ObjectiveTo investigate the therapeutic effects and mechanisms of modified Huangqi Jianzhong decoction （HQJZ） on gastric precancerous conditions （GPC）. MethodsIn the cell experiment， human gastric mucosal epithelial cells underwent malignant transformation induced by N-methyl-N′-nitro-N-nitrosoguanidine （MNNG） for the modeling of GPC （MC cells）. The cells were allocated into four groups： control ， model， low-dose HQJZ （HQJZ-L）， and high-dose HQJZ （HQJZ-H）. The control and model groups were cultured with the complete medium， while HQJZ-L and HQJZ-H groups received additional interventions with HQJZ at low （0.5 g·L^-1） and high （1.0 g·L^-1） doses， respectively. Cell counting kit-8 （CCK-8） assay was used to evaluate cytotoxicity， Transwell assay to assess cell invasion， Annexin V-FITC/PI staining to detect apoptosis， immunofluorescence assay to analyze reactive oxygen species （ROS） expression and mitochondrial autophagy， and Western blot to verify expression of proteins in key pathways. In the animal experiment， the GPC model was established in healthy BALB/c mice through MNNG induction. Twenty-four mice were allocated into four groups： control， model， HQJZ-L， and HQJZ-H. Control and model groups received normal saline （10 mL·kg^-1·d^-1） orally， while HQJZ-L and HQJZ-H groups were administrated with low-dose （6.24 g·kg^-1·d^-1） and high-dose （12.48 g·kg^-1·d^-1） HQJZ， respectively. After treatment， hematoxylin‑eosin （HE） staining and AB-PAS staining were performed to observe histopathological changes in the gastric tissue. Immunofluorescence assay was used to detect reactive oxygen species （ROS） and microtubule-associated protein 1 light chain 3 （LC3） levels in the gastric mucosa， TdT-mediated dUTP nick-end labeling （TUNEL） staining to assess apoptosis rates， and Western blotting and immunohistochemistry （IHC） to analyze the expression levels of Ki67， proliferating cell nuclear antigen （PCNA）， and foxhead box O3 （FoxO3）. ResultsCell viability assays showed that HQJZ dose-dependently reduced MC cell viability compared with the control group （P<0.05， P<0.01）. Transwell assays revealed that the model group exhibited enhanced cell invasion compared with the control group （P<0.05）. Compared with the model group， HQJZ treatment attenuated the cell invasion （P<0.05）. Gastric mucosal pathology in mice demonstrated that compared with the control group， the model group showed elevated HE and AB-PAS pathological scores （P<0.05）， while HQJZ treatment reduced these scores （P<0.05）. Transmission electron microscopy revealed increased mitochondrial number and volume in the model group compared with the control group. HQJZ treatment resulted in abnormal mitochondrial structure and significant alterations in rough endoplasmic reticulum morphology and distribution， presenting as dilated and hollow forms. Mitochondrial and apoptosis assessments indicated that compared with the control group， the model group exhibited enhanced Mito Tracker Green fluorescence （P<0.05）， no significant change in DCFH-DA fluorescence， Mito Tracker Red CMXRos fluorescence， ROS immunofluorescence， or malondialdehyde （MDA） level， increased GSH level （P<0.05）， enhanced LC3 fluorescence （P<0.05）， no significant change in apoptosis rate， and elevated ATP content in cells and mouse serum （P<0.05）. Compared with the model group， HQJZ treatment reduced Mito Tracker Green fluorescence （P<0.05）， increased DCFH-DA fluorescence， Mito Tracker Red fluorescence， MDA level， LC3 fluorescence， and apoptosis rate （P<0.05）， and decreased cellular ATP content （P<0.05）. The HQJZ-L group showed no significant change in ROS immunofluorescence or GSH level， whereas the HQJZ-H group demonstrated enhanced ROS immunofluorescence and glutathione （GSH） level （P<0.05）. Immunohistochemistry and Western blotting revealed that compared with the control group， the model group exhibited increased numbers of PCNA- and Ki67-positive cells （P<0.05） and elevated protein levels of FoxO3， sirtuin 1 （SIRT1）， and B-cell lymphoma 6 （Bcl-6） （P<0.05）. HQJZ treatment reduced the numbers of PCNA- and Ki67-positive cells （P<0.05） and lowered the protein levels of FoxO3， SIRT1， and Bcl-6 （P<0.05）. ConclusionHQJZ alleviates the progression of gastric precancerous lesions by regulating mitochondrial function via the FoxO3/ROS pathway and promoting apoptosis of GPC-malignant cells.

In order to establish a method for the detection of serum antibodies to donkey-derived e-quine coronavirus(ECoV),recombinant ECoV N protein was expressed in E.coli system,purified by nickel column affinity chromatography and identified by Western blot.After optimizing the re-action conditions,the indirect ELISA(iELISA)detection method was established using the puri-fied recombinant protein as coating antigen and used to detect 143 clinical serum samples.The re-sults showed that the recombinant N protein,which has good reaction activity with serum antibod-y,was successfully expressed.The optimum conditions of the established iELISA method were as follows:the amount of antigen coated was 0.2 μg/well and overnight at 4 ℃,10％skimmed milk powder solution was sealed at 37℃ for 1.5 h,the dilution concentration of serum was 1∶200,and the enzyme-labeled secondary antibody diluted at 1∶10 000.The sensitivity test results showed that the positive serum could be diluted to 1∶6 400.The specificity test results showed that all an-tibodies to several donkey pathogens were negative.The repetitive test results showed that the in-tra-and inter-batch coefficients of variation were 2.90％-6.12％and 2.29％-7.88％respectively.The positive rate of clinical donkey serum was 57.3％.The iELISA established in this study pro-vides a technical support for epidemiological investigation and antibody surveillance.

ObjectiveThe theory of homotherapy for heteropathy is one of the classical rules in traditional Chinese medicine. Taking this theory as a breakthrough point， this study employed gas chromatography-mass spectrometry （GC-MS） to elucidate the mechanism underlying the therapeutic effects of Zhuyuwan on both intrahepatic cholestasis （IC） and ulcerative colitis （UC） from the viewpoint of serum metabolic homeostasis. MethodsThe rat models of α-naphthylisothiocyanate （ANIT）-induced cholestasis and 2，4，6-trinitro-benzenesulfonic acid （TNBS）-induced UC were treated with low （0.6 g·kg^-1） and high （1.2 g·kg^-1） doses of Zhuyuwan by gavage. In the experiment regarding IC， 24 Sprague-Dawley （SD） rats were randomly assigned into four groups： normal， ANIT model， low-dose Zhuyuwan， and high-dose Zhuyuwan. In the experiment regarding UC， 24 SD rats were randomly allocated into four groups： normal， TNBS model， low-dose Zhuyuwan， and high-dose Zhuyuwan. Firstly， the two disease models and the intervention effects of Zhuyuwan on the two diseases were evaluated based on serum levels of biochemical indicators ［alanine aminotransferase （ALT）， aspartate transaminase （AST）， γ-glutamyltranspeptidase （γ-GT）， and total bile acid （TBA）］， colon damage score， colon weight index， disease activity index， and histopathological changes in rats. Secondly， the rat serum samples were analyzed by gas chromatography-mass spectrometry （GC-MS） to screen the common core pathways of the two disease models， and the expression of core genes in the pathways was determined by Real-time PCR， on the basis of which the biological mechanism of the treatment of the two disease models by Zhuyuwan was ultimately elucidated. ResultsThe results of the experiment regarding IC showed that the ANIT model group had higher ALT， AST， γ-GT， and TBA levels than the normal group （P<0.01）. Compared with the ANIT model group， the low-dose Zhuyuwan group showed declined ALT and TBA levels （P<0.01） and the high-dose Zhuyuwan group showed lowered ALT， TBA， AST， and γ-GT levels （P<0.01）. The results of the experiment regarding UC showed that compared with the normal group， the TNBS model group presented increases in the colonic damage score， colon weight index， and disease activity index （P<0.01）. Compared with the TNBS model group， the low-dose Zhuyuwan group showcased declines in colon weight index （P<0.01） and disease activity index （P<0.05）， and the high-dose Zhuyuwan group showed reductions in the colon damage score， colon weight index， and disease activity index （P<0.01）. GC-MS metabolomics analysis combined with qRT-PCR demonstrated that Zhuyuwan had a similar inverse regulatory effect on arginine metabolism disruption in the above two disease models. ConclusionZhuyuwan exhibited definite therapeutic effects on both IC and UC， and the regulation of arginine biosynthesis pathway is the core mechanism for the treatment of both diseases by Zhuyuwan.

ObjectiveThe theory of homotherapy for heteropathy is one of the classical rules in traditional Chinese medicine. Taking this theory as a breakthrough point， this study employed gas chromatography-mass spectrometry （GC-MS） to elucidate the mechanism underlying the therapeutic effects of Zhuyuwan on both intrahepatic cholestasis （IC） and ulcerative colitis （UC） from the viewpoint of serum metabolic homeostasis. MethodsThe rat models of α-naphthylisothiocyanate （ANIT）-induced cholestasis and 2，4，6-trinitro-benzenesulfonic acid （TNBS）-induced UC were treated with low （0.6 g·kg^-1） and high （1.2 g·kg^-1） doses of Zhuyuwan by gavage. In the experiment regarding IC， 24 Sprague-Dawley （SD） rats were randomly assigned into four groups： normal， ANIT model， low-dose Zhuyuwan， and high-dose Zhuyuwan. In the experiment regarding UC， 24 SD rats were randomly allocated into four groups： normal， TNBS model， low-dose Zhuyuwan， and high-dose Zhuyuwan. Firstly， the two disease models and the intervention effects of Zhuyuwan on the two diseases were evaluated based on serum levels of biochemical indicators ［alanine aminotransferase （ALT）， aspartate transaminase （AST）， γ-glutamyltranspeptidase （γ-GT）， and total bile acid （TBA）］， colon damage score， colon weight index， disease activity index， and histopathological changes in rats. Secondly， the rat serum samples were analyzed by gas chromatography-mass spectrometry （GC-MS） to screen the common core pathways of the two disease models， and the expression of core genes in the pathways was determined by Real-time PCR， on the basis of which the biological mechanism of the treatment of the two disease models by Zhuyuwan was ultimately elucidated. ResultsThe results of the experiment regarding IC showed that the ANIT model group had higher ALT， AST， γ-GT， and TBA levels than the normal group （P<0.01）. Compared with the ANIT model group， the low-dose Zhuyuwan group showed declined ALT and TBA levels （P<0.01） and the high-dose Zhuyuwan group showed lowered ALT， TBA， AST， and γ-GT levels （P<0.01）. The results of the experiment regarding UC showed that compared with the normal group， the TNBS model group presented increases in the colonic damage score， colon weight index， and disease activity index （P<0.01）. Compared with the TNBS model group， the low-dose Zhuyuwan group showcased declines in colon weight index （P<0.01） and disease activity index （P<0.05）， and the high-dose Zhuyuwan group showed reductions in the colon damage score， colon weight index， and disease activity index （P<0.01）. GC-MS metabolomics analysis combined with qRT-PCR demonstrated that Zhuyuwan had a similar inverse regulatory effect on arginine metabolism disruption in the above two disease models. ConclusionZhuyuwan exhibited definite therapeutic effects on both IC and UC， and the regulation of arginine biosynthesis pathway is the core mechanism for the treatment of both diseases by Zhuyuwan.

In order to establish a method for the detection of serum antibodies to donkey-derived e-quine coronavirus(ECoV),recombinant ECoV N protein was expressed in E.coli system,purified by nickel column affinity chromatography and identified by Western blot.After optimizing the re-action conditions,the indirect ELISA(iELISA)detection method was established using the puri-fied recombinant protein as coating antigen and used to detect 143 clinical serum samples.The re-sults showed that the recombinant N protein,which has good reaction activity with serum antibod-y,was successfully expressed.The optimum conditions of the established iELISA method were as follows:the amount of antigen coated was 0.2 μg/well and overnight at 4 ℃,10％skimmed milk powder solution was sealed at 37℃ for 1.5 h,the dilution concentration of serum was 1∶200,and the enzyme-labeled secondary antibody diluted at 1∶10 000.The sensitivity test results showed that the positive serum could be diluted to 1∶6 400.The specificity test results showed that all an-tibodies to several donkey pathogens were negative.The repetitive test results showed that the in-tra-and inter-batch coefficients of variation were 2.90％-6.12％and 2.29％-7.88％respectively.The positive rate of clinical donkey serum was 57.3％.The iELISA established in this study pro-vides a technical support for epidemiological investigation and antibody surveillance.

Objective:To explore the clinical application of preimplantation genetic testing for monogenic disorders (PGT-M) in an unique case with Spinal muscular atrophy (SMA) type 2+ 0.Methods:A special SMA family presented at the Third Affiliated Hospital of Guangzhou Medical University on October 19, 2020 was selected as the study subject. Multiple ligation-dependent probe amplification (MLPA) and molecular tagging linkage analysis were carried out to identify the SMN1 genotype of the couple and their fetus. Subsequently, next-generation sequencing (NGS), molecular tagging linkage analysis, and chromosomal microarray analysis were employed to determine the haplotypes and validate the result of PGT-M on the 11 embryos derived for the couple. Results:The female partner was identified as a carrier of the rare SMN1[2+ 0] variant, and prenatal diagnosis confirmed the fetus to be affected by SMA. Ultimately, PGT-M has successfully selected four embryos free from the pathogenic SMN1 variants and X chromosome deletion. Conclusion:PGT-M can effectively prevent the transmission of rare genetic variants such as the SMA 2+ 0 subtype in the families. Above finding has provided guidance for genetic counseling and family planning for the couple.

Objective To investigate the protective mechanism of Wenyang Fuyuan Prescription on nerve injury by improving brain iron metabolism in rats with cerebral ischemia-reperfusion injury(CIRI)based on ferroptosis.Methods A total of 72 SD rats were randomly divided into sham-operation group,CIRI model group,Wenyang Fuyuan Prescription group(18.0 g·kg-1,gavage),ferroptosis inducer group(100 mg·kg-1,intraperitoneal injection),Wenyang Fuyuan Prescription(18.0 g·kg-1,gavage)+ ferroptosis inducer group(intraperitoneal injection)and ferroptosis inhibitor group(5 mg·kg-1,intraperitoneal injection),12 rats in each group.All the procedures adopted in the sham group were the same as those in the model group.But nylon thread was inserted into the internal carotid artery at a depth of 9 mm and un-plugged middle cerebral artery.The rest of the groups were used to construct middle cerebral artery occlusion/reperfusion(MCAO/R)model by thread embolism method.Ferroptosis inducer(100 mg·kg-1)and ferroptosis inhibitor(5 mg·kg-1)were administered intraperitoneally to rats according to the grouping 24 hours before modeling.Wenyang Fuyuan Prescription(18.0 g·kg-1)was administered by gavage 2 hours after anesthesia and awakening.All intervention were given once daily for 7 consecutive days.The Longa scoring standard was used to evaluate the neurological deficit on 1,3,and 7 days after MCAO/R surgery,respectively.At the end of the treatment period,brain tissues were taken to observe the morphological changes of rat neurons in each group by hematoxylin eosin staining(HE).The ultrastructural changes of neuron mitochondria in each group were observed by transmission electron microscope.The biochemical kit was used to detect the content of iron ions(Fe2+)and reduced glutathione(GSH)in brain tissue.The protein and mRNA expressions of transferrin receptor 1(TFR1),iron regulatory protein 1(IRP1)and ferroportin(FPN)were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot.Results① Compared with sham group,the neurological deficit scores of rats in model group increased at each time point(P＜0.01).HE staining showed neurons were sparse and disordered,the nuclei underwent pyknosis,and vacuoles appeared at the edges.Under electron microscopy,it was observed that the number of neuronal mitochondria decreased,the density of mitochondrial membranes increased,massive numbers of mitochondrial membranes ruptured and dissolved,and mitochondrial cristae disappeared.The content of Fe2+,both mRNA and protein expressions of TFR1 were significantly increased(P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN were significantly decreased(P＜0.05,P＜0.01).② Compared with the model group,the neurological deficit scores of rats in the Wenyang Fuyuan Prescription group decreased at various time points(P＜0.05).The number of neurons increased,their arrangement was relatively neat,the morphology of the nucleus is complete and clear,the mitochondrial structure of neurons was relatively complete,the mitochondrial membrane was relatively intact,and the mitochondrial cristae were clear.The content of Fe2+,both mRNA and protein expressions of TFR1 were decreased(P＜0.05,P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN increased(P＜0.05,P＜0.01).③ Compared with the Wenyang Fuyuan Prescription group,the neurological deficit scores of rats in ferroptosis inducer group and the Wenyang Fuyuan Prescription + ferroptosis inducer group increased at all time points(P＜0.05).Distribution of neurons was in disorder,the nucleus shrinked,and vacuoles appeared at the edges.The density of mitochondrial membranes increased,some ruptured and dissolved mitochondrial membranes were found.The number of mitochondria decreased and mitochondrial cristae disappeared.The content of Fe2+,both TFR1 mRNA and protein expression increased(P＜0.05,P＜0.01),while the content of GSH,as well as expressions of mRNA and protein for IRP1 and FPN decreased(P＜0.05,P＜0.01).However,there was no statistically significant difference in all observed indicators between the ferroptosis inhibitor group and the Wenyang Fuyuan Prescription group(P＞0.05).Conclusion Wenyang Fuyuan Prescription can improve the neurological function and pathological damage of CIRI rats.Its mechanism may be related to regulating the expression of IRP1 protein,improving the brain iron metabolism pathway,and inhibiting ferroptosis.

COVID-19 represents the most serious public health event in the past few decades of the 21^st century. The development of vaccines, neutralizing antibodies, and small molecule chemical agents have effectively prevented the rapid spread of COVID-19. However, the continued emergence of SARS-CoV-2 variants have weakened the efficiency of these vaccines and antibodies, which brought new challenges for searching novel anti-SARS-CoV-2 drugs and methods. In the process of SARS-CoV-2 infection, the virus firstly attaches to heparan sulphate on the cell surface of respiratory tract, then specifically binds to hACE2. The S protein of SARS-CoV-2 is a highly glycosylated protein, and glycosylation is also important for the binding of hACE2 to S protein. Furthermore, the S protein is recognized by a series of lectin receptors in host cells. These finding implies that glycosylation plays important roles in the invasion and infection of SARS-CoV-2. Based on the glycosylation pattern and glycan recognition mechanisms of SARS-CoV-2, it is possible to develop glycan inhibitors against COVID-19. Recent studies have shown that sulfated polysaccharides originated from marine sources, heparin and some other glycans display anti-SARS-CoV-2 activity. This review summarized the function of glycosylation of SARS-CoV-2, discoveries of glycan inhibitors and the underpinning molecular mechanisms, which will provide guidelines to develop glycan-based new drugs against SARS-CoV-2.
Antibodies, Neutralizing ; Glycosylation ; Heparin ; Heparitin Sulfate ; Humans ; Polysaccharides/chemistry* ; Receptors, Mitogen/metabolism* ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus/metabolism* ; COVID-19 Drug Treatment

Objective:To investigate the efficacy and safety of hydromorphone for postoperative analgesia in children with congenital heart disease, and provide a suitable reference dose for postoperative analgesia in children.Methods:Using a prospective study, 157 patients with congenital heart disease(ASA Ⅱ- Ⅳ) admitted to pediatric intensive care unit at Children′s Hospital of Chongqing Medical University from November 2019 to November 2021 were randomly divided into five groups.Low-dose hydromorphone group (H1 group, 30 cases): hydromorphone dose ≥2 and <3 μg/(kg·h), hydromorphone medium-dose group (H2 group, 30 cases): hydromorphone dose ≥3 and <4 μg/(kg·h), high-dose hydromorphone group (H3 group, 31 cases): hydromorphone dose ≥4 and ≤5 μg/(kg·h), sufentanil group (S group, 36 cases): the dose of sufentanil was 0.08 μg/(kg·h), morphine group (M group, 30 cases): the dose of morphine was 20 μg/(kg·h). The five groups of children received midazolam 2 μg/(kg·min) intravenously at the same time as sedative therapy.Pain score and sedation score were scored at 1 h, 4 h, 8 h, 12 h, and 24 h after operation.Heart rate, mean arterial pressure, blood glucose, lactate, and serum cortisol levels were also monitored and detected, and the occurrence of adverse reactions, the number of cases requiring additional analgesic and sedative drugs, as well as the duration of mechanical ventilation were compared.Results:(1) There were no significant differences regarding the age, body weight, cardiopulmonary bypass time, pediatric critical illness score and ASA score among five groups (all P>0.05). (2) There was no significant difference in the levels of respiration, heart rate, blood sugar, lactate and serum cortisol among five groups after operation.There was no significant difference in preoperative mean arterial pressure among the groups, but there was significant difference in the postoperative mean arterial pressure among the groups at 4 h and 8 h( P<0.05). (3) The analgesic satisfaction of H1 group, H2 group and H3 group at 1 h, 4 h, 12 h and 24 h after operation was significantly higher than that in M group ( P<0.05). There was no significant difference in analgesic satisfaction among H1 group, H2 group and H3 group at each time point.(4) The sedation satisfaction of H1 group, H2 group and H3 group at 4 h and 24 h after operation was significantly higher than that in M group ( P<0.05). There was no significant difference in sedation satisfaction among H1 group, H2 group and H3 group at each time point.(5) There was no significant difference in postoperative analgesia satisfaction and sedation satisfaction between H1 group, H2 group, H3 group and S group.(6) Children in H1 group[1(0, 2)], H2 group[1(0, 2)], H3 group[1(0, 2)] had fewer additional doses within 24 hours than that in M group[2(2, 3)]( P<0.05), and fewer children in H1 group, H2 group and H3 group had been given analgesic sedatives than that in M group ( P<0.05); The extubation time was shortest in H2 group and S group[H2 group(88.3±2.9) h, S group(85.9±3.0) h]. (7) There were no adverse reactions in H1 group, H2 group, H3 group and S group, and there were two cases of apnea in M group. Conclusion:The analgesic effect of hydromorphone in children with congenital heart disease after surgery is better than that of morphine, and the effect of hydromorphone is comparable to that of sufentanil.Hydromorphone 3-4 μg/(kg·h)+ midazolam 2 μg/(kg·min) can achieve satisfactory analgesic and sedative effects in children after congenital heart surgery, with few adverse reactions, safe and reliable, which is an excellent choice for postoperative analgesia and sedation in children.

Objective: To explore the clinical features and risk factors of hepatic injury due to immune checkpoint inhibitors (CPI) therapy in malignant tumor. Methods: Data of 112 patients (64 men and 48 women) who received CPI between January 2016 and March 2019 in Chinese Academy of Medical Sciences and Peking Union Medical College Shenzhen Hospital, and Huazhong University of Science and Techology Union Shenzhen Hospital were retrospectively collected. The median age of these patients was 60 years. Results: Hepatic adverse events were observed in 30 patients out of 112 patients (26.8%). Among them, the incidence of grade 3-5 hepatic adverse events were 7.14% (8/112). The median time of hepatic adverse event occurrence was 3 weeks (2-30) after undergoing therapy. The results of univariate and multivariate analyses showed that liver cancer was attributed to the CPI induced hepatitis (P<0.05). Patients with severe hepatic injury got almost complete resolution after receiving methlprednisolone for 4 to 6 weeks. Conclusion Live cancer is the risk factor of CPI-related hepatic adverse events.