ObjectiveTo study the effect of Qizhu Kang'ai prescription （QZAP） on the gluconeogenesis enzyme phosphoenolpyruvate carboxykinase 1 （PCK1） in the liver of mouse model of liver cancer induced by diethylnitrosamine （DEN） combined with carbon tetrachloride （CCl₄） and Huh7 cells of human liver cancer， so as to explore the mechanism on regulating metabolic reprogramming and inhibiting cell proliferation of liver cancer cells. MethodDEN combined with CCl₄ was used to construct a mouse model of liver cancer via intraperitoneal injection. A normal group， a model group， and a QZAP group were set up， in which QZAP （3.51 g·kg^-1） or an equal volume of normal saline was administered daily by gavage， respectively. Serum and liver samples were collected after eight weeks of intervention. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyltransferase （γ-GT）， and alpha-fetoprotein （AFP） in mice were detected to evaluate liver function changes of mice in each group. Hematoxylin-eosin （HE） staining and Sirius red staining were used to observe pathological changes in liver tissue. In the cell experiment， Huh7 cells were divided into blank group， QZAP low， medium， and high dose groups and/or PCK1 inhibitor （SKF-34288 hydrochloride） group， and Sorafenib group. The corresponding drug-containing serum and drug treatment were given， respectively. Cell counting kit-8 （CCK-8） method， colony formation experiment， Edu fluorescent labeling detection， intracellular adenosine triphosphate （ATP） content detection， and cell cycle flow cytometry detection were used to evaluate the proliferation ability， energy metabolism changes， and change in the cell cycle of Huh7 cells in each group. Western blot was used to detect the protein expression levels of PCK1， serine/threonine kinase （Akt）， phosphorylated Akt （p-Akt）， and cell cycle-dependent protein kinase inhibitor 1A （p21）. ResultCompared with the model group， the pathological changes such as cell atypia， necrosis， and collagen fiber deposition in liver cancer tissue of mice in the QZAP group were alleviated， and the number of liver tumors was reduced （P<0.01）. The serum ALT， AST， γ-GT， and AFP levels were reduced （P<0.01）. At the cell level， compared with the blank group， low， medium， and high-dose groups of QZAP-containing serum and the Sorafenib group could significantly reduce the survival rate of Huh7 cells （P<0.01） and the number of positive cells with Edu labeling （P<0.01） and inhibit clonal proliferation ability （P<0.01）. The QZAP groups could also reduce the intracellular ATP content （P<0.05） and increase the distribution ratio of the G₀/G₁ phase of the cell cycle （P<0.05） in a dose-dependent manner. Compared with the model group and blank group， PCK1 and p21 protein levels of mouse liver cancer tissue and Huh7 cells in the QZAP groups were significantly reduced （P<0.05，P<0.01）， and the p-Akt protein level was significantly increased （P<0.01）. Compared with the blank group， the ATP content and cell survival rate of Huh7 cells in the SKF-34288 hydrochloride group were significantly increased （P<0.05）， but there was no statistical difference in the ratio of Edu-positive cells and the proportion of G₀/G₁ phase distribution. Compared with the SKF-34288 hydrochloride group， the QZAP combined with the SKF-34288 hydrochloride group significantly reduced the ATP content， cell survival rate， and Edu-positive cell ratio of Huh7 cells （P<0.05） and significantly increased the G₀/G₁ phase distribution proportion （P<0.05）. ConclusionQZAP may induce the metabolic reprogramming of liver cancer cells by activating PCK1 to promote Akt/p21-mediated tumor suppression， thereby exerting an anti-hepatocellular carcinoma proliferation mechanism.

ObjectiveTo investigate the changes in the pathogen spectrum of viral diarrhea in local pediatric inpatients as well as any variations in genotypes of major pathogens during the COVID-19 control period. MethodsFecal samples were collected from the children <5 years who were hospitalized due to acute gastroenteritis in a pediatric hospital in Shanghai. PCR test was carried out to detect rotavirus， norovirus， sapovirus， astrovirus and enteric adenovirus， and then genotyping was performed for major pathogens. ResultsOut of 546 samples， 37.55% tested positive for virus with the following positive rate ranking： norovirus GⅡ （22.16%）， group A rotavirus （16.12%）， astrovirus （2.93%）， enteric adenovirus （2.38%）， sapovirus （0.92%） and norovirus GⅠ （0.18%）. The predominant genotype within norovirus GⅡ were GⅡ.4［P31］ and GⅡ.4［P16］ with a proportion of 24.79% and 14.05% respectively. The detection rate of GⅡ.4［P31］ dropped significantly over the 2-year period （χ²＝16.140，P<0.001）. In addition， an emerging rotavirus genotype G8P ［8］， which was rarely found nationally， was discovered for the first time locally with an increasing proportion， accounting for 7.95% of all rotavirus positive cases. Phylogenic analysis demonstrated that the representative strains of this genotype were genetically closer to the DS-1-like G8P ［8］ strain found in Southeast Asia. ConclusionThe changes in the prevalence of various norovirus genotypes together with the emergence of rare rotavirus genotype in the local area illustrate the importance of continuous monitoring of viral diarrhea and genotyping of key pathogens. Increased local activity of the rare genotype also adds new parameters in the efficacy evaluation of marketed vaccines and development of potential new vaccines in near future.

Objective:To establish UPLC fingerprint method and 2 contents determination methods of Buddleja officinalis; To provide a reference for improving the quality control standard and evaluation of Buddleja officinalis from different habitats.Methods:UPLC method was used to establish the fingerprints of 17 batches of Buddleja officinalis. The similarity evaluation, clustering analysis, principal component analysis and orthogonal partial least squares discriminant analysis were used to compare the quality differences of Buddleja officinalis from different habitats. The contents of acteoside and linarin in Buddleja officinalis were determined.Results:There were 12 common peaks in UPLC fingerprints of Buddleja officinalis, six of which were identified as echinacoside, acteoside, cynaroside, isoacteoside, linarin, and apigenin. The fingerprint similarity of 17 batches of Buddleja officinalis was more than 0.9; Buddleja officinalis from different habitats were classified into 2 groups. Five differential markers were determined by OPLS-DA analysis. The order of significance was acteoside > peak 3 > echinacoside > isoacteoside > linarin. Edgeworthia chrysantha was identified by the method of fingerprint as counterfeit. The results of content determination showed that the content of Buddleja officinalis in Hubei and Sichuan was the high and stable.Conclusion:The method can effectively analyze the differences of Buddleja officinalis from different habitats, and provide reference for the quality control of Buddleja officinalis.

BACKGROUND:Numerous basic and clinical trials have confirmed that the low survival rate after bone marrow mesenchymal stem cell transplantation is a serious constraint on its long-term therapeutic effect.Previous studies have shown that apoptosis-related factors play an important role in the apoptosis of bone marrow mesenchymal stem cells,of which apoptosis-inducing factor may be a key factor. OBJECTIVE:Bone marrow mesenchymal stem cells,of which apoptosis-inducing factor was knocked down,were transplanted into infarcted myocardium of mice,aiming to certify the importance of apoptosis-inducing factor in the survival of bone marrow mesenchymal stem cells to further recover cardiac function after infarction. METHODS:Firstly,bone marrow mesenchymal stem cells were infected with LV-AIF-shRNA lentivirus to down-regulate the expression of apoptosis-inducing factor protein.Flow cytometry,western blot assay,and RT-qPCR were used to detect the infection efficiency of lentivirus.CCK-8 assay was used to detect the cell viability of bone marrow mesenchymal stem cells with apoptosis-inducing factor knockdown under hypoxic and ischemic conditions.Then,with the mouse model of acute myocardial infarction constructed,the normal bone marrow mesenchymal stem cells and bone marrow mesenchymal stem cells with apoptosis-inducing factor gene knockdown were transplanted into the model,respectively.The expression of apoptosis-inducing factor was examined by fluorescence immunoassay.Serum brain natriuretic peptide levels were detected by ELISA.Cardiac ultrasound was used to detect cardiac function.Myocardial fibrosis was observed by Masson staining.The expression of SRY gene was detected by RT-qPCR in apoptosis-inducing factor-knocked bone marrow mesenchymal stem cells after transplantation,reflecting cell survival. RESULTS AND CONCLUSION:(1)Bone marrow mesenchymal stem cells with apoptosis-inducing factor gene knockdown were successfully established by LV-AIF-shRNA lentivirus infection,following 97.7%of infection efficiency,and notably decline of the expression of apoptosis-inducing factor(P<0.001).(2)Under ischemia and hypoxia,the cell viability of apoptosis-inducing factor knockdown bone marrow mesenchymal stem cells was significantly increased compared with normal bone marrow mesenchymal stem cells.(3)Compared with normal bone marrow mesenchymal stem cells after transplantation,the survival number of bone marrow mesenchymal stem cells in the infarcted myocardium after apoptosis-inducing factor gene knockdown was significantly increased to 3.71 times(P<0.001),and the apoptosis-inducing factor protein expression and myocardial fibrosis degree in the infarcted area were significantly reduced.(4)Compared with normal bone marrow mesenchymal stem cells,the serum brain natriuretic peptide level of bone marrow stem cells with apoptosis-inducing factor gene knockdown after transplantation was significantly decreased(P<0.05),and left ventricular ejection fraction and left ventricular shortening fraction were significantly improved(P<0.05).(5)These findings confirm that apoptosis-inducing factor gene knockdown can reduce myocardial fibrosis and improve cardiac function after acute myocardial infarction via enhancing the bone marrow mesenchymal stem cell viability and increasing the bone marrow mesenchymal stem cell survival after transplantation in the donor.

Doxorubicin(DOX)is a commonly administered chemotherapy drug for treating hematological malignancies and solid tumors;however,its clinical application is limited by significant cardiotoxicity.Cynaroside(Cyn)is a flavonoid glycoside distributed in honeysuckle,with confirmed potential biological functions in regulating inflammation,pyroptosis,and oxidative stress.Herein,the effects of Cyn were evaluated in a DOX-induced cardiotoxicity(DIC)mouse model,which was established by intraperitoneal injections of DOX(5 mg/kg)once a week for three weeks.The mice in the treatment group received dexrazoxane,MCC950,and Cyn every two days.Blood biochemistry,histopathology,immunohistochemistry,reverse transcription-quantitative polymerase chain reaction(RT-qPCR),and western blotting were conducted to investigate the cardioprotective effects and potential mechanisms of Cyn treatment.The results demonstrated the significant benefits of Cyn treatment in mitigating DIC;it could effectively alleviate oxidative stress to a certain extent,maintain the equilibrium of cell apoptosis,and enhance the cardiac function of mice.These effects were realized via regulating the transcription levels of pyroptosis-related genes,such as nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),caspase-1,and gasdermin D(GSDMD).Mechanistically,for DOX-induced myocardial injury,Cyn could significantly modulate the expression of pivotal genes,including adenosine monophosphate-activated protein kinase(AMPK),peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α),sirtuin 3(SIRT3),and nuclear factor erythroid 2-related factor 2(Nrf2).We attribute it to the mediation of AMPK/SIRT3/Nrf2 pathway,which plays a central role in preventing DOX-induced cardiomyocyte injury.In conclusion,the present study confirms the therapeutic potential of Cyn in DIC by regulating the AMPK/SIRT3/Nrf2 pathway.

BACKGROUND: Hair follicles are easily accessible and contain stem cells with different developmental origins, including mesenchymal stem cells (MSCs), that consequently reveal the potential of human hair follicle (hHF)-derived MSCs in repair and regeneration. However, the role of hHF-MSCs in Achilles tendinopathy (AT) remains unclear. The present study investigated the effects of hHF-MSCs on Achilles tendon repair in rabbits. METHODS: First, we extracted and characterized hHF-MSCs. Then, a rabbit tendinopathy model was constructed to analyze the ability of hHF-MSCs to promote repair in vivo . Anatomical observation and pathological and biomechanical analyses were performed to determine the effect of hHF-MSCs on AT, and quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical staining were performed to explore the molecular mechanisms through which hHF-MSCs affects AT. Furthermore, statistical analyses were performed using independent sample t test, one-way analysis of variance (ANOVA), and one-way repeated measures multivariate ANOVA as appropriate. RESULTS: Flow cytometry, a trilineage-induced differentiation test, confirmed that hHF-derived stem cells were derived from MSCs. The effect of hHF-MSCs on AT revealed that the Achilles tendon was anatomically healthy, as well as the maximum load carried by the Achilles tendon and hydroxyproline proteomic levels were increased. Moreover, collagen I and III were upregulated in rabbit AT treated with hHF-MSCs (compared with AT group; P  < 0.05). Analysis of the molecular mechanisms revealed that hHF-MSCs promoted collagen fiber regeneration, possibly through Tenascin-C (TNC) upregulation and matrix metalloproteinase (MMP)-9 downregulation. CONCLUSIONS hHF-MSCs can be a treatment modality to promote AT repair in rabbits by upregulating collagen I and III. Further analysis revealed that treatment of AT using hHF-MSCs promoted the regeneration of collagen fiber, possibly because of upregulation of TNC and downregulation of MMP-9, thus suggesting that hHF-MSCs are more promising for AT.
Animals ; Humans ; Rabbits ; Hair Follicle ; Achilles Tendon/pathology* ; Tendinopathy/pathology* ; Proteomics ; Collagen Type I ; Mesenchymal Stem Cells

【Objective】 To investigate the predictive value of high preoperative neutrophile-lymphocyte ratio (NLR) for the prognosis of nonurothelial carcinoma of the bladder (NUBC) after radical cystectomy (RC). 【Methods】 Clinical and follow-up data of NUBC patients undergoing RC during Jan.2005 and Dec.2020 were collected. The optimal cut-off value of NLR was determined with the receiver operating characteristic (ROC) curve. The survival curve was drawn with Kaplan-Meier method to compare the differences in cancer specific survival (CSS) and overall survival (OS) between the high-NLR and low-NLR groups. The independent risk factors of CSS and OS were screened with Cox proportional hazard regression model. 【Results】 Of the 62 eligible cases,34 (54.8%) were diagnosed with adenocarcinoma,17 (27.4%) with squamous cell carcinoma, 6 (9.7%) with small cell carcinoma and 5 (8.1%) with sarcoma. Kaplan-Meier analysis results showed high NLR was associated with poor CSS (P=0.001) and OS (P<0.001). Cox regression results indicated that high NLR (HR=2.42, 95%CI: 1.12-5.23, P=0.025) and advanced pathologic tumor stage (HR=3.21, 95%CI:1.53-6.74,P=0.002) were independent risk factors of unfavorable CSS. Similarly, high NLR (HR=2.75, 95%CI: 1.35-5.56, P=0.005) and advanced pathologic tumor stage (HR=2.81, 95%CI:1.43-5.57, P=0.003) were independent risk factors of unfavorable OS. 【Conclusion】 As an independent risk factor of unfavorable CSS and OS in NUBC patients undergoing RC, high preoperative NLR is of great value in the prediction of long-term prognosis and may help to optimize individualized treatment.

Objective:To comprehensively evaluated the quality of Sargentodoxae Caulis from different habitats with a combination of indexes and characteristic chromatogram method from Chinese Pharmcopoeia (Edition 2020). Methods:The contents of water content, total ash, ethanolic extract, sulfur dioxide residue, heavy metals and harmful elements, total phenols, chlorogenic acid, salidroside and characteristic chromatogram of 17 batches of Sargentodoxae Caulis were determined. The quality of Sargentodoxae Caulis was comprehensively evaluated by combining chemical pattern recognition method. Results:The water content, total ash content, extracts, and content determination of 17 batches of Sargentodoxae Caulis from different habitats complyed with the provisions of the Chinese Pharmcopoeia (Edition 2020). There were differences in the contents of extracts, chlorogenic acid, and salidroside, among which the content of Anhui origin was higher. A total of 8 common peaks were identified from the 17 batches samples. Conclusion:Comprehensive evaluation of multiple indicators can demonstrate the quality of Sargentodoxae Caulis more correctly, and shows that the quality of Sargentodoxae Caulis from different habitats is different. The quality of Sargentodoxae Caulis from Anhui is better than that from other habitats.

Objective:Based on radiomics characteristics, different machine learning classification models are constructed to predict the gamma pass rate of dose verification in intensity-modulated radiotherapy for pelvic tumors, and to explore the best prediction model.Methods:The results of three-dimensional dose verification based on phantom measurement were retrospectively analyzed in 196 patients with pelvic tumor intensity-modulated radiotherapy plans. The gamma pass rate standard was 3%/2 mm and 10% dose threshold. Prediction models were constructed by extracting radiomic features based on dose documentation. Four machine learning algorithms, random forest, support vector machine, adaptive boosting, and gradient boosting decision tree were used to calculate the AUC value, sensitivity, and specificity respectively. The classification performance of the four prediction models was evaluated.Results:The sensitivity and specificity of the random forest, support vector machine, adaptive boosting, and gradient boosting decision tree models were 0.93, 0.85, 0.93, 0.96, 0.38, 0.69, 0.46, and 0.46, respectively. The AUC values were 0.81 and 0.82 for the random forest and adaptive boosting models, respectively, and 0.87 for the support vector machine and gradient boosting decision tree models.Conclusions:Machine learning method based on radiomics can be used to construct a prediction model of gamma pass rate for specific dosimetric verification of pelvic intensity-modulated radiotherapy. The classification performance of the support vector machine model and gradient boosting decision tree model is better than that of the random forest model and adaptive boosting model.

Objective:To investigate the value of cystatin C (Cys-C) in the early diagnosis of acute kidney injury (AKI) after radical nephrectomy and the predictive value for the prognosis of Cys-C based estimated glomerular filtration rate (eGFR Cys-C) after surgery. Methods:The clinical data of 118 patients who underwent unilateral radical nephrectomy in our hospital from January 2019 to December 2020 were retrospectively analyzed. According to the diagnostic criteria of AKI, they were divided into AKI group of 75 cases and no-AKI group of 43 cases. AKI group was (62.7±10.7) years old, with 49 males and 26 females. The no-AKI group was (62.3±12.8) years old, with 21 males and 22 females. The urea nitrogen was (4.9±1.3) mmol/L, creatinine (75.7±14.5)μmol/L, Cys-C (0.85±0.22) mg/L, eGFR Cr(76.3±11.2)ml/(min·1.73m 2), and eGFR Cys-C(101.4±17.4)ml/(min·1.73m 2)in AKI group before operation.In no-AKI group, preoperative urea nitrogen was (4.9±1.5) mmol/L, creatinine (74.5±13.1)μmol/L, Cys-C (0.81±0.29) mg/L, eGFR Cr(78.6±12.5)ml/(min·1.73m 2), and eGFR Cys-C(99.3±18.8)ml/(min·1.73m 2), and there were no significant differences in the values of urea nitrogen, creatinine, Cys-C and eGFR between the two groups before surgery ( P>0.05). ROC curve was used to analyze the diagnostic value of urea nitrogen, creatinine, Cys-C, eGFR calculated based on creatinine and Cys-C at 48h after surgery, and binary Logistic regression was used to analyze the risk factors for AKI. The creatinine status of patients diagnosed with SPS was evaluated 6 months after surgery, based on the definition of Cys-C based eGFR being less than 70% of creatinine-based eGFR(SPS=eGFR Cys-C/ eGFR Cr≤0.7). Results:In AKI group, creatinine was(115.2±22.1)μumol/L, Cys-C (1.8±0.27) mg/L, eGFR Cr (51.6±9.6)ml/(min·1.73m 2), and eGFR Cys-C(43.4±8.5)ml/(min·1.73m 2)48 h after operation. The creatinine was(92.7±13.3)μmol/L, Cys-C(1.3±0.23) mg/L, eGFR Cr(62.2±11.3)ml/(min·1.73m 2), and eGFR Cys-C(61.5±9.5)ml/(min·1.73m 2) in no-AKI group, and difference were statistically significant between the two groups ( P<0.01). ROC curve was used to analyze the diagnosis of AKI. Creatinine, Cys-C, eGFR Cr and eGFR Cys-Cwere all of diagnostic value for AKI (all P<0.01), and AUC(Area under curve) were 0.809, 0.889, 0.761 and 0.925 respectively. The sensitivity, specificity and area under the curve of eGFR Cys-C were 93.3%, 74.4% and 92.5% respectively. Binary Logistic regression analysis showed that creatinine( OR=10.851, 95% CI 2.322-50.688, P=0.004), Cys-C( OR=10.016, 95% CI 2.306-43.362, P=0.001), eGFR Cr( OR=17.923, 95%CI 3.216-53.172, P=0.001) and eGFR Cys-C( OR=19.817, 95% CI 3.367-55.263, P=0.001)were all independent risk factors for AKI. The predictive accuracy of eGFR Cys-C, creatinine, Cys-C, eGFR Cr were 91.6%, 85.7%, 90.2%, 88.5%, respectively. There were 15 cases were confirmed SPS in the AKI group, and only 2 cases were confirmed SPS in the no-AKI group, indicating patients in the AKI group developed more SPS than those in the no-AKI group, with statistically significant difference(Kappa value was 5.22, P=0.02). The 6-month follow-up showed that the creatinine of confirmed SPS was (103.8±23.4)μmol/L and the creatinine of unconfirmed SPS was (86.8±27.2)μmol/L, with statistically significant difference ( P<0.01). Conclusions:eGFR Cys-C calculated based on Cys-C has high sensitivity in diagnosing AKI and has early diagnostic value. Patients diagnosed with SPS based on eGFR Cys-C had higher creatinine 6 months after surgery.