Peroxide derivatives as SARS-CoV-2 entry inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Host cell invasion is mediated by the interaction of the viral spike protein (S) with human angiotensin-converting enzyme 2 (ACE2) through the receptor-binding domain (RBD). In this work, bio-layer interferometry (BLI) was used to screen a series of fifty-two peroxides, including aminoperoxides and bridged 1,2,4 - trioxolanes (ozonides), with the aim of identifying small molecules that interfere with the RBD-ACE2 interaction. We found that two compounds, compound 21 and 29, exhibit the activity to inhibit RBD-ACE2. They are further demonstrated to inhibit SARS-CoV-2 cell entry, as shown in pseudovirus assay and experiment with authentic SARS-CoV-2. A comprehensive in silico analysis was carried out to study the physicochemical and pharmacokinetic properties, revealing that both compounds have good physicochemical properties as well as good bioavailability. Our results highlight the potential of small molecules targeting RBD inhibitors as potential therapeutic drugs for COVID-19.

Mutant p53R211* ameliorates inflammatory arthritis in AIA rats via inhibition of TBK1-IRF3 innate immune response.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune inflammation disease characterized by imbalance of immune homeostasis. p53 mutants are commonly described as the guardian of cancer cells by conferring them drug-resistance and immune evasion. Importantly, p53 mutations have also been identified in RA patients, and this prompts the investigation of its role in RA pathogenesis. METHODS: The cytotoxicity of disease-modifying anti-rheumatic drugs (DMARDs) against p53 wild-type (WT)/mutant-transfected RA fibroblast-like synoviocytes (RAFLSs) was evaluated by MTT assay. Adeno-associated virus (AAV) was employed to establish p53 WT/R211* adjuvant-induced arthritis (AIA) rat model. The arthritic condition of rats was assessed by various parameters such as micro-CT analysis. Knee joint samples were isolated for total RNA sequencing analysis. The expressions of cytokines and immune-related genes were examined by qPCR, ELISA assay and immunofluorescence. The mechanistic pathway was determined by immunoprecipitation and Western blotting in vitro and in vivo. RESULTS: Among p53 mutants, p53R213* exhibited remarkable DMARD-resistance in RAFLSs. However, AAV-induced p53R211* overexpression ameliorated inflammatory arthritis in AIA rats without Methotrexate (MTX)-resistance, and our results discovered the immunomodulatory effect of p53R211* via suppression of T-cell activation and T helper 17 cell (Th17) infiltration in rat joint, and finally downregulated expressions of pro-inflammatory cytokines. Total RNA sequencing analysis identified the correlation of p53R211* with immune-related pathways. Further mechanistic studies revealed that p53R213*/R211* instead of wild-type p53 interacted with TANK-binding kinase 1 (TBK1) and suppressed the innate immune TBK1-Interferon regulatory factor 3 (IRF3)-Stimulator of interferon genes (STING) cascade. CONCLUSIONS: This study unravels the role of p53R213* mutant in RA pathogenesis, and identifies TBK1 as a potential anti-inflammatory target.

Cyclophilin D-mediated angiotensin II-induced NADPH oxidase 4 activation in endothelial mitochondrial dysfunction that can be rescued by gallic acid.

Vascular endothelial dysfunction plays a central role in the most dreadful human diseases, including stroke, tumor metastasis, and the coronavirus disease 2019 (COVID-19). Strong evidence suggests that angiotensin II (Ang II)-induced mitochondrial dysfunction is essential for endothelial dysfunction pathogenesis. However, the precise molecular mechanisms remain obscure. Here, polymerase-interacting protein 2 (Poldip 2) was found in the endothelial mitochondrial matrix and no effects on Poldip 2 and NADPH oxidase 4 (NOX 4) expression treated by Ang II. Interestingly, we first found that Ang II-induced NOX 4 binds with Poldip 2 was dependent on cyclophilin D (CypD). CypD knockdown (KD) significantly inhibited the binding of NOX 4 to Poldip 2, and mitochondrial ROS generation in human umbilical vein endothelial cells (HUVECs). Similar results were also found in cyclosporin A (CsA) treated HUVECs. Our previous study suggested a crosstalk between extracellular regulated protein kinase (ERK) phosphorylation and CypD expression, and gallic acid (GA) inhibited mitochondrial dysfunction in neurons depending on regulating the ERK-CypD axis. Here, we confirmed that GA inhibited Ang II-induced NOX 4 activation and mitochondrial dysfunction via ERK/CypD/NOX 4/Poldip 2 pathway, which provide novel mechanistic insight into CypD act as a key regulator of the NOX 4/Poldip 2 axis in Ang II-induced endothelial mitochondrial dysfunction and GA might be beneficial in the treatment of wide variety of diseases, such as COVID-19, which is worthy further research.

Astragalus saponins and its main constituents ameliorate ductular reaction and liver fibrosis in a mouse model of DDC-induced cholestatic liver disease.

Cholestatic liver disease (CLD) is a chronic liver disease characterized by ductular reaction, inflammation and fibrosis. As there are no effective chemical or biological drugs now, majority of CLD patients eventually require liver transplantation. Astragali radix (AR) is commonly used in the clinical treatment of cholestatic liver disease and its related liver fibrosis in traditional Chinese medicine, however its specific active constituents are not clear. Total astragalus saponins (ASTs) were considered to be the main active components of AR. The aim of this study is to investigate the improvement effects of the total astragalus saponins (ASTs) and its main constituents in cholestatic liver disease. The ASTs from AR was prepared by macroporous resin, the content of saponins was measured at 60.19 ± 1.68%. The ameliorative effects of ASTs (14, 28, 56 mg/kg) were evaluated by 3, 5-Diethoxycarbonyl-1, 4-dihydrocollidine (DDC)-induced CLD mouse model. The contents of hydroxyproline (Hyp), the mRNA and protein expression of cytokeratin 19 (CK19) and α-smooth muscle actin (α-SMA) in liver tissue were dose-dependently improved after treatment for ASTs. 45 astragalus saponins were identified in ASTs by UHPLC-Q-Exactive Orbitrap HRMS, including astragaloside I, astragaloside II, astragaloside III, astragaloside IV, isoastragaloside I, isoastragaloside II, cycloastragenol, etc. And, it was found that ductular reaction in sodium butyrate-induced WB-F344 cell model were obviously inhibited by these main constituents. Finally, the improvement effects of astragaloside I, astragaloside II, astragaloside IV and cycloastragenol (50 mg/kg) were evaluated in DDC-induced CLD mice model. The results showed that astragaloside I and cycloastragenol significantly improved mRNA and protein expression of CK19 and α-SMA in liver tissue. It suggested that astragaloside I and cycloastragenol could alleviate ductular reaction and liver fibrosis. In summary, this study revealed that ASTs could significantly inhibit ductular reaction and liver fibrosis, and astragaloside I and cycloastragenol were the key substances of ASTs for treating cholestatic liver disease.

SNS-032 attenuates liver fibrosis by anti-active hepatic stellate cells via inhibition of cyclin dependent kinase 9.

Liver fibrosis is a common pathological process of all chronic liver diseases. Hepatic stellate cells (HSCs) play a central role in the development of liver fibrosis. Cyclin-dependent kinase 9 (CDK9) is a cell cycle kinase that regulates mRNA transcription and elongation. A CDK9 inhibitor SNS-032 has been reported to have good effects in anti-tumor. However, the role of SNS-032 in the development of liver fibrosis is unclear. In this study, SNS-032 was found to alleviate hepatic fibrosis by inhibiting the activation and inducing the apoptosis of active HSCs in carbon tetrachloride-induced model mice. In vitro, SNS-032 inhibited the activation and proliferation of active HSCs and induced the apoptosis of active HSCs by downregulating the expression of CDK9 and its downstream signal transductors, such phosphorylated RNA polymerase II and Bcl-2. CDK9 short hairpin RNA was transfected into active HSCs to further elucidate the mechanism of the above effects. Similar results were observed in active HSCs after CDK9 knockdown. In active HSCs with CDK9 knockdown, the expression levels of CDK9, phosphorylated RNA polymerase II, XIAP, Bcl-2, Mcl-1, and ɑ-SMA significantly decreased, whereas those of cleaved-PARP1 and Bax decreased prominently. These results indicated that SNS-032 is a potential drug and CDK9 might be a new prospective target for the treatment of liver fibrosis.

Rubus chingii Hu. unripe fruits extract ameliorates carbon tetrachloride-induced liver fibrosis and improves the associated gut microbiota imbalance.

BACKGROUND: The unripe fruits of Rubus chingii Hu. ("Fu-peng-zi" in Chinese) is a well-known herbal tonic in traditional Chinese medicine (TCM) for tonifying liver and kidney. However, little is known regarding its therapeutic efficacy against liver fibrosis and the underlying mechanism. METHODS: The current research aims to explore the potential of Rubus chingii Hu. unripe fruits extract (RF) in the treatment of liver fibrosis and explore the underlying mechanism. RF was administered (450 and 900 mg·kg- 1 of body weight per day) orally to male C57BL/6 mice with CCl4-induced liver fibrosis for 3 weeks. The histopathological changes and fibrosis stage in liver tissue were assessed using hematoxylin and eosin (H&E) and Sirius red staining. The distribution of α-SMA and Col1A1 in the liver was analyzed to determine the hepatic stellate cells (HSCs) activation using immunohistochemistry and immunofluorescent analysis. Various biochemical markers in serum (ALT, AST) and liver (Hyp, IL1-ß, IL6, TNF-α and MCP-1) were observed to assess the liver's injury, fibrosis, and inflammation. In liver tissue, fibrosis-associated proteins including α-SMA, TGF-ß1, Smad2/3, p-Smad2/3, and Smad4 were detected through a Western blot assay. Pyrosequencing-based analysis of bacterial 16 S ribosomal RNA from variable regions V3-V4 of fecal samples characterized the gut microbiota. Spearman's rank correlation analysis was performed for the association between altered bacterial genera by RF and pharmacodynamics parameters. RESULTS: Three weeks of RF treatment can significantly lower liver inflammatory levels, pathological abnormalities, and collagen fibrous deposition in mice with CCl4-induced liver fibrosis. The expressions of α-SMA and Col1A1 were lowered by RF, while the expression levels of TGF-ß/Smads signaling pathway-related proteins, including TGF-ß1, p-Smad2/3, and Smad4, were dramatically decreased by RF. The RF treatment significantly increased or reduced 18 different bacterial species, restoring the CCl4-induced gut microbiota imbalance to the normal group's levels. According to correlation analysis, the bacterial genera Bifidobacterium and Turicibacter were the most significant in restoring CCl4-induced liver fibrosis. CONCLUSIONS: RF can reduce liver damage and delay the onset of liver fibrosis through modulating TGF-ß/Smads signaling pathway. Furthermore, RF's anti-liver fibrosis effect was related to balancing the gut microbial community, partly attained by increasing Bifidobacterium and Turicibacter in liver fibrosis.

Pharmacokinetics and anti-liver fibrosis characteristics of amygdalin: Key role of the deglycosylated metabolite prunasin.

BACKGROUND: Amygdalin (Amy) is a cyanoside and is one of the chief active ingredients in Persicae Semen, Armeniacae Semen Amarum, and Pruni Semen. Amy has extensive and remarkable pharmacological activities, including against anti-hepatic fibrosis. However, the pharmacokinetic and anti-liver fibrosis effects of Amy and its enzyme metabolite prunasin (Pru) in vivo have not been studied and compared, and studies on Pru are limited. PURPOSE: To investigate the pharmacokinetic characteristics and anti-liver fibrosis effect of Amy and its metabolite Pru in vivo and in vitro, and elucidate whether the metabolism of Amy in vivo for Pru is activated. METHODS: Pru was prepared from Amy via the enzymatic hydrolysis of ß-glucosidase, and isolated by silica gel column chromatography. An efficient and sensitive ultrahigh-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry was developed and validated to determine simultaneously Amy and Pru in rat plasma after dosing intravenously and orally for pharmacokinetic studies. The affinities of Amy and Pru for ß-glucosidase were compared by enzyme kinetic experiments to explain the possible reasons for the differences in pharmacokinetic behavior. In vitro, the inhibitory effects of Amy and Pru on hepatic stellate cell activation and macrophage inflammation on JS1 and RAW 264.7 cells were determined. In vivo, the ameliorative effects of Amy and Pru on liver fibrosis effects were comprehensively evaluated by CCl4-induced liver fibrosis model in mice. RESULTS: The standard curves of Amy and Pru in rat plasma showed good linearity within the concentration range of 1.31-5000.00 ng/ml, with acceptable selectivity, carry-over, detection limit and quantification limits, intra- and inter-day precision, accuracy, matrix effect, and stability. The Cmax and AUC(0-∞) of Pru (Cmax = 1835.12 ± 268.09 ng/ml, AUC(0-∞) = 103,913.17 ± 14,202.48 ng•min/ml) were nearly 79.51- and 66.22-fold higher than those of Amy (Cmax = 23.08 ± 5.08 ng/ml, AUC(0-∞) = 1569.22 ± 650.62 ng•min/ml) after the oral administration of Amy. The oral bioavailability of Pru (64.91%) was higher than that of Amy (0.19%). The results of enzyme hydrolysis kinetics assay showed that the Vmax and Km of Pru were lower than those of Amy in commercial ß-glucosidase and intestinal bacteria. In vitro cellular assays showed that Amy and Pru were comparable in inhibiting the NO production in the RAW264.7 cell supernatant and the mRNA expression of α-SMA and Col1A1 in JS1 cells. Amy and Pru were also showed comparable activity in ameliorating CCl4-induced liver fibrosis in mice. CONCLUSION: The pharmacokinetic characteristics of Amy and Pru in rat plasma were significantly different. After the separate gavage of Amy and Pru, Amy was absorbed predominantly as it's metabolite Pru, whereas Pru was absorbed predominantly as a prototype. The anti-liver fibrosis effects of Amy and its deglycosylated metabolite Pru were comparable in vivo and in vitro. The deglycosylated activated metabolite Pru of Amy plays an important role in anti-liver fibrosis. These findings will facilitate the further exploitation of Amy and Pru.

Research Progress in Chinese Medicine Preparations for Promoting Blood Circulation and Removing Blood Stasis for Cirrhotic Patients with Portal Vein Thrombosis Following Splenectomy.

This article presented an overview of the therapeutic effects of Chinese medicine (CM) preparations for promoting blood circulation and removing blood stasis for patients with portal vein thrombosis (PVT) after splenectomy. Based on published clinical researches of CM preparations for PVT after splenectomy in patients with cirrhotic portal hypertension (CPH), this paper evaluated the incidence of PVT, and explored potential active components and mechanisms of CM preparations. Safflower Yellow Injection, Danshen Injection () Danhong Injection (), and Compound Danshen Dropping Pill () achieved good curative effect alone or combined with anticoagulant therapy. In addition, Compound Biejia Ruangan Tablet () and Anluo Huaxian Pill () can also significantly improve the hemodynamic disorders of portal vein system in patients with cirrhosis. Considering the role of CM preparations in ameliorating the incidence of PVT after splenectomy in patients with CPH, we suggested that future research should provide more attention to CM alone or CM combined with anticoagulant for cirrhosis with PVT.

The Novel Chinese Medicine JY5 Formula Alleviates Hepatic Fibrosis by Inhibiting the Notch Signaling Pathway.

Advanced liver fibrosis can lead to cirrhosis, resulting in an accelerated risk of hepatocellular carcinoma and liver failure. Fuzheng Huayu formula (FZHY) is a traditional Chinese medicine formula treated liver fibrosis in China approved by a Chinese State Food and Drug Administration (NO: Z20050546), composed of Salvia Miltiorrhiza bge., Prunus davidiana (Carr.) Franch., cultured Cordyceps sinensis (BerK.) Sacc. Mycelia, Schisandra chinensis (Turcz.) Baill., Pinus massoniana Lamb., and Gynostemma pentaphyllum (Thunb.) Makino. However, the main active substances and mechanism of FZHY are unclear. The aim of this study is to identify a novel anti-fibrotic compound, which consists of the main active ingredients of FZHY, and investigate its mechanism of pharmacological action. The main active ingredients of FZHY were investigated by quantitative analysis of FZHY extracts and FZHY-treated plasma and liver in rats. The anti-fibrotic composition of the main active ingredients was studied through uniform design in vivo, and its mechanism was evaluated in carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced liver fibrosis models in rats and mice, and transforming growth factor beta 1-induced LX-2 cell activation model in vitro. A novel Chinese medicine, namely JY5 formula, consisting of salvianolic acid B, schisantherin A, and amygdalin, the main active ingredients of FZHY, significantly alleviated hepatic hydroxyproline content and collagen deposition in CCl4-and BDL-induced fibrotic liver in rats and mice. In addition, JY5 inhibited the activation of hepatic stellate cells (HSCs) by inactivating Notch signaling in vitro and in vivo. In this study, we found a novel JY5 formula, which exerted anti-hepatic fibrotic effects by inhibiting the Notch signaling pathway, consequently suppressing HSCs activation. These results provide an adequate scientific basis for clinical research and application of the JY5 formula, which may be a potential novel therapeutic candidate for liver fibrosis.

Identification of natural compounds as SARS-CoV-2 entry inhibitors by molecular docking-based virtual screening with bio-layer interferometry.

Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enter the host cells through the interaction between its receptor binding domain (RBD) of spike glycoprotein with angiotensin-converting enzyme 2 (ACE2) receptor on the plasma membrane of host cell. Neutralizing antibodies and peptide binders of RBD can block viral infection, however, the concern of accessibility and affordability of viral infection inhibitors has been raised. Here, we report the identification of natural compounds as potential SARS-CoV-2 entry inhibitors using the molecular docking-based virtual screening coupled with bilayer interferometry (BLI). From a library of 1871 natural compounds, epigallocatechin gallate (EGCG), 20(R)-ginsenoside Rg3 (RRg3), 20(S)-ginsenoside Rg3 (SRg3), isobavachalcone (Ibvc), isochlorogenic A (IscA) and bakuchiol (Bkc) effectively inhibited pseudovirus entry at concentrations up to 100 µM. Among these compounds, four compounds, EGCG, Ibvc, salvianolic acid A (SalA), and isoliensinine (Isl), were effective in inhibiting SARS-CoV-2-induced cytopathic effect and plaque formation in Vero E6 cells. The EGCG was further validated with no observable animal toxicity and certain antiviral effect against SARS-CoV-2 pseudovirus mutants (D614G, N501Y, N439K & Y453F). Interestingly, EGCG, Bkc and Ibvc bind to ACE2 receptor in BLI assay, suggesting a dual binding to RBD and ACE2. Current findings shed some insight into identifications and validations of SARS-CoV-2 entry inhibitors from natural compounds.

Hepatoprotective effect of genistein against dimethylnitrosamine-induced liver fibrosis in rats by regulating macrophage functional properties and inhibiting the JAK2/STAT3/SOCS3 signaling pathway.

BACKGROUND: Liver fibrosis is a dysregulated wound-healing process in response to diverse liver injuries, and an effective drug therapy is not yet available. Genistein, which is one of the most active natural flavonoids mainly derived from soybean products (e.g., Cordyceps sinensis mycelium), exhibits various biological effects, including hepatoprotective and anti-inflammatory properties. However, the anti-hepatic fibrosis mechanisms of genistein are poorly understood. The aim of our research is to explore the effect and the possible mechanism of genistein against liver fibrosis. MATERIALS AND METHODS: Cell counting kit-8, EdU, and flow cytometry assays were applied to evaluate the effects of genistein on cell viability, proliferation, and cell cycle arrest in human hepatic stellate cell (HSC) line LX2 cells. HSC activation was induced by transforming growth factor-ß1 in LX2 cells and liver fibrosis model was established by the intraperitoneal injection of dimethylnitrosamine (DMN) in rats to assess the anti-fibrosis effects of genistein in vivo and in vitro models. HSC activation was assessed by qRT-PCR, Western blot, immunohistochemistry, and immunofluorescent assay. Liver injury and collagen deposition were evaluated by histopathological assay, serum biochemistry, and hepatic hydroxyproline content assays. The mRNA expressions of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and inflammation related-factors were assessed by qRT-PCR assay. Furthermore, the functional properties of macrophage in the liver were assessed by immunohistochemistry assay. The expression levels of the JAK2/STAT3/SOCS3 signaling pathway related-protein were assessed by Western blot analysis. RESULTS: Genistein significantly inhibited cell viability and proliferation and induced cell cycle arrest at G0/G1 phase in LX2 cells, respectively. Furthermore, oral administration of genistein significantly ameliorated liver injury and the collagen deposition in rats with DMN-induced fibrosis model. Genistein suppressed the expression levels of HSC activation marker α-smooth muscle actin and collagen type I alpha 1 in vivo and in vitro. Genistein significantly decreased the mRNA expression levels of extracellular matrix degradation genes MMP2/9 and TIMP1 in rats. Genistein alleviated the mRNA expression levels of IL-1ß, IL-6, TNF-α, and MCP-1 and regulated the protein expressions of CD68, CD163, and CD206 in the liver. Moreover, genistein attenuated the expressions of p-JAK2/JAK2, p-STAT3/STAT3, and SOCS3 protein both in vivo and in vitro. CONCLUSION: Taken together, our results showed that genistein could be improved liver fibrosis both in vivo and in vitro, probably through regulating the functional properties of macrophage and inhibiting the JAK2/STAT3/SOCS3 signaling pathway.

Hepatoprotective effect of Xiayuxue decoction ethyl acetate fraction against carbon tetrachloride-induced liver fibrosis in mice via inducing apoptosis and suppressing activation of hepatic stellate cells.

CONTEXT: Xiayuxue decoction (XYXD), a traditional Chinese medicine, is used for treating liver disease. However, the potential active constituents and mechanisms are still unclear. OBJECTIVE: To explore the main active fraction extracts, active ingredients and possible mechanisms of XYXD for anti-hepatic fibrosis. MATERIALS AND METHODS: Different fractions including ethyl acetate fraction (EF) were prepared from XYXD. These fractions, especially EF, were used to evaluate cell viability, proliferation, cell cycle, cytotoxicity and activation in hepatic stellate cells (HSCs). Liver fibrosis model was established by CCl4 in C57BL/6 mice, and allocated to CCl4 group, XYXD group and EF group with normal mice as control. Further, mitochondrial apoptosis-related proteins of HSCs, destruction and angiogenesis of liver sinusoidal endothelial cells (LSECs) and active ingredients of EF were evaluated. RESULTS: The inhibition of proliferation, increase of S or/and G2/M phase population and suppression of α-SMA and COL-1 expression were obeserved in EF treated-JS1 and -LX2. Liver fibrosis-related indicators were improved by EF similar to XYXD in vivo. EF induced the apoptosis of HSCs in CCl4-induced fibrosis, and inhibited the expression of HSCs apoptosis pathway-related proteins (JNK and p38-MAPKs), and LSECs destruction and angiogenesis. Multiple ingredients (emodin, rhein, aloe-emodin, prunasin) in EF have shown inhibited the activation of JS1. DISCUSSION AND CONCLUSION: EF was the main active fraction extracts of XYXD, and the underlying mechanisms might relate to induction of HSCs apoptosis. Emodin, rhein, aloe-emodin and prunasin were main active ingredients of EF, which provides a potential drug for the treatment of liver fibrosis.

Metabolism, Transport and Drug-Drug Interactions of Silymarin.

Silymarin, the extract of milk thistle, and its major active flavonolignan silybin, are common products widely used in the phytotherapy of liver diseases. They also have promising effects in protecting the pancreas, kidney, myocardium, and the central nervous system. However, inconsistent results are noted in the different clinical studies due to the low bioavailability of silymarin. Extensive studies were conducted to explore the metabolism and transport of silymarin/silybin as well as the impact of its consumption on the pharmacokinetics of other clinical drugs. Here, we aimed to summarize and highlight the current knowledge of the metabolism and transport of silymarin. It was concluded that the major efflux transporters of silybin are multidrug resistance-associated protein (MRP2) and breast cancer resistance protein (BCRP) based on results from the transporter-overexpressing cell lines and MRP2-deficient (TR-) rats. Nevertheless, compounds that inhibit the efflux transporters MRP2 and BCRP can enhance the absorption and activity of silybin. Although silymarin does inhibit certain drug-metabolizing enzymes and drug transporters, such effects are unlikely to manifest in clinical settings. Overall, silymarin is a safe and well-tolerated phytomedicine.

[Effect of electrical stimulation at auricular points combined with sound masking on the expression of CREB, BDNF and TrkB in the auditory cortex of tinnitus rats].

OBJECTIVE: To explore the effect of electrical stimulation at auricular points (EAS) combined with sound masking on the expression of cAMP-response element binding protein (CREB), brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB) in the auditory cortex of tinnitus rats. METHODS: A total of 27 adult male SD rats were randomly divided into a control group, a model group and an EAS group. The rats in the model group and the EAS group were intervened with intraperitoneal injection of sodium salicylate to induce tinnitus model, while the rats in the control group were intervened with injection of 0.9% NaCl solution. After the model was successfully established, the rats in the EAS group were treated with electrical stimulation at "Shenmen" (TF4) and "Yidan" (CO11), combined with sound masking; the treatment was given once a day for 15 days. The gap prepulse inhibition of acoustic startle (GPIAS) and prepulse inhibition (PPI) testing were performed using the acoustic startle reflex starter package for rats. The expression of BDNF, TrkB, CREB and p-CREB in the auditory cortex of each group were measured with Western Blot analysis. RESULTS: ① Compared with the control group, the GPIAS values in 12 kHz, 16 kHz, 20 kHz and 28 kHz were significantly decreased in the model group (all P<0.05); compared with the model group, GPIAS values in 12 kHz, 16 kHz, 20 kHz and 28 kHz were significantly increased in the EAS group (all P<0.05). ② Compared with the control group, the expression of BDNF and p-CREB in the model group was significantly increased (P<0.01), and the expression of TrkB in the model group was significantly increased (P<0.05); the differences of expression of BDNF, TrkB, CREB and p-CREB between the model group and the EAS group had no statistics significance (all P>0.05). CONCLUSION: EAS could improve the GPIAS values of high-frequency background sound in tinnitus rats, which may be related with the upregulation of the BDNF/TrkB/CREB signaling pathway in the auditory cortex, leading to the reversion of the maladaptive plasticity.

[Effects of electrical stimulation at acupoints in the distribution area of auricular vagus nerve combined with sound masking method on auditory brainstem response and neurotransmitters of inferior colliculus in rats of tinnitus].

OBJECTIVE: To explore the effects of electrical stimulation at acupoints in the distribution area of auricular vagus nerve combined with sound masking on auditory brainstem response (ABR) and contents of neurotransmitters of γ-aminobutyric acid (γ-GABA), 5-hydroxytryptamine (5-HT) and acetyl choline (Ach) in inferior colliculus of tinnitus rats. METHODS: Twenty-four male adult SD rats were randomized into a control group, a model group, a 7-d treatment group and a 15-d treatment group. Except the control group, rats in the remaining groups were treated with intraperitoneal injection of 10% salicylate sodium at a dose of 350 mg/kg to establish tinnitus model. Rats in the control group were treated with injection of 0.9% NaCl. Rats in the 7-d treatment group and 15-d treatment group were treated with electrical stimulation at "Shenmen (TF4)" and "Yidan (CO11)" in the distribution area of auricular vagus nerve combined with sound masking, once a day, for 7 days and 15 days. The SigGenRP software of TDT system was applied to provide voice for single ear and collect the signal, and the voice threshold of ABR was tested. The levels of γ-GABA, 5-HT and Ach in inferior colliculus of rats were detected by enzyme linked immunosorbent assay (ELISA) and compared. RESULTS: Compared with the model group, the threshold values of ABR in 12 kHz and 16 kHz voice stimulation in the 7-d treatment group were significantly lower all P < 0.05); the threshold values of ABR from 4 kHz to 28 kHz voice stimulation in the 15-d treatment group were signally reduced (P < 0.05, P < 0.01), which was more significant than those in the 7-d treatment group. The level of γ-GABA in the model group was significantly lower than that in the control group (P < 0.05), and that in the 15-d treatment group was apparently higher than that in the model group (P < 0.05). The level of 5-HT in the model group was markedly higher than that in the control group (P < 0.05), and that in the 7-d treatment group was lower than that in the model group (P < 0.05), while that in the 15-d treatment group was apparently higher than that in the model group (P < 0.05). The level of Ach in the model group was obviously; lower than that in the control group (P < 0.05), and that in the 7-d treatment group was higher than that in the model group (P < 0.05). CONCLUSION: Electrical stimulation at auricular vagus nerve combined with sound masking) could regulate the threshold of ABR, especially in the 15-d treatment group. This may be ascribed to modulating the levels of neurotransmitter of γ-GABA, 5-HT and Ach in inferior colliculus.

Transcutaneous electrical stimulation at auricular acupoints innervated by auricular branch of vagus nerve pairing tone for tinnitus: study protocol for a randomized controlled clinical trial.

BACKGROUND: Subjective tinnitus is a phantom sensation experienced in the absence of any source of sound. Its mechanism remains unclear, and no approved drugs are available. Vagus nerve stimulation (VNS) is an exciting new method to treat tinnitus, but direct electrical stimulation of the cervical vagus has disadvantages. This randomized controlled clinical trial aims to overcome these limitations by stimulating the auricular branch of vagus nerve (ABVN) on the outer ear. Since the ABVN is the only peripheral branch of the vagus nerve distributed on the ear's surface, it should be possible to achieve analogous efficacy to VNS by activating the central vagal pathways. However, researches have indicated that the curative effect lies in a combination of auditory and vagal nerve stimulation. Moreover, from traditional Chinese theory, auricular acupoints used to treat tinnitus are mainly in the regions supplied by the ABVN. Whether stimulation at the auricular acupoints is due to unintentional stimulation of vagal afferent fibers also needs evidence. METHODS/DESIGN: A total of 120 subjects with subjective tinnitus are randomized equally into four groups: (1) electrical stimulation at auricular acupoints (CO10, CO11, CO12, and TF4) innervated by the ABVN; (2) electrical stimulation at auricular acupoints (CO10, CO11, CO12, and TF4) innervated by ABVN pairing tones; (3) electrical stimulation at auricular acupoints innervated by non-ABVN pairing tones; (4) electrical acupuncture. Patients will be treated for 30 minutes every other day for 8 weeks. The primary outcome measure is the Tinnitus Handicap Inventory. The secondary outcome measure combines a visual analogue scale to measure tinnitus disturbance and loudness with the Hospital Anxiety and Depression Scale. Assessment is planned at baseline (before treatment) and in the 4th and 8th week, with further follow-up visits after termination of the treatment at the 12th week. Any adverse events will be promptly documented. DISCUSSION: Completion of this trial will help to confirm whether ABVN or the combination of ABVN and sound stimulus plays a more important role in treating tinnitus. Moreover, the result of this clinical trial will enhance our understanding of specific auricular acupoints. TRIAL REGISTRATION: Chinese Clinical Trials Register ChiCTR-TRC-14004940.