Animal experiments are essential tools in biomedical research, serving as a bridge between basic research and clinical trials. Systematic reviews and meta-analyses (SRs/MAs) of animal experiments are crucial methods for integrating evidence from animal experiment, which can facilitate the translation of findings into clinical research, reduce translational risks, and promote resource integration in basic research. With the continuous development of the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) methodology, its application in SRs/MAs of animal experiments has gained increasing attention. This article first outlines the principles and specific applications of the GRADE methodology in SRs/MAs of animal experiments, including qualitative descriptive systematic reviews, meta-analyses, and network meta-analyses. It then deeply analyzes the misuse of the GRADE methodology in practice, including incorrect evidence grading, improper classification of evidence, misapplication in qualitative systematic reviews, inconsistencies between the documentation of the upgrading and downgrading process and results, and inappropriate use for making recommendations. Furthermore, this article comprehensively discusses the factors influencing the grading of evidence certainty in SRs/MAs of animal experiments, including the impact of bias risk, indirectness, inconsistency, imprecision, and publication bias on evidence downgrading, as well as the role of large effect sizes and cross-species consistency in evidence upgrading. Finally, in response to the issues discussed, improvement strategies are proposed, including further research and optimization of the GRADE methodology for SRs/MAs of animal experiments, the development of reporting guidelines tailored to the characteristics of SRs/MAs in animal experiment research, and enhanced professional training for researchers in the GRADE methodology. This article aims to improve the quality of evidence in SRs/MAs of animal experiments, strengthen their reliability in clinical decision-making, and promote the more efficient translation of findings from animal experiment research into clinical practice.

ObjectiveTo evaluate the clinical efficacy and safety of Qidan Tangshen Granules （芪丹糖肾颗粒， QTG） in the treatment of early diabetic kidney disease （DKD） with qi deficiency， blood stasis， and kidney deficiency syndrome， and to explore its mechanism. MethodsA double-blind， double-simulated method was used to enroll 200 patients with early DKD and qi deficiency， blood stasis， and kidney deficiency syndrome. Patients were randomly assigned in a 1∶1 ratio to the treatment group （100 cases） and the control group （100 cases）. The treatment group received QTG plus a valsartan capsule simulant， while the control group received valsartan capsules plus a QTG simulant， both for 12 weeks. The primary outcome was the urinary albumin-to-creatinine ratio （UACR）. Secondary outcomes included estimated glomerular filtration rate （eGFR）， fasting blood glucose （FBG）， 2-hour postprandial blood glucose （PBG）， glycated hemoglobin （HbA1c）， and traditional Chinese medicine （TCM） syndrome scores （including individual symptom scores for fatigue， dull complexion， soreness and weakness of the waist and knees， headache and chest pain， irritability， spontaneous sweating， thirst and polydipsia， polyphagia， polyuria， numbness of the limbs， and the total TCM syndrome score）. Oxidative stress markers including serum 8-hydroxy-2'-deoxyguanosine （8-OHDG）， 3-nitrotyrosine （3-NT）， and superoxide dismutase （SOD） were also assessed. Clinical efficacy and TCM syndrome efficacy were evaluated after treatment， and routine blood tests， urinalysis， and liver function tests were conducted and adverse reaction during the tria was recorded to assess safety. ResultsA total of 191 patients completed the study （95 in the treatment group and 96 in the control group）. The treatment group showed significant reductions in UACR， FBG， PBG， and HbA1c levels after treatment （P＜0.05 or P＜0.01）. The single TCM symptom scores except for polyphagia and total TCM syndrome scores significantly decreased （P＜0.05 or P＜0.01）. Compared to the control group， the treatment group had signi-ficantly lower UACR， FBG， PBG levels， and total TCM syndrome scores， sinlge symptoms scores except for polyphagia and limb numbness （P＜0.05 or P＜0.01）. Among 40 randomly selected patients （21 cases in the treatment group and 19 cases in the control group） for oxidative stress analysis， there were no significant differences in SOD， 3-NT， and 8-OHDG levels before and after treatment within or between groups （P＞0.05）. The overall effective rate in the treatment group was 64.2% （61/95） and 39.6% （38/96） in the control group， while the TCM syndrome efficacy rates were 80.0% （76/95） and 24.0% （23/96）， respectively， with the treatment group showing superior efficacy （P＜0.01）. No significant differences were observed in routine blood tests， urinalysis， or liver function indices before and after treatment in either group （P＞0.05）. The incidence of adverse reactions was 8.4% （8/95） in the treatment group and 9.4% （9/96） in the control group， with no statistically significant difference （P＞0.05）. ConclusionQTG can effectively reduce UACR and blood glucose levels， alleviate clinical symptoms， and improve clinical efficacy in patients with early DKD with qi deficiency， blood stasis， and kidney deficiency syndrome. The treatment is well-tolerated and safe， with no significant impact on oxidative stress markers.

To monitor and analyze the molecular variation of the H3N2 influenza virus in Guangzhou during the COVID-19 pandemic, respiratory samples of influenza-like cases from influenza monitoring sentinel hospitals were collected from influenza monitoring sentinel hospitals for virus isolation and whole genome sequencing. The results showed that during COVID-19, there was only one peak of H3N2 influenza in the second quarter of 2022 in Guangzhou (the positive rate was 52.23%), and the epidemic intensity and duration were both higher than those in 2019. The HA gene and NA gene of the epidemic strain in Guangzhou in 2022 belonged to the 3C.2a1b. 2a. 1a. 1 branch, which had a good antigenic site matching with the vaccine strain (A/Cambodia/e0826360/2020) from 2021 to 2022 and had no antigen drift. In 2022 strains, the variation of antigen determinant mainly occurred in the I48T of C region, while no variation occurred in the A, B, D, and E regions. The binding site of the HA protein receptor was consistent with the vaccine strain (A/Cambodia/e0826360/2020). Most of the strains in 2022 carried 13 glycosylation sites on the HA protein, but an outbreak of strains caused a loss of glycosylation sites at 24-NST. In conclusion, the strains that caused the epidemic of H3N2 influenza in Guangzhou in 2022 were not evolved or transmitted from the local strains in 2019 during the COVID-19 pandemic.

Background::Although some well-established oncogenes are involved in cancer initiation and progression such as prostate cancer (PCa), the long tail of cancer genes remains to be defined. Goosecoid ( GSC) has been implicated in cancer development. However, the comprehensive biological role of GSC in pan-cancer, specifically in PCa, remains unexplored. The aim of this study was to investigate the role of GSC in PCa development. Methods::We performed a systematic bioinformatics exploration of GSC using datasets from The Cancer Genome Atlas, Genotype-Tissue Expression, Gene Expression Omnibus, German Cancer Research Center, and our in-house cohorts. First, we evaluated the expression of GSC and its association with patient prognosis, and identified GSC-relevant genetic alterations in cancers. Further, we focused on the clinical characterization and prognostic analysis of GSC in PCa. To understand the transcriptional regulation of GSC by E2F transcription factor 1 ( E2F1), we performed chromatin immunoprecipitation quantitative polymerase chain reaction (qPCR). Functional experiments were conducted to validate the effect of GSC on the tumor cellular phenotype and sensitivity to trametinib. Results::GSC expression was elevated in various tumors and significantly correlated with patient prognosis. The alterations of GSC contribute to the progression of various tumors especially in PCa. Patients with PCa and high GSC expression exhibited worse progression-free survival and biochemical recurrence outcomes. Further, GSC upregulation in patients with PCa was mostly accompanied with higher Gleason score, advanced tumor stage, lymph node metastasis, and elevated prostate-specific antigen (PSA) levels. Mechanistically, the transcription factor, E2F1, stimulates GSC by binding to its promoter region. Detailed experiments further demonstrated that GSC acted as an oncogene and influenced the response of PCa cells to trametinib treatment. Conclusions::GSC was highly overexpressed and strongly correlated with patient prognosis in PCa. We found that GSC, regulated by E2F1, acted as an oncogene and impeded the therapeutic efficacy of trametinib in PCa.

To monitor and analyze the molecular variation of the H3N2 influenza virus in Guangzhou during the COVID-19 pandemic, respiratory samples of influenza-like cases from influenza monitoring sentinel hospitals were collected from influenza monitoring sentinel hospitals for virus isolation and whole genome sequencing. The results showed that during COVID-19, there was only one peak of H3N2 influenza in the second quarter of 2022 in Guangzhou (the positive rate was 52.23%), and the epidemic intensity and duration were both higher than those in 2019. The HA gene and NA gene of the epidemic strain in Guangzhou in 2022 belonged to the 3C.2a1b. 2a. 1a. 1 branch, which had a good antigenic site matching with the vaccine strain (A/Cambodia/e0826360/2020) from 2021 to 2022 and had no antigen drift. In 2022 strains, the variation of antigen determinant mainly occurred in the I48T of C region, while no variation occurred in the A, B, D, and E regions. The binding site of the HA protein receptor was consistent with the vaccine strain (A/Cambodia/e0826360/2020). Most of the strains in 2022 carried 13 glycosylation sites on the HA protein, but an outbreak of strains caused a loss of glycosylation sites at 24-NST. In conclusion, the strains that caused the epidemic of H3N2 influenza in Guangzhou in 2022 were not evolved or transmitted from the local strains in 2019 during the COVID-19 pandemic.

Objective:To identify a novel reassortant H3N2 avian influenza virus using nanopore sequencing technology and analyze its genetic characteristics.Methods:The positive samples of the H3N2 avian influenza virus, collected from the external environment in the farmers' market of Guangzhou, were cultured in chicken embryos. The whole genome was sequenced by targeted amplification and nanopore sequencing technology. The genetic characteristics were analyzed using bioinformatics software.Results:The phylogenetic trees showed that each gene fragment of the strain belonged to the Eurasian evolutionary branch, and the host source was of avian origin. The HA gene was closely related to the origin of the H3N6 virus. The NA gene was closely related to the H3N2 avian influenza virus from 2017 to 2020. The PB1 gene was closely related to the H5N6 avian influenza virus in Guangxi Zhuang Autonomous Region and Fujian Province from 2016 to 2022 and was not related to the PB1 gene of the H5N6 avian influenza epidemic strain in Guangzhou. The other internal gene fragments had complex sources with significant genetic diversity. Molecular characteristics indicated that the strain exhibited the molecular characteristics of a typical low pathogenic avian influenza virus and tended to bind to the receptors of avian origin. On important protein sites related to biological characteristics, this strain had mutations of PB2-L89V, PB1-L473V, NP-A184K, M1-N30D/T215A, and NS1-P42S/N205S.Conclusions:This study identified a novel reassortant H3N2 avian influenza virus by nanopore sequencing, with the PB1 gene derived from the H5N6 avian influenza virus. The virus had a low ability to spread across species, but further exploration was needed to determine whether its pathogenicity to the host was affected.

Objective:To study the effects of fluoride on apoptosis and oxidative stress levels of spinal cord nerve cells in rats.Methods:A total of 54 6-week-old Sprague-Dawley female rats, weighing 150 - 200 g, were selected and fed for 1 week. They were divided into a control group [given deionized water containing 0 mg/L sodium fluoride (NaF)], a low fluoride group (given deionized water containing 50 mg/L NaF), and a high fluoride group (given deionized water containing 100 mg/L NaF) using a random number table method, with 18 rats in each group. All groups received standard feed. After 4, 8, and 12 weeks of fluoride exposure, six rats were selected from each group to observe the occurrence of dental fluorosis, and the motor function of hind limbs in rats was evaluated based on the Basso-Beattie-Bresnahan (BBB) score. Then the rats were anesthetized with 5% chloral hydrate via intraperitoneal injection and euthanized by cardiac puncture. Spinal cord tissue of the rats was collected to detect the activities of oxidative stress factors such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as the contents of malondialdehyde (MDA) and catalase (CAT). After 12 weeks of fluoride exposure, morphologic changes in rat spinal cord neurons were observed using Nissl staining, and apoptosis of spinal cord nerve cells was detected using the TdT mediated dUTP nick end labeling (TUNEL) cell apoptosis detection kit. The Western blotting was used to detect the expression of B-lymphoblastoma-2 (Bcl-2) gene related X protein (Bax), Bcl-2 promoter (Bad), and Bcl-2 protein in rat spinal cord tissue; immunofluorescence staining was used to observe the expression of Bax and Bcl-2 protein in spinal cord neurons.Results:After 12 weeks of fluoride exposure, rats in both the low fluoride and high fluoride groups developed varying degrees of dental fluorosis; the differences of BBB scores of rats in the control, low fluoride, and high fluoride groups were statistically significant ( F = 14.09, P < 0.001). The differences of SOD [(124.04 ± 4.87), (96.66 ± 15.01), (91.12 ± 15.87) U/mg prot] and GSH-Px activitives [(561.92 ± 59.65), (456.83 ± 29.51), (385.07 ± 74.87) U/mg prot], MDA [(9.96 ± 1.50), (16.64 ± 2.05), (20.80 ± 3.37) nmol/mg prot] and CAT contents [(8.97 ± 1.05), (6.39 ± 0.97), (6.42 ± 0.83) nmol/mg prot] among the control, low fluoride, and high fluoride groups were statistically significant ( F = 11.17, 14.19, 30.12, 14.52, P < 0.05). Among them, the SOD, GSH-Px activities, and CAT content in the low fluoride and high fluoride groups were lower than those in the control group, while the MDA content was higher than that in the control group ( P < 0.05). The GSH-Px activity in the high fluoride group was lower than that in the low fluoride group, and MDA content was higher than that in the low fluoride group ( P < 0.05). The intact neuronal structures and clear visible nuclei were seen, and Nissl bodies were uniformly stained in the spinal cord neurons of the control group rats, with more numbers, and no apoptotic cells were observed; the staining of Nissl bodies in the spinal cord neurons of rats was uneven in the low fluoride and high fluoride groups, with fewer numbers, and more apoptotic cells. There were statistically significant differences in the apoptosis rate of spinal cord nerve cells and the expression levels of Bax, Bad, and Bcl-2 protein in the spinal cord tissues of rats in the control, low fluoride, and high fluoride groups ( F = 272.81, 35.53, 17.57, 92.50, P < 0.05). The results of immunofluorescence staining showed that there were statistically significant differences in the fluorescent intensity of Bax and Bcl-2 proteins in the spinal cord neurons of rats in the control, low fluoride, and high fluoride groups ( F = 12.67, 22.14, P < 0.05). Conclusion:Chronic fluorosis induces a decrease in antioxidant enzyme activity, an increase in lipid peroxidation levels, and an increase in neuronal apoptosis in the spinal cord of rats.

ObjectiveTo predict the potential nephrotoxic components in traditional Chinese medicine health food products based on the Traditional Chinese Medicine Toxicity Alert System and Basic Toxicology Database （TCMTAS-BTD）， screen and validate the predicted components by cell and animal experiments， and decipher the mechanism of nephrotoxicity by network pharmacology. MethodTCMTAS-BTD was utilized to predict the toxicity of 3 540 compounds found in the catalogue of traditional Chinese health food ingredients. In the cell experiment， the top 5 compounds with high toxicity probability were screened by measurement of cell proliferation and viability （CCK-8） and high-content screening. ICR mice were randomized into a control group， a low-dose （2.91 mg·kg^-1·d^-1） ecliptasaponin A， and a high-dose （29.1 mg·kg^-1·d^-1） ecliptasaponin A group， with 10 mice in each group， and treated continuously for 28 days. During the experiment， the general conditions of the rats were observed， and the kidney index was calculated. The levels of serum creatinine （SCr） and blood urea nitrogen （BUN） in the serum as well as the content of malondialdehyde （MDA） and superoxide dismutase （SOD） in the renal tissue were measured. The pathological changes of the kidney were observed. Network pharmacology was employed to predict the potential pathways of nephrotoxicity. Finally， the pathway-associated proteins were validated by Western blot. ResultThe top 5 compounds with high probability of nephrotoxicity were ecliptasaponin A， chrysophanol， rutaecarpine， tanshinoneⅠ， and geniposidic acid. In the cell experiment， CCK-8 results showed that 10 μmol·L^-1 ecliptasaponin A， 60 μmol·L^-1 chrysophanol， 40 μmol·L^-1 rutaecarpine， and 20 μmol·L^-1 tanshinone I altered the viability of HK-2 cells. High-content analysis showed that 10 μmol·L^-1 ecliptasaponin A， chrysophanol， rutaecarpine， and tanshinone Ⅰ reduced the cell number （P<0.05， P<0.01）. The animal experiment showed that the mice in the high-dose ecliptasaponin A group presented slow movement， slow weight gain （P<0.01）， increased kidney index （P<0.01）， elevated SCr， BUN， and MDA levels （P<0.01）， and lowered SOD level （P<0.01）. Mild histopathological changes were observed in the high-dose ecliptasaponin A group. The network pharmacology results showed that the key targets of nephrotoxicity induced by ecliptasaponin A were mainly enriched in the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway， prostatic cancer and lipid and atherosclerosis pathways. Western blot results verified that high dose of ecliptasaponin A raised the phosphorylation levels of PI3K and Akt （P<0.01）. ConclusionOn day 28 of administration， 29.1 mg·kg^-1 ecliptasaponin A was found to induce renal injury in rats. The mechanism may be related with the PI3K/Akt signaling pathway， which implied that excessive and prolonged usage of Ecliptae Herba may increase the incidence of adverse drug reactions.

ObjectiveTo study the effect of Tanreqing injection （TRQ） on the pulmonary flora in the rat model of chronic obstructive pulmonary disease （COPD）. MethodWistar rats were randomized into control， model， and TRQ groups. The rats in other groups except the control group were treated by smoking combined with intratracheal instillation of lipopolysaccharide for the modeling of COPD. The TRQ group was intraperitoneally injected with TRQ （2 g·kg^-1）. At the end of the experiment， after blood collection from the abdominal aorta of the rats， the lung tissue was collected for hematoxylin-eosin and picric sirius red staining to reveal the pathological changes. The lung lavage fluid was collected， and the diversity and relative abundance of lung flora in different groups were analyzed by 16S rDNA amplicon sequencing. ResultThe lungs of the control group were normal， and those of the model group showed neutrophil infiltration， telangiectasia， lung hemorrhage and emphysema in individual cases， and thickening of collagen fibers in the trachea. Compared with the model group， the TRQ group showed significantly improved lungs and recovered collagen fibers. The MLI analysis showed that compared with the control group， the model group showcased increased alveolar space （P<0.01）， which was reduced in the TRQ group （P<0.05）. Compared with the control group， the model group showed increased wall thickness （P<0.01）， and the increase was attenuated in the TRQ group （P<0.01）. TRQ increased the Simpson index and altered the α diversity of pulmonary flora. The results of principal co-ordinate analysis showed that TRQ changed the β diversity and reduced the β diversity index of pulmonary flora. At the genus level， the model group showed increased relative abundance of g_Bacillus and g_Brevundimonas and decreased relative abundance of g_Pseudomonas， compared with the control group. After treatment with TRQ， the relative abundance of g_Stenotrophomonas increased， and that of g_Bacillus decreased. The LEfSe of differential taxa between groups showed that the modeling increased the relative abundance of g_Lachnospiraceae_NK4A136_group， and TRQ treatment increased the relative abundance of g_Rhodococcus and g_Stenotrophomonas. ConclusionTRQ can regulate the diversity of pulmonary flora and restore the balance of bacterial genera in the rat model of COPD， which may be one of the mechanisms of the prevention and treatment of COPD with TRQ.