The multiple systemic symptoms appearing during the convalescence of novel coronavirus infection(COVID-19)are called long-term symptoms after COVID-19,also named as long COVID.Traditional Chinese medicine(TCM)has unique advantages in the treatment of long COVID.In the view of Professor ZHANG Zhong-De,the novel coronavirus infection belongs to the category of damp-toxin pestilence in TCM.During the convalescence of novel coronavirus infection,the damp-toxin pathogen causes the dysfunction of qi,blood,yin and yang of zang-fu organs,and then results in the consumption of yang qi or the depletion of fluid and blood.Damp-turbidity obstructing qi movement and entering collaterals and then developing into blood stasis is the key pathogenesis of long COVID.Therefore,the therapeutic principle of harmonizing yin,yang,qi and blood of the five zang-organs is proposed,and the methods for transporting and transforming damp-toxin and phlegm-turbidity,regulating qi,activating blood and removing stasis should be adopted.According to the exuberance or decline of qi,blood and body fluid,and yin and yang of the five zang-organs,the Fuzheng Series Formulas(series formulas for supporting healthy qi)were recommended for the specific syndrome types:Fuzheng Shengbai Formula can be used for the syndrome of deficiency of original qi;Fuzheng Yifei Formula can be used for lung-spleen-kidney deficiency syndrome;Fuzheng Anshen Formula can be used for the syndrome of liver qi stagnation and heart-spleen deficiency.During the treatment,Professor ZHANG Zhong-De makes the modification of drugs indicated for the retention sites of damp-toxin and turbid qi,and addresses the importance of spleen and stomach in the whole treatment process by protecting spleen and stomach,so as to promote the rehabilitation of patients after novel coronavirus infection and improve their quality of life.

Humans ; Consensus ; Hypersensitivity ; Desensitization, Immunologic/adverse effects* ; Immunotherapy/adverse effects* ; Injections, Subcutaneous ; Allergens

The global COVID-19 coronavirus pandemic has infected over 109 million people, leading to over 2 million deaths up to date and still lacking of effective drugs for patient treatment. Here, we screened about 1.8 million small molecules against the main protease (M^pro) and papain like protease (PL^pro), two major proteases in severe acute respiratory syndrome-coronavirus 2 genome, and identified 1851M^pro inhibitors and 205 PL^pro inhibitors with low nmol/l activity of the best hits. Among these inhibitors, eight small molecules showed dual inhibition effects on both M^pro and PL^pro, exhibiting potential as better candidates for COVID-19 treatment. The best inhibitors of each protease were tested in antiviral assay, with over 40% of M^pro inhibitors and over 20% of PL^pro inhibitors showing high potency in viral inhibition with low cytotoxicity. The X-ray crystal structure of SARS-CoV-2 M^pro in complex with its potent inhibitor 4a was determined at 1.8 Å resolution. Together with docking assays, our results provide a comprehensive resource for future research on anti-SARS-CoV-2 drug development.
Humans ; Antiviral Agents/chemistry* ; COVID-19 ; COVID-19 Drug Treatment ; High-Throughput Screening Assays ; Molecular Docking Simulation ; Protease Inhibitors/chemistry* ; SARS-CoV-2/enzymology* ; Viral Nonstructural Proteins

Microglial surveillance plays an essential role in clearing misfolded proteins such as amyloid-beta, tau, and α-synuclein aggregates in neurodegenerative diseases. However, due to the complex structure and ambiguous pathogenic species of the misfolded proteins, a universal approach to remove the misfolded proteins remains unavailable. Here, we found that a polyphenol, α-mangostin, reprogrammed metabolism in the disease-associated microglia through shifting glycolysis to oxidative phosphorylation, which holistically rejuvenated microglial surveillance capacity to enhance microglial phagocytosis and autophagy-mediated degradation of multiple misfolded proteins. Nanoformulation of α-mangostin efficiently delivered α-mangostin to microglia, relieved the reactive status and rejuvenated the misfolded-proteins clearance capacity of microglia, which thus impressively relieved the neuropathological changes in both Alzheimer's disease and Parkinson's disease model mice. These findings provide direct evidences for the concept of rejuvenating microglial surveillance of multiple misfolded proteins through metabolic reprogramming, and demonstrate nanoformulated α-mangostin as a potential and universal therapy against neurodegenerative diseases.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.
Humans ; Salvia miltiorrhiza/metabolism* ; Biosynthetic Pathways ; Quinones/metabolism* ; Plant Roots/metabolism* ; Gene Expression Regulation, Plant

Aim To investigate whether Linggui Zhugan Decoction ( LGZGD) can inhibit ventricular remodeling and prevent heart failure in rats after myocardial infarction by regulating Nrf2/BNIP3 pathway. Methods The model of heart failure after myocardial infarction was established by left coronary artery ligation in rats. Two weeks after modeling, all rats were randomly divided into model group, LGZGD group, and captopril group. Meanwhile sham operation group was set up. The rats were given continuous intragastric administration with drug or distilled water for 28 days, once a day. The behavioral signs of rats in each group were observed. The cardiac function of rats in each group was examined by echocardiography. Serum BNP and NT-ProBNP content were detected by enzyme -linked immunoassay; The changes of myocardial his-topathological and collagen fibers in rats were detected using sirius staining. The contents of oxidative stress index including ROS, SOD in myocardial tissue of rats in each group were observed by DCFH-DA fluorescent probe and Enzyme-linked immunoassay. The ultra-structure of mitochondria was observed by transmission electron microscopy. Expressions of apoptotic proteins ( mitochondrial CytC, cytoplasmic CytC) were detected by Western blot. Expression of proteins related to the Nrf2/BNIP3 pathway were examined by immunofluorescence and Western blot. Results LGZGD could significantly improve the cardiac function of rats, reduce the contents of BNP and NT-ProBNP, inhibit the excessive deposition of collagen in myocardial interstiti-um, reduce ROS, increase the content of SOD, improve mitochondrial structure damage, up-regulate the expression of Nrf2 and nuclear translocation, and reduce the expression of BNIP3. Conclusions LGZGD can inhibit the ventricular remodeling and prevent the occurrence of heart failure after myocardial infarction. Its pharmacological effects are mainly related to regulating the Nrf2/BNIP3 pathway, activating Nrf2, promoting its nuclear transfer, and further down-regulating BNIP3, protecting mitochondrial function, and reducing cardiomyocyte apoptosis.

This study aims to investigate the effect of atractylenolide Ⅲ(ATL-Ⅲ) on hydrogen peroxide(H_2O_2)-induced endoplasmic reticulum stress and apoptosis of H9 c2 cells via the ROS/GRP78/caspase-12 signaling pathway.The binding activity of ATL-Ⅲ to GRP78 was determined by molecular docking.The result showed that ATL-Ⅲ had a good binding activity to GRP78, and the binding activity of ATL-Ⅲ was stronger than that of its specific inhibitor.The endoplasmic reticulum stress model of H9 c2 was established by H_2O_2(100 μmol·L~(-1)) treatment.Five groups were designed: blank control group, model group, and ATL-Ⅲ(15, 30, and 60 μmol·L~(-1)) groups.Apoptosis was detected by Hoechst/PI double staining and flow cytometry.The levels of superoxide dismutase(SOD), malondialdehyde(MDA), and lactate dehydrogenase(LDH) were measured by colorimetry.The levels of reactive oxygen species(ROS) and calcium(Ca~(2+)) in cytoplasm were determined by the fluorescence probe DCFH-DA and the calcium fluorescence probe Flou-4, respectively.The protein levels of GRP78, caspase-12, and caspase-3 were determined by Western blot, and the mRNA levels of GRP78 and caspase-12 by RT-qPCR.N-acetyl-L-cysteine(NAC) and 4-phenylbutyric acid(4-PBA) were respectively used to inhibit ROS and GRP78, and then the mechanism of ATL-Ⅲ in protecting the cells from endoplasmic reticulum stress induced by H_2O_2 were deduced.ATL-Ⅲ(15, 30, and 60 μmol·L~(-1)) decreased the apoptosis rate and ROS, MDA, and LDH levels(P<0.01), increased the SOD activity(P<0.01), and down-regulated the protein levels of GRP78, caspase-12, and caspase-3 and the mRNA levels of GRP78 and caspase-12(P<0.05).The addition of NAC decreased the apoptosis rate and ROS, MDA, GRP78, caspase-12, and caspase-3 levels(P<0.01), while it elevated the SOD level(P<0.01).The addition of 4-PBA also decreased the apoptosis rate and the levels of GRP78, caspase-12, caspase-3, and Ca~(2+)(P<0.01).The effect of inhibitors were consistent with that of ATL-Ⅲ.In conclusion, ATL-Ⅲ can protect H9 c2 cardiomyocytes by regulating ROS/GRP78/caspase-12 signaling pathway to inhibit H_2O_2-induced endoplasmic reticulum stress and apoptosis.
Apoptosis ; Calcium/pharmacology* ; Caspase 12/metabolism* ; Caspase 3/metabolism* ; Endoplasmic Reticulum Chaperone BiP ; Endoplasmic Reticulum Stress ; Lactones ; Molecular Docking Simulation ; RNA, Messenger ; Reactive Oxygen Species/metabolism* ; Sesquiterpenes ; Signal Transduction ; Superoxide Dismutase/metabolism*

Objective: To analyze the clinical epidemiological characteristics including composition of pathogens , clinical characteristics, and disease prognosis acute bacterial meningitis (ABM) in Chinese children. Methods: A retrospective analysis was performed on the clinical and laboratory data of 1 610 children <15 years of age with ABM in 33 tertiary hospitals in China from January 2019 to December 2020. Patients were divided into different groups according to age,<28 days group, 28 days to <3 months group, 3 months to <1 year group, 1-<5 years of age group, 5-<15 years of age group; etiology confirmed group and clinically diagnosed group according to etiology diagnosis. Non-numeric variables were analyzed with the Chi-square test or Fisher's exact test, while non-normal distrituction numeric variables were compared with nonparametric test. Results: Among 1 610 children with ABM, 955 were male and 650 were female (5 cases were not provided with gender information), and the age of onset was 1.5 (0.5, 5.5) months. There were 588 cases age from <28 days, 462 cases age from 28 days to <3 months, 302 cases age from 3 months to <1 year of age group, 156 cases in the 1-<5 years of age and 101 cases in the 5-<15 years of age. The detection rates were 38.8% (95/245) and 31.5% (70/222) of Escherichia coli and 27.8% (68/245) and 35.1% (78/222) of Streptococcus agalactiae in infants younger than 28 days of age and 28 days to 3 months of age; the detection rates of Streptococcus pneumonia, Escherichia coli, and Streptococcus agalactiae were 34.3% (61/178), 14.0% (25/178) and 13.5% (24/178) in the 3 months of age to <1 year of age group; the dominant pathogens were Streptococcus pneumoniae and the detection rate were 67.9% (74/109) and 44.4% (16/36) in the 1-<5 years of age and 5-<15 years of age . There were 9.7% (19/195) strains of Escherichia coli producing ultra-broad-spectrum β-lactamases. The positive rates of cerebrospinal fluid (CSF) culture and blood culture were 32.2% (515/1 598) and 25.0% (400/1 598), while 38.2% (126/330)and 25.3% (21/83) in CSF metagenomics next generation sequencing and Streptococcus pneumoniae antigen detection. There were 4.3% (32/790) cases of which CSF white blood cell counts were normal in etiology confirmed group. Among 1 610 children with ABM, main intracranial imaging complications were subdural effusion and (or) empyema in 349 cases (21.7%), hydrocephalus in 233 cases (14.5%), brain abscess in 178 cases (11.1%), and other cerebrovascular diseases, including encephalomalacia, cerebral infarction, and encephalatrophy, in 174 cases (10.8%). Among the 166 cases (10.3%) with unfavorable outcome, 32 cases (2.0%) died among whom 24 cases died before 1 year of age, and 37 cases (2.3%) had recurrence among whom 25 cases had recurrence within 3 weeks. The incidences of subdural effusion and (or) empyema, brain abscess and ependymitis in the etiology confirmed group were significantly higher than those in the clinically diagnosed group (26.2% (207/790) vs. 17.3% (142/820), 13.0% (103/790) vs. 9.1% (75/820), 4.6% (36/790) vs. 2.7% (22/820), χ²=18.71, 6.20, 4.07, all P<0.05), but there was no significant difference in the unfavorable outcomes, mortility, and recurrence between these 2 groups (all P>0.05). Conclusions: The onset age of ABM in children is usually within 1 year of age, especially <3 months. The common pathogens in infants <3 months of age are Escherichia coli and Streptococcus agalactiae, and the dominant pathogen in infant ≥3 months is Streptococcus pneumoniae. Subdural effusion and (or) empyema and hydrocephalus are common complications. ABM should not be excluded even if CSF white blood cell counts is within normal range. Standardized bacteriological examination should be paid more attention to increase the pathogenic detection rate. Non-culture CSF detection methods may facilitate the pathogenic diagnosis.
Adolescent ; Brain Abscess ; Child ; Child, Preschool ; Escherichia coli ; Female ; Humans ; Hydrocephalus ; Infant ; Infant, Newborn ; Male ; Meningitis, Bacterial/epidemiology* ; Retrospective Studies ; Streptococcus agalactiae ; Streptococcus pneumoniae ; Subdural Effusion ; beta-Lactamases

ObjectiveTo observe the inhibitory effect of modified Xiao Xianxiongtang on epithelial-mesenchymal transition （EMT） of human gastric cancer MGC803 cells and its relationship with secretory glycoprotein Wnt/β-catenin pathway. MethodThe BALB/c nude mice were implanted with human gastric cancer MGC803 cell suspension in the heterotopic subcutaneous position for inducing tumor. After successful modeling， they were randomly divided into the model group， low-， medium-， and high-dose （16.0，32.0，and 64.0 g·kg^-1） groups of modified Xiao Xianxiongtang， and capecitabine （400 mg·kg^-1） group， with eight mice in each group， and gavaged with the corresponding drugs， once per day， for 28 consecutive days. Those in the capecitabine group received one-week discontinuation after every two weeks of treatment. The general state and body weight of the nude mice were observed， and the transplanted tumor volume was measured. After being killed， they were weighed and the tumor inhibition rate was calculated. Hematoxylin-eosin （HE） staining was carried out for observing the pathological changes in transplanted tumor tissues. The gene and protein expression levels of Wnt1 and β-catenin were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， followed by the determination of matrix metalloproteinase-9 （MMP-9）， vascular endothelial growth factor （VEGF）， N-cadherin， E-cadherin， Vimentin， and Snail protein expression by Western blot. The expression levels of cyclooxygenase 2 （COX2） and prostaglandin E₂ （PGE₂） were detected by enzyme-linked immunosorbent assay （ELISA）. ResultIt was found that the transplanted tumor in each group showed different growth trends with time， with the most obvious growth observed in the model group. Compared with the model group， the low-， medium-， and high-dose modified Xiao Xianxiongtang groups exhibited reduced tumor volume and slowed growth to varying degrees over time. After medication for days 7，14，21，and 28， the tumor volumes in the low- and high-dose modified Xiao Xianxiongtang groups and capecitabine group declined （P<0.05， P<0.01）， and that in the medium-dose Xiao Xianxiongtang group was also remarkably reduced after medication for days 14，21，and 28 （P<0.01）. Compared with the model group， the high-dose modified Xiao Xianxiongtang group and capecitabine group showed a significant reduction in the relative tumor volume after treatment for days 7，14，21，28 （P<0.01）， and the low- and medium-dose modified Xiao Xianxiongtang groups also presented with decreased relative tumor volume after treatment for days 14，21，28 （P<0.05， P<0.01）. Compared with the model group， the modified Xiao Xianxiongtang at low， medium， and high doses and capecitabine all increased the tumor inhibition rate to varying degrees （P<0.01）， down-regulated the mRNA and protein expression levels of Wnt1 and β-catenin in tumor tissue （P<0.05， P<0.01） and protein expression levels of MMP-9， VEGF， N-cadherin， Vimentin， and Snail （P<0.05， P<0.01）， up-regulated E-cadherin protein expression （P<0.05， P<0.01）， and reduced COX2 and PGE₂ contents （P<0.05， P<0.01）. ConclusionModified Xiao Xianxiongtang inhibits the EMT of human gastric cancer MGC803 cell-transplanted tumor， which may be related to Wnt/β-catenin pathway.

ObjectiveTo explore the effect of Xiao Xianxiongtang （XXXT） on the transforming growth factor （TGF）-β₁-induced invasion， metastasis， and epithelial-mesenchymal transition （EMT） of gastric cancer MGC-803 cells and the underlying mechanism. MethodThe molecular docking between XXXT and nuclear factor of activated T cells （NFAT） was performed by CB-DOCK （http：//clab.labshare.cn/cb-dock/）. The invasion and metastasis model of MGC-803 cells was established with 10 μg·L^-1 TGF-β₁. MGC-803 cells were classified into blank group， model group， 0.1 g·L^-1 XXXT group， 0.2 g·L^-1 XXXT group， and 0.4 g·L^-1 XXXT group. For further clarifying the key role of Wnt5a/Ca²⁺/NFAT signaling pathway in the inhibition of XXXT on gastric cancer， MGC-803 cells were transfected with Wnt5a overexpression plasmid， and then the cells were classified into blank plasmid group， Wnt5a-OE group， blank plasmid + XXXT （0.4 g·L^-1） group， and Wnt5a-OE + XXXT （0.4 g·L^-1） group. Cell viability was determined by cell counting kit-8 （CCK-8） assay， cell invasion and migration ability by Transwell invasion assay and wound healing assay， expression of EMT-related proteins （E-cadherin， N-cadherin， Vimentin， Snail） and Wnt5a/Ca²⁺/NFAT signaling pathway-related key proteins ［Wnt5a， calcineurin （CaN）， NFAT1， and p-NFAT1］ by Western blot， and changes in intracellular Ca²⁺ concentration by immunofluorescence assay. ResultMolecular docking suggested that XXXT acted on Wnt5a/Ca²⁺/NFAT signaling pathway. XXXT （0.1， 0.2， 0.4 g·L^-1） significantly promoted the loss of MGC-803 cell viability （P<0.05，P<0.01）. It inhibited cells from invading the transwell lower chamber and slowed down the healing of cell wounds in a dose-dependent manner （P<0.05， P<0.01）. Moreover， it promoted the expression of E-cadherin while suppressed the expression of N-cadherin， Vimentin， and Snail （P<0.05， P<0.01）. Further experiments showed that XXXT could inhibit the expression of Wnt5a， CaN， NFAT1， and p-NFAT1， and reduce the nuclear expression of NFAT1 and the transcription activity mediated by NFAT1， so as to reduce the cellular Ca²⁺ concentration （P<0.05， P<0.01）. XXXT can reverse the effect of Wnt5a （P<0.05， P<0.01）. ConclusionXXXT can attenuate the invasion， metastasis， and EMT of MGC-803 cells via the Wnt5a/Ca²⁺/NFAT pathway， thereby weakening the tumor-promoting effect of TGF-β₁. In summary， XXXT may exert therapeutic effect on gastric cancer by regulating the invasion， metastasis， and EMT of gastric cancer cells.