meta-analysis was performed using RevMan 5.3. A total of 604 articles were obtained from the retrieval, and 824 patients in 9 studies were finally selected through screening. The course of treatment was about 1 month, and all of the studies were completed in China. Meta-analysis showed that the efficacy of Shentong Zhuyu Decoction in the treatment of lumbar disc herniation was better than that of the control group(RR=1.19, 95%CI[1.12, 1.26], P<0.000 01), including the comparison with Yaotongning Capsules(RR=1.10, 95%CI[1.02, 1.19], P=0.01) and diclofenac sodium tablets(RR=1.26, 95%CI[1.11, 1.44], P=0.000 4). In VAS pain score, Shentong Zhuyu Decoction had also obvious advantages(MD=-3.70, 95%CI[-6.31,-1.09], P=0.005). In addition, the number of adverse events in the Shentong Zhuyu Decoction group was lower than that in the control group. Shentong Zhuyu Decoction can effectively alleviate discomfort symptoms of patients with LDH. It has an obvious clinical efficacy and less adverse reactions. However, restricted by the quality of the included literatures, prospective, multi-center, large-sample-size randomized controlled trials are still needed to further improve the clinical evidence of the effect of Shentong Zhuyu Decoction in the treatment of LDH.

Study on optimization of UPLC fingerprint of classic prescription of Shentong Zhuyu Decoction based on principle of information entropy maximization / 中草药

Objective: To optimize the condition of the fingerprint of the classic prescription Shentong Zhuyu Decoction (SZD) by the principle of information entropy maximization. Methods: The chromatographic fingerprint was obtained with Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) and gradient eluted with acetonitrile and 0.4% phosphoric acid aqueous solution; The flow rate was 0.6 mL/min, and the column temperature was maintained at 40 ℃; The detection wavelengths were set at 190-400 nm, and the injection volume was 1 μL, and calculate the information entropy to optimize the wavelength of maximizing the information entropy of the fingerprint of SZD, under the optimal wavelength, investigate the mobile phase, column temperature, flow rate, sample volume, select the optimal conditions based on the principle of maximizing the information entropy, and carry out the fingerprint study of 15 batches of SZD under the optimal conditions. Results : Under the principle of information entropy maximization, the optimal conditions of the fingerprint of SZD were as follows: the detection wavelength was 235 nm, and gradient eluted with acetonitrile and 0.4% phosphoric acid aqueous solution; The flow rate was 0.4 mL/min, and the column temperature was maintained at 40 ℃, the injection volume was 3 μL; The absolute and relative entropy was the largest. The total statistical moment similarity of 15 batches of SZD fingerprints ranged from 0.882 8 to 0.995 3. The RSD values of the total first-order moment and information entropy of the total amount were 5.40% and 2.25% respectively, all of which were less than 10%, indicating that the imprinting effect of the overall chemical structure of the multi-component system of traditional Chinese medicine was stable. Conclusion: When the number and area of chromatographic peaks reach the best characterization, the information entropy is the largest. At this time, the number of peaks is high, the shape of peaks is better, the resolution is good, the number of theoretical plates is higher, and the best condition of fingerprint can be determined. The principle of information entropy maximization can provide scientific basis for fingerprint optimization of traditional Chinese medicine and its compound prescription, and lay a foundation for quality control of classical formulas.

Efficacy and safety of Shentong Zhuyu Decoction for lumbar disc herniation: systematic review and Meta-analysis / 中国中药杂志

To evaluate the clinical efficacy and safety of Shentong Zhuyu Decoction in the treatment of lumbar disc herniation, in order to provide evidence for its clinical application. PubMed, Cochrane Library, EMbase, CNKI, WanFang, VIP, CBM and Clinical Trials.gov databases were sysmatically retrieved. Two researchers independently searched, screened and extracted data. Randomized controlled trials in line with the inclusion criteria were included in the study. The article quality was assessed with the bias risk tool in the Cochrane Handbook. Meta-analysis was performed using RevMan 5.3. A total of 604 articles were obtained from the retrieval, and 824 patients in 9 studies were finally selected through screening. The course of treatment was about 1 month, and all of the studies were completed in China. Meta-analysis showed that the efficacy of Shentong Zhuyu Decoction in the treatment of lumbar disc herniation was better than that of the control group(RR=1.19, 95%CI[1.12, 1.26], P<0.000 01), including the comparison with Yaotongning Capsules(RR=1.10, 95%CI[1.02, 1.19], P=0.01) and diclofenac sodium tablets(RR=1.26, 95%CI[1.11, 1.44], P=0.000 4). In VAS pain score, Shentong Zhuyu Decoction had also obvious advantages(MD=-3.70, 95%CI[-6.31,-1.09], P=0.005). In addition, the number of adverse events in the Shentong Zhuyu Decoction group was lower than that in the control group. Shentong Zhuyu Decoction can effectively alleviate discomfort symptoms of patients with LDH. It has an obvious clinical efficacy and less adverse reactions. However, restricted by the quality of the included literatures, prospective, multi-center, large-sample-size randomized controlled trials are still needed to further improve the clinical evidence of the effect of Shentong Zhuyu Decoction in the treatment of LDH.

Effects of Chinese Medicine on Chemotherapy-induced Peripheral Neuropathy:A Randomized, Double-blind, Placebo-controlled Clinical Trial / 中国康复理论与实践

Objective To investigate the efficacy of a Chinese Medicine formula on chemotherapy-induced peripheral neuropathy (CIPN).Methods From April,2013 to December,2014,56 patients were randomized blindly to receive granulas of Chinese Medicine of modified Huangqi-Guizhi Wuwu and Shentong-Zhuyu(treatment group,n=28)or placebo(control group,n=28),for a month.They were as-sessed with Number Rating Scale (NRS) of symptoms of CIPN, National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0, and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-30 (EORTC QLQ-C30)before,and four weeks,eight weeks and twelve weeks after treatment.Results The score of NRS(F>9.518,P<0.01) and grade of NCI-CTCAE(Z>3.887,P<0.05)improved in the treatment group compared with those in the control group eight weeks and twelve weeks after treatment.The scores of EORTC QLQ-C30 also improved twelve weeks after treatment, but only significantly in the symptom of fatigue(t=-2.971,P<0.01).Conclusion The Chinese Medicine can release the symptoms of CIPN,decrease the grade of neuro-toxicity,and may improve the quality of life somewhat.

Effects of Ultrafiltration Process on Activating Blood and Removing Stasis Efficacy of Shentong Zhuyu Decoction / 中国中医药信息杂志

Objective To study the effects of ultrafiltration process on activating blood and removing blood stasis efficacy ofShentong Zhuyu Decoction; To investigate the feasibility of applying ultrafiltration technology in purifying Shentong Zhuyu Decoction.Methods The mice micro artery and vein diameter, clotting time and opening capillary of auricle microcirculation of mice were used as indexes to observe the effects of different ultrafiltration process on activating blood and removing blood stasis efficacy ofShentong Zhuyu Decoction.ResultsShentong Zhuyu Decoction showed satisfying efficacy of activating blood and removing blood stasis. There was no significant difference between the non-ultrafiltration process and ultrafiltration processed by 20 and 50 nm ultrafiltration membranes.Conclusion Ultrafiltration technology can be applied to purifying Shentong Zhuyu Decoction, and the membrane pore size must be more than 20 nm.