The in vitro and in vivo antiviral effects of aloperine against Zika virus infection.

Zika virus (ZIKV) infection poses a significant threat to global public health and is associated with microcephaly. There are no approved ZIKV-specific vaccines or drugs for the clinical treatment of the infection. Currently, there are no approved ZIKV-specific vaccines or drugs for the clinical treatment of the infection. In this study, we investigated the antiviral potential of aloperine, a quinolizidine alkaloid, against ZIKV infection in vivo and in vitro. Our results demonstrate that aloperine effectively inhibits ZIKV infection in vitro, with a low nanomolar half maximal effective concentration (EC50 ). Specifically, aloperine strongly protected cells from ZIKV multiplication, as indicated by decreased expression of viral proteins and virus titer. Our further investigations using the time-of-drug-addition assay, binding, entry, and replication assays, detection of ZIKV strand-specific RNA, the cellular thermal shift assay, and molecular docking revealed that aloperine significantly inhibits the replication stage of the ZIKV life cycle by targeting the domain RNA-dependent RNA polymerase (RDRP) of ZIKV NS5 protein. Additionally, aloperine reduced viremia in mice and effectively lowered the mortality rate in infected mice. These findings highlight the potency of aloperine and its ability to target ZIKV infection, suggesting its potential as a promising antiviral drug against ZIKV.

ENT characteristics and therapeutic results in multisystemic disorders of mitochondrial encephalomyopathy.

Here we report the evaluation of the frequency of subjective and objective otolaryngologic findings and therapeutic results in 32 patients with mitochondrial encephalomyopathy (MEM) from September 2001 to June 2021. Our analysis included studying the patients' family histories, the clinical manifestations of MEM, and the therapeutic effects of treatments. The patients' ages ranged from 2 to 77 years, with a median age of 12.3 years. We found that MEM ENT symptoms were characterized by hearing loss, dysphagia, and facial weakness. Most cases of sensorineural hearing loss were bilateral symmetrical progressive or sudden deafness since adolescence, which were often underestimated. Associated neuromuscular symptoms required mtDNA testing. Dysphagia and facial weakness occurred preferentially in middle-aged patients, and muscle biopsies were advised. Distortion product otoacoustic emissions and auditory brainstem responsetesting were more sensitive and reliable than pure tone averages for objective monitoring of pathogenesis. Administration of the mitochondrial synthase complex benefited patients with acute episodes. If patients did not fully recover and exhibitedresidual language deficits, hearing aids or cochlear implants were recommended. Counsel was given regarding synthetical treatments for facial weakness, endoscopic circopharyngealmyotomy for dysphagia, and surgical correction of ptosis. This study demonstrates that increased awareness of these symptoms is important to address appropriate interventions and avoid complications such as ablepsia, aphasia, social isolation, malnutrition, aspiration pneumonia, and heart failure in the setting of MEM.

Physcion, a novel inhibitor of 5α-reductase that promotes hair growth in vitro and in vivo.

Androgenic alopecia (AGA) has a high incidence. Excess dihydrotestosterone in blood capillaries, which is converted from testosterone by 5α-reductase, is an AGA causative factor. We identified the inhibitory activity of four Polygonum multiflorum compounds against 5α-reductase via high-performance liquid chromatography, and the results showed that Physcion was a potent 5α-reductase inhibitor. Additionally, we found that through inhibiting 5α-reductase expression, Physcion could shorten the time of dorsal skin darkening and hair growth, improve hair follicle morphology, and significantly increase hair follicle count. Eventually, through molecular docking study, we found the binding energy and molecular interactions between Physcion and 5α-reductase type II. These results suggested that Physcion is a potent 5α-reductase inhibitor, as well as a new natural medicine for treating AGA.

Inhibitory effects of Patchouli alcohol on the early lifecycle stages of influenza A virus.

Background: The antiviral activity and underlying mechanism of Patchouli alcohol remain unclear. Methods: This study evaluated the cytotoxicity, optimal methods for drug administration, anti-influenza A activity of Patchouli alcohol. The antiviral mechanism of Patchouli alcohol was also assessed via qRT-PCR, western blot, hemagglutination inhibiting (HAI) assay, and hemolysis inhibiting assay. Results: Patchouli alcohol was shown to have low cytotoxicity and its strongest antiviral effect was associated with premixed administration. Patchouli alcohol inhibited virus replication during the early lifecycle stages of influenza A virus infection and specifically prevented expression of the viral proteins, HA and NP. In both the HAI and hemolysis inhibiting assays, Patchouli alcohol was able to block HA2-mediated membrane fusion under low pH conditions. Patchouli alcohol had lower binding energy with HA2 than HA1. Conclusion: These findings suggest that Patchouli alcohol could be a promising membrane fusion inhibitor for the treatment of influenza A infection.

Modified Baihu decoction therapeutically remodels gut microbiota to inhibit acute gouty arthritis.

Background: Acute gouty arthritis (AGA) is the most common first symptom of gout, and the development of gout as a metabolic and immune inflammatory disease is also correlated with the gut microbiota. However, the mechanism of the effect of changes in the gut microbiota on AGA remains unclear. The intestinal flora can not only affect purine metabolism or regulate inflammation, but also influence the therapeutic effect of drugs on AGA. The aim of this study was to investigate the exact mechanism of modified Baihu decoction (MBD) in the treatment of AGA and whether it is related to the regulation of the structure of the intestinal flora. Methods: On the 21st day of MBD administration by continuous gavage, a rat acute gouty arthritis model was constructed using sodium urate (0.1 mL/rat, 50 mg/mL), and the ankle joint swelling was measured before and 4 h, 8 h, 24 h, and 48 h after the injection of sodium urate. After 48 h of sodium urate injection, serum, liver, kidney, ankle synovial tissue and feces were collected from rats. The collected samples were examined and analyzed using H&E, Elisa, Immunohistochemistry, Histopathology, 16S rDNA, and Biochemical analysis. To investigate the mechanism of MBD to alleviate AGA using pro-inflammatory factors and intestinal flora. Results: MBD (5.84, 35 g/kg) was administered orally to AGA rats and diclofenac sodium tablets (DS-tablets) were used as standard treatment control. Serum biochemical assessment confirmed that MBD is a safe drug for the treatment of AGA. In addition, our findings confirmed that MBD relieved AGA-related symptoms, such as toe swelling. Lowering serum levels of uric acid, IL-1ß, and TGF-ß1 immunohistochemical results also confirmed that MBD reduced the expression of inflammatory elements such as IL-1ß, NLRP3, ASC, and Caspase-1 in synovial tissue.Furthermore, compared with control group, the 16s rDNA sequencing of AGA rat faeces revealed an increase in the relative abundance of Lachnospiraceae, Muribaculaceae, and Bifidobacteriaceae species. While the relative abundance of Lactobacillaceae, Erysipelotrichaceae, Ruminococcaceae, Prevotellaceae and Enterobacteriaceae showed a relative decrease in species abundance. Of these, the reduction in species abundance of Enterobacteriaceae was associated with a reduction in amino acid metabolism and environmental perception. After MBD therapeutic intervention, the disturbance of the intestinal flora caused by AGA was restored. Conclusion: In summary, MBD is an effective agent for the treatment of AGA, with the potential mechanism being the regulation of intestinal flora to control inflammation. This would help to promote the therapeutic effect of MBD on AGA.

[Mitochondrial encephalomyopathy involves ophthalmology otorhinolaryngology neurology and their clinical features].

Objective:This study aimed to provide better understanding of the otolaryngologic features, combined with ophthalmologic and neurologic characteristics in mitochondrial encephalomyopathy（MEM）, and to help ENT and auditory practitioner making correct diagnosis as well. Methods:Twenty-eight patients with MEM were enrolled between September 2001 and January 2020. Information about family histories and clinical symptoms was retrospectively analyzed. All patients underwent otorhinolaryngological, ophthalmological and neurological examinations, including: pure-tone audiometry, acoustic immittance（AI）, distortion-product otoacoustic emissions（DPOAE）, auditory brainstem response（ABR）, cochlear micropotential（CM）, speech discrimination score（SDS）, electroneurography（ENoG）, computed tomography（CT） of the temporal bone and cranial magnetic resonance weighted imaging scan（MRI）, muscle biopsy and mtDNA gene testing. Results:ENT subjective manifestations were present in 15 cases （53.6%） with sensorineural hearing loss（SNHL）, 4（14.3%） with tinnitus, 4（14.3%） with facial weakness, 3（10.7%） with dysphagia, 1（3.6%） with auditory agnosia. Ophthalmological and neurological symptoms included ptosis in 16 cases （57.1%）, exercise intolerance in 16（57.1%）, optic atrophy in 15（53.6%）, muscular atrophy in 6（21.4%）, and stroke-like episodes in 5（17.9%）. The results of objective examinations were as follows: DPOAE were not elicited in 18（64.3%） cases, ABR abnormalities in 18（64.3%） cases, hearing threshold shift in 15（53.6%） cases, AI normal and CM was not detected in all cases, SDS decreased in 6（21.4%） cases, facial ENoG abnormalities in 4（14.3%） cases, laryngeal ENoG abnormalities in 3（10.7%） cases, EMG abnormalities in 6（21.4%） cases, and ECG abnormalities in 8（28.6%） cases. Temporal CT were normal, but cranial MRI abnormalities were found in 19 cases（67.9%）, including central nerve demyelination, white matter hyperintensities, generalized cerebellar and cerebral atrophy, multiple cortical/subcortical infarct-like lesions, basal ganglia calcification. Conclusion:Multisystemic syndromes in MEM can present as a variety of otolaryngological, ophthalmological and neurological abnormalities, such as ptosis, audio-visual disturbance, exercise intolerance and stroke-like episodes etc. SNHL, tinnitus, auditory agnosia, facial weakness and dysphagia were ENT specific manifestations. SNHL in MEM is bilateral symmetrical progressive or of sudden onset since teenage. mtDNA testing may be helpful for adolescent patient whose SNHL was associated with neuromuscular symptoms. Muscle biopsy should be considered when middle-aged patients developed facial weakness and dysphagia. DPOAE and ABR are the optimal objective audiometric tests to monitor the progression of MEM associated with SNHL.

Ganghuo Kanggan Decoction in Influenza: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation.

Background: Ganghuo Kanggan decoction (GHKGD) is a clinical experience prescription used for the treatment of viral pneumonia in the Lingnan area of China, and its clinical effect is remarkable. However, the mechanism of GHKGD in influenza is still unclear. Objective: To predict the active components and signaling pathway of GHKGD and to explore its therapeutic mechanism in influenza and to verified it in vivo using network pharmacology. Methods: The potential active components and therapeutic targets of GHKGD in the treatment of influenza were hypothesized through a series of network pharmacological strategies, including compound screening, target prediction and pathway enrichment analysis. Based on the target network and enrichment results, a mouse model of influenza A virus (IAV) infection was established to evaluate the therapeutic effect of GHKGD on influenza and to verify the possible molecular mechanism predicted by network pharmacology. Results: A total of 116 candidate active compounds and 17 potential targets were identified. The results of the potential target enrichment analysis suggested GHKGD may involve the RLR signaling pathway to reduce inflammation in the lungs. In vivo experiments showed that GHKGD had a protective effect on pneumonia caused by IAV-infected mice. Compared with the untreated group, the weight loss in the GHKGD group in the BALB/c mice decreased, and the inflammatory pathological changes in lung tissue were reduced (p < 0.05). The expression of NP protein and the virus titers in lung were significantly decreased (p < 0.05). The protein expression of RIG-I, NF-kB, and STAT1 and the level of MAVS and IRF3/7 mRNA were remarkably inhibited in GHKGD group (p < 0.05). After the treatment with GHKGD, the level of Th1 cytokines (IFN-γ, TNF-α, IL-2) was increased, while the expression of Th2 (IL-5, IL4) cytokines was reduced (p < 0.05). Conclusion: Through a network pharmacology strategy and in vivo experiments, the multi-target and multi-component pharmacological characteristics of GHKGD in the treatment of influenza were revealed, and regulation of the RLR signaling pathway during the anti-influenza process was confirmed. This study provides a theoretical basis for the research and development of new drugs from GHKGD.

Antiviral activity of lycorine against Zika virus in vivo and in vitro.

The emergence and re-emergence of Zika virus (ZIKV), is a cause for international concern. These highly pathogenic arboviruses represent a serious health burden in tropical and subtropical areas worldwide. Despite these burdens, antiviral therapies do not exist, and inhibitors of ZIKV are therefore urgently needed. To elucidate the anti-ZIKV effect of lycorine, we used reverse transcription-quantitative real-time PCR (qRT-PCR), immunofluorescence, Westernwestern blot, and plaque forming assay to analyse viral RNA (vRNA), viral protein, progeny virus counts, and validated inhibitors in vitro using a variety of cell lines. Additionally, we found that lycorine acts post-infection according to time-of-addition assay, and inhibits RdRp activity. Lycorine protected AG6 mice against ZIKV-induced lethality by decreasing the viral load in the blood. Due to its potency and ability to target ZIKV infection in vivo and in vitro, lycorine might offer promising therapeutic possibilities for combatting ZIKV infections in the future.

Inhibition of Histone Methyltransferase G9a Attenuates Noise-Induced Cochlear Synaptopathy and Hearing Loss.

Posttranslational modification of histones alters their interaction with DNA and nuclear proteins, influencing gene expression and cell fate. In this study, we investigated the effect of G9a (KMT1C, EHMT2), a major histone lysine methyltransferase encoded by the human EHMT2 gene and responsible for histone H3 lysine 9 dimethylation (H3K9me2) on noise-induced permanent hearing loss (NIHL) in adult CBA/J mice. The conditions of noise exposure used in this study led to losses of cochlear synapses and outer hair cells (OHCs) and permanent auditory threshold shifts. Inhibition of G9a with its specific inhibitor BIX 01294 or with siRNA significantly attenuated these pathological features. Treatment with BIX 01294 also prevented the noise-induced decrease of KCNQ4 immunolabeling in OHCs. Additionally, G9a was increased in cochlear cells, including both outer and inner sensory hair cells, some spiral ganglion neurons (SGNs), and marginal cells, 1 h after the completion of the noise exposure. Also subsequent to noise exposure, immunoreactivity for H3K9me2 appeared in some nuclei of OHCs following a high-to-low frequency gradient with more labeled OHCs in the 45-kHz than the 32-kHz region, as well as in the marginal cells and in some SGNs of the basal turn. These findings suggest that epigenetic modifications of H3K9me2 are involved in NIHL and that pharmacological targeting of G9a may offer a strategy for protection against cochlear synaptopathy and NIHL.

Salvianolic acid B, an antioxidant derived from Salvia militarize, protects mice against γ­radiation­induced damage through Nrf2/Bach1.

Salvianolic acid B (SB) is an antioxidant derived from Salvia militarize, and is one of the most widely used herbs in traditional Chinese medicine. SB is a potent antioxidant that has been well documented as a scavenger of oxygen free radicals, and has been used for the prevention and treatment of atherosclerosis­associated disorders. To explore its potential therapeutic effects in treating radiation damage, in this study, mice were treated with SB at different doses of 5, 12.5 and 20 mg/kg, subsequent to receiving γ­irradiation. The effects of SB on peripheral blood, bone marrow nucleated cells, spleen and thymus indices, and oxidation resistance were evaluated in both radiated mice and control groups. The results indicated that SB significantly increased the counts of peripheral white blood cells, red blood cells and platelets. The number of nucleated cells in the bone marrow and the level of protein increased as well. In addition, improved spleen and thymus indices in the bone marrow were observed. SB treatment additionally reversed the deterioration of both the thymus and spleen indices, which is associated with increased serum superoxide dismutase activity and decreasing malondialdehyde levels via nuclear factor (erythroid­derived 2)­like 2 protein/BTB and CNC homology 1 mediated antioxidant effect. Furthermore, ROS levels and Bax protein expression were also suppressed by SB. The data suggested that SB is effective in protecting mice from γ­radiation injury, and could potentially be applicable for clinical use. Notably, the present study identified a promising candidate drug for enhancing the hematopoietic and immune systems.

Mitochondrial Calcium Transporters Mediate Sensitivity to Noise-Induced Losses of Hair Cells and Cochlear Synapses.

Mitochondria modulate cellular calcium homeostasis by the combined action of the mitochondrial calcium uniporter (MCU), a selective calcium entry channel, and the sodium calcium exchanger (NCLX), which extrudes calcium from mitochondria. In this study, we investigated MCU and NCLX in noise-induced hearing loss (NIHL) using adult CBA/J mice and noise-induced alterations of inner hair cell (IHC) synapses in MCU knockout mice. Following noise exposure, immunoreactivity of MCU increased in cochlear sensory hair cells of the basal turn, while immunoreactivity of NCLX decreased in a time- and exposure-dependent manner. Inhibition of MCU activity via MCU siRNA pretreatment or the specific pharmacological inhibitor Ru360 attenuated noise-induced loss of sensory hair cells and synaptic ribbons, wave I amplitudes, and NIHL in CBA/J mice. This protection was afforded, at least in part, through reduced cleavage of caspase 9 (CC9). Furthermore, MCU knockout mice on a hybrid genetic CD1 and C57/B6 background showed resistance to noise-induced seizures compared to wild-type littermates. Owing to the CD1 background, MCU knockouts and littermates suffer genetic high frequency hearing loss, but their IHCs remain intact. Noise-induced loss of IHC synaptic connections and reduction of auditory brainstem response (ABR) wave I amplitude were recovered in MCU knockout mice. These results suggest that cellular calcium influx during noise exposure leads to mitochondrial calcium overload via MCU and NCLX. Mitochondrial calcium overload, in turn, initiates cell death pathways and subsequent loss of hair cells and synaptic connections, resulting in NIHL.

[Expression of NR2A in rat auditory cortex after sound insulation and auditory plasticity].

OBJECTIVE: To study the changes of N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) expression at local synapses in auditory cortices after early postnatal sound insulation and tone exposure. METHOD: We prepared highly purified synaptosomes from primary auditory cortex by Optiprep flotation gradient centrifugations, and compared the differences of NR2A expression in sound insulation PND14, PND28, PND42 and Tone exposure after sound insulation for 7 days by Western blotting. RESULT: The results showed that the NR2A protein expression of PND14 and PND28 decreased significantly (P<0.05). Tone exposure after sound insulation for 7 days, mSIe NR2A protein level increased significantly (P<0.05). It showed bidirectional regulation of NR2A protein. No significant effects of sound insulation and lone exposure were found on the relative expression level of NR2A of PND42 (P>0.05). CONCLUSION: The results indicate that sound insulation and experience can modify the protein expression level of NR2A during the critical period of rat postnatal development. These findings provide important data for the study on the mechanisms of the developmental plasticity of sensory functions.

[The changes of phosphorylated c-Jun expression in spiral ganglion after exposed to noise].

OBJECTIVE: To investigate the mechanism of hearing pathway NMDAR regulating the changes of phosphorylated c-Jun expression in spiral ganglion after exposed Kunming mice to neural injury noise stimulation to induce permanent or temporary threshold shift. METHOD: To compare the different expressions of the key components (phosphorylated C-Jun) of NMDAR signal pathway during neural injury stimulation by Immunohistochemistry in CG. RESULT: The levels of phosphorylated c-Jun remarkably, increased in the spiral ganglion after 8 h, 48 h, 7 d and 14 d following noise trauma induced permanent threshold shift (PTS), and the numbers of positive cells reduced gradually. The similar changes occur in mice treated with MK-801 30 minutes before and after 3 h trauma induced PTS. After 48 h of noise induced TTS, the expression of Phosphorylated c-Jun return the level of normal control. CONCLUSION: The expressions of phosphorylated c-Jun are time-related and uniform in the time and position in CG after noise trauma. MK-801 can alleviate the damage of noise trauma by altered the NMDA receptor-mediated calcium influx. Therefore, the NMDA receptors may involved in the damage of inner ear in common.

Anatomic structural study of cerebellopontine angle via endoscope.

BACKGROUND: Minimally invasive surgery in skull base relying on searching for possible anatomic basis for endoscopic technology is controversial. The objective of this study was to observe the spatial relationships between main blood vessels and nerves in the cerebellopontine angle area and provide anatomic basis for lateral and posterior skull base minimally invasive surgery via endoscopic retrosigmoid keyhole approach. METHODS: This study was conducted on thirty dried adult skulls to measure the spatial relationships among the surface bony marks of posterior cranial fossa, and to locate the most appropriate drilling area for retrosigmoid keyhole approach. In addition, we used 10 formaldehyde-fixed adult cadaver specimens for simulating endoscopic retrosigmoid approach to determine the visible scope. RESULTS: The midpoint between the mastoid tip and the asterion was the best drilling point for retrosigmoid approach. A hole centered on this point with the 2.0 cm in diameter was suitable for exposing the related structures in the cerebellopontine angle. Retrosigmoid keyhole approach can decrease the pressure on the cerebellum and expose the related structures effectively which include facial nerve, vestibulocochlear nerve, trigeminal nerve, glossopharyngeal nerve, vagus nerve, accessory nerve, hypoglossal nerve, anterior inferior cerebellar artery, posterior inferior cerebellar artery and labyrinthine artery, etc. CONCLUSIONS: Exact location on endoscope retrosigmoid approach can avoid dragging cerebellum during the minimally invasive surgery. The application of retrosigmoid keyhole approach will extend the application of endoscopic technology.

[Long-term surgical results for congenital aural atresia and hearing reconstruction].

OBJECTIVE: To evaluate the stability of hearing results and complications in long-term following-up who underwent reconstruction surgery. METHODS: Six hundreds and seventy five cases (700 ears) of congenital aural atresia were reviewed from January 1984 to January 2001 at the Department of Otorhinolaryngology Head and Neck Surgery, Tongren hospital. Except 40 ears undone hearing reconstruction, 635 cases (660 ears) underwent long-term following-up for 3 to 19 years, with an average of 7.9 years. RESULTS: Stenosis and recurrent infection of the external auditory canal (EAC) were the most frequent complications. Stenosis was seen in 120 ears, and 2 ears re-atresia, with an incidence of 18.48% (122/660). Recurrent infection of the cavity and canal skin happened in 6 ears. Closure of the air-bone gap (ABG) post-operation were gained in all cases, and ABG gains 20 dB or more occurred in 512 ears (77.57%), but 30 dB or more in 231 ears (35%). Following-up results: Stable hearing results gained in 450 ears over the length of following-up; the hearing worsened than that of 3 weeks postoperatively occurred in 160 ears, including 2 ears with sensorineural hearing loss. Hearing deteriorated more than 20 dB happened in 35 ears, and 10-15 dB in others cases but still be improved compared with that of preoperation. CONCLUSIONS: Atresiaplasty surgery in individuals with congenital aural atresia can yield reliable, lasting hearing results in 68.2% (450/660), with a low incidence of complications; the initial improved hearing deteriorated gradually over the first 6 months post-operation, which are related with the stenosis and infection of canal. Cavity adhesion, bony EAC re-growth, ossicular chain re-fixation or displace may affect the hearing results in some cases. Even unilateral aural atresia may benefit from the reconstruction surgery and achieve serviceable hearing results.