Objective To investigate the mechanism by which Shenqi Xiaozheng Decoction-medicated serum regulates c-Myc/SLC1A5/GLS1 signaling axis to inhibit glutamine(Gln)metabolism and activate immunogenic cell death(ICD)in non-small cell lung cancer(NSCLC)cells.Methods A549 cells were divided into control group,model group,Shenqi Xiaozheng Decoction-medicated serum low-,medium-and high-dosage groups and positive control group.A Gln-dependent growth model was established,and cells were treated with different concentrations of Shenqi Xiaozheng Decoction-medicated serum or the SLC1A5 inhibitor V-9302.Cell proliferation was assessed by CCK-8,EdU and colony formation assays;Cell invasion and migration were evaluated using Transwell and wound-healing assays;intracellular Gln,glutathione(GSH),and α-ketoglutarate(α-KG)contents were determined by colorimetric assay;reactive oxygen species(ROS)contents were measured with fluorescent probes;Western blot was used to detect the protein expressions of E-cadherin,N-cadherin,c-Myc,SLC1A5 and GLS1;c-Myc/SLC1A5 colocalization and high mobility group box 1(HMGB1)expression were assessed by dual immunofluorescence staining;flow cytometry was used to evaluate calreticulin(CRT)exposure on the cell surface,and ATP and HMGB1 contents in cell supernatants were quantified by ELISA.Results Compared with the control group,the model group showed significantly increased A549 cell viability,EdU-positive rate and migration rate(P<0.05),as well as higher colony counts and invasion cell numbers(P<0.05);cellular Gln,GSH and α-KG contents were significantly elevated(P<0.05,P<0.01),while ROS content were not significantly different(P>0.05),E-cadherin protein expression significantly decreased,whereas the protein expressions of N-cadherin,c-Myc,SLC1A5 and GLS1 significantly increased(P<0.05,P<0.01).c-Myc and SLC1A5 colocalization was enhanced,HMGB1 expression was significantly increased(P<0.01),CRT exposure significantly increased(P<0.01),and ATP and HMGB1 contents in cell supernatant were significantly elevated(P<0.05,P<0.01).Compared with the model group,Shenqi Xiaozheng Decoction-medicated serum at different concentrations significantly inhibited Gln-stimulated A549 cell proliferation,migration and invasion in a dosage-dependent manner.Mechanistic studies indicated that Shenqi Xiaozheng Decoction could reduce Gln uptake and synthesis of its metabolic products GSH and α-KG,induce ROS accumulation,up-regulate protein expression of E-cadherin,down-regulate the protein expressions of N-cadherin,c-Myc,SLC1A5 and GLS1(P<0.05,P<0.01),and enhance CRT exposure,ATP release and HMGB1 secretion(P<0.01).Conclusion Shenqi Xiaozheng Decoction may exert a synergistic"metabolism-immunity"antitumor effect by inhibiting c-Myc/SLC1A5/GLS1 axis-mediated Gln uptake,inducing ROS accumulation,and activating ICD signaling.

The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37 ℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100％agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.

Objective To evaluate the efficacy and safety of Qixian Tongluo Formula in the treatment of post-cerebral infarction paralysis with kidney deficiency and blood stasis syndrome,and to preliminarily explore the molecular mechanism of Qixian Tongluo Formula in improving impaired motor function from the perspective of cross-kingdom regulation of Chinese medicine microRNA(miRNA).Methods A pragmatic randomized controlled trial was conducted with 102 patients in the recovery period of post-cerebral infarction paralysis with kidney deficiency and blood stasis syndrome in our hospital.Patients were randomly divided into trial group and control group,with 51 cases in each group.The control group received standard Western medicine standard treatment,while the trial group received Qixian Tongluo Formula in addition to the standard treatment,with one dose per day,boiled in water,and taken warm after breakfast and dinner for a course of 2 months.The disability rate was used as the main efficacy indicator,and the incidence of adverse reactions was used as a safety indicator.miRNA from patient serum and Qixian Tongluo decoction were extracted respectively,and high-throughput sequencing was performed.The two sequences were compared to screen out the cross-kingdom gene transfer of Chinese medicine miRNA.Finally,its target genes of miRNA were predicted,and GO function and KEGG pathway enrichment analysis were carried out.Results A total of 67 patients completed the clinical trial,including 36 cases in the trial group and 31 cases in the control group;The disability rate in the trial group(13.9%)was lower than that in the control group(35.5%)(P<0.05);The incidence of adverse reactions was similar between the trial group(7.69%)and the control group(6.06%)(P>0.05);A total of 9530 Qixian Tongluo decoction miRNA sequences were screened,with 150 potentially involved in cross-kingdom gene transfer,including families such as miR-15 and miR-17;According to the target gene prediction of the top 10 miRNAs in cross-kingdom gene transfer of Chinese medicine,345 overlapping target genes were obtained;GO functional enrichment analysis revealed 16 biological processes,7 cellular components,and 2 molecular functions among the top 25 enriched functions,while KEGG pathway analysis mainly focused on the transforming growth factor-βsignaling pathway,neurotrophin signaling pathway,which are closely related to neural repair and functional recovery processes such as glial scar formation and synaptic plasticity after cerebral ischemia.Conclusion Qixian Tongluo Formula can significantly improve the functional independence level of patients with kidney deficiency and blood stasis syndrome in the recovery period of paralysis after cerebral infarction,offering a safe and effective treatment option for these patients;There were a large number of miRNAs in Qixian Tongluo decoction,some of which could cross-kingdom transferred into the human blood circulation,and promote the recovery of motor function in patients with cerebral infarction through multi-target,multi link and multi pathway gene network regulation.This study provides a new idea for subsequent clinical and basic research.

Colorectal cancer(CRC)is a common malignancy of the digestive system,with increasing morbidity and mortality worldwide.The standard treatment for CRC has,in the past,been surgery,chemotherapy,and radiotherapy.However,these treatments have significant side effects and are particularly challenging for patients with advanced metastatic CRC(mCRC).Therefore,it is crucial to explore alternative and more effective treatments.In recent years,with the development of molecular technology,numerous biomarkers have been identified,and targeted therapy and immunotherapy have achieved surprisingly positive results in the treatment of mCRC.Patients receiving targeted ther-apy and immunotherapy have an improved prognosis and quality of life.On this basis,this article reviews the latest research progress in tar-geted therapy and immunotherapy for mCRC,providing a reference for precision treatment of mCRC.

BACKGROUND:Studies have reported that alendronate intake significantly increases bone mineral density in patients with osteoporosis. OBJECTIVE:To analyze and compare the changes in metabolites before and after alendronate intervention in ovariectomized rats by chromatography-mass spectrometry,and to further explore the specific mechanism and target of alendronate in the treatment of osteoporosis. METHODS:A total of 36 female Sprague-Dawley rats were randomly divided into model group,alendronate sodium group and sham operation group.The osteoporosis model was established by ovariectomy in the first two groups.Four weeks after modeling,the rats in the alendronate group were intragastrically given alendronate sodium,while those in the sham operation group and model group were given equal volume of normal saline.After 12 weeks of continuous gavage,the metabolites of the lumbar spine were analyzed by chromatography-mass spectrometry,and the common differential metabolites were obtained,which were analyzed by bioinformatics such as Kyoto Gene and Genome Encyclopedia pathway. RESULTS AND CONCLUSION:Totally 17 different metabolites were obtained in the three groups.The enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes showed that alendronate sodium could regulate unsaturated fatty acid biosynthesis,linoleic acid metabolism and other pathways to protect ovariectomized rats.These results indicate that alendronate sodium may exert its anti-osteoporosis effect by interfering with unsaturated fatty acid bioanabolism and linoleic acid metabolism,so as to achieve the purpose of preventing osteoporosis

BACKGROUND:Liuwei Dihuang Wan takes"three tonifying and three reducing effects"as its compatibility feature to nourish yin and tonify the kidneys,while Zuogui Wan takes"seeking yin in yang"as its compatibility feature to nourish yin and tonify the kidneys by promoting yang.Both of them belong to the same method of nourishing yin and tonifying the kidneys,and have better curative effects at the symptomatic and cellular molecular levels. OBJECTIVE:To observe the effects of Liuwei Dihuang Wan and Zuogui Wan in bone metabolism,and to explore their mechanism of action in the osteoprotegerin(OPG)/receptor activator of nuclear factor-κB ligand(RANKL)osteoblastic pathway. METHODS:Thirty-two Sprague-Dawley rats were randomized into model,Liuwei Dihuang Wan,Zuogui Wan,and sham operation group,with eight rats in each group.Osteoporosis models were prepared using removal of both ovaries in the first three groups.Starting at 30 days postoperatively,rats in the Liuwei Dihuang Wan group were gavaged with Liuwei Dihuang Wan 1.125 g/kg/d;rats in the Zuoqui Wan group were gavaged with Zuogui Wan 2.25 g/kg/d;and rats in the sham operation group and the model group were gavaged with saline 10 mL/kg/d.After 12 weeks of gavage,the rat tibia was taken to measure bone mineral density.The serum levels of estrogen,bone alkaline phosphatase,and cAMP/cGMP were measured using ELISA,and the expression of OPG/RANKL in the femur was detected using western blot. RESULTS AND CONCLUSION:Compared with the sham operation group,the model group showed a decrease in bone mineral density and levels of estrogen and bone alkaline phosphatase(P＜0.05)and an increase in cAMP/cGMP level(P＜0.05).Compared with the model group,the Liuwei Dihuang Wan group and the Zuogui Wan group significantly increased bone mineral density(P＜0.05)and bone alkaline phosphatase levels(P＜0.05);the Zuogui Wan group significantly decreased cAMP/cGMP levels(P＜0.05)and upregulated OPG expression(P＜0.05);the Liuwei Dihuang Wan group upregulated OPG expression and downregulated RANKL expression(P＜0.05);and both groups were unable to significantly increase estrogen levels(P＞0.05).To conclude,Zuogui Wan,which seeks yin from yang,can effectively increase the expression of OPG but cannot downregulate the expression of RANKL.However,Liuwei Dihuang Wan,which has three tonifying and three reducing effects,can bidirectionally regulate the expression of OPG and RANKL.This result suggests that Liuwei Dihuang Wan can significantly inhibit osteoclastic function compared with Zuogui Wan,and further research is needed to verify this conclusion.

Objective To investigate the mechanism by which Shenqi Xiaozheng Decoction-medicated serum regulates c-Myc/SLC1A5/GLS1 signaling axis to inhibit glutamine(Gln)metabolism and activate immunogenic cell death(ICD)in non-small cell lung cancer(NSCLC)cells.Methods A549 cells were divided into control group,model group,Shenqi Xiaozheng Decoction-medicated serum low-,medium-and high-dosage groups and positive control group.A Gln-dependent growth model was established,and cells were treated with different concentrations of Shenqi Xiaozheng Decoction-medicated serum or the SLC1A5 inhibitor V-9302.Cell proliferation was assessed by CCK-8,EdU and colony formation assays;Cell invasion and migration were evaluated using Transwell and wound-healing assays;intracellular Gln,glutathione(GSH),and α-ketoglutarate(α-KG)contents were determined by colorimetric assay;reactive oxygen species(ROS)contents were measured with fluorescent probes;Western blot was used to detect the protein expressions of E-cadherin,N-cadherin,c-Myc,SLC1A5 and GLS1;c-Myc/SLC1A5 colocalization and high mobility group box 1(HMGB1)expression were assessed by dual immunofluorescence staining;flow cytometry was used to evaluate calreticulin(CRT)exposure on the cell surface,and ATP and HMGB1 contents in cell supernatants were quantified by ELISA.Results Compared with the control group,the model group showed significantly increased A549 cell viability,EdU-positive rate and migration rate(P<0.05),as well as higher colony counts and invasion cell numbers(P<0.05);cellular Gln,GSH and α-KG contents were significantly elevated(P<0.05,P<0.01),while ROS content were not significantly different(P>0.05),E-cadherin protein expression significantly decreased,whereas the protein expressions of N-cadherin,c-Myc,SLC1A5 and GLS1 significantly increased(P<0.05,P<0.01).c-Myc and SLC1A5 colocalization was enhanced,HMGB1 expression was significantly increased(P<0.01),CRT exposure significantly increased(P<0.01),and ATP and HMGB1 contents in cell supernatant were significantly elevated(P<0.05,P<0.01).Compared with the model group,Shenqi Xiaozheng Decoction-medicated serum at different concentrations significantly inhibited Gln-stimulated A549 cell proliferation,migration and invasion in a dosage-dependent manner.Mechanistic studies indicated that Shenqi Xiaozheng Decoction could reduce Gln uptake and synthesis of its metabolic products GSH and α-KG,induce ROS accumulation,up-regulate protein expression of E-cadherin,down-regulate the protein expressions of N-cadherin,c-Myc,SLC1A5 and GLS1(P<0.05,P<0.01),and enhance CRT exposure,ATP release and HMGB1 secretion(P<0.01).Conclusion Shenqi Xiaozheng Decoction may exert a synergistic"metabolism-immunity"antitumor effect by inhibiting c-Myc/SLC1A5/GLS1 axis-mediated Gln uptake,inducing ROS accumulation,and activating ICD signaling.

Objective To evaluate the efficacy and safety of Qixian Tongluo Formula in the treatment of post-cerebral infarction paralysis with kidney deficiency and blood stasis syndrome,and to preliminarily explore the molecular mechanism of Qixian Tongluo Formula in improving impaired motor function from the perspective of cross-kingdom regulation of Chinese medicine microRNA(miRNA).Methods A pragmatic randomized controlled trial was conducted with 102 patients in the recovery period of post-cerebral infarction paralysis with kidney deficiency and blood stasis syndrome in our hospital.Patients were randomly divided into trial group and control group,with 51 cases in each group.The control group received standard Western medicine standard treatment,while the trial group received Qixian Tongluo Formula in addition to the standard treatment,with one dose per day,boiled in water,and taken warm after breakfast and dinner for a course of 2 months.The disability rate was used as the main efficacy indicator,and the incidence of adverse reactions was used as a safety indicator.miRNA from patient serum and Qixian Tongluo decoction were extracted respectively,and high-throughput sequencing was performed.The two sequences were compared to screen out the cross-kingdom gene transfer of Chinese medicine miRNA.Finally,its target genes of miRNA were predicted,and GO function and KEGG pathway enrichment analysis were carried out.Results A total of 67 patients completed the clinical trial,including 36 cases in the trial group and 31 cases in the control group;The disability rate in the trial group(13.9%)was lower than that in the control group(35.5%)(P<0.05);The incidence of adverse reactions was similar between the trial group(7.69%)and the control group(6.06%)(P>0.05);A total of 9530 Qixian Tongluo decoction miRNA sequences were screened,with 150 potentially involved in cross-kingdom gene transfer,including families such as miR-15 and miR-17;According to the target gene prediction of the top 10 miRNAs in cross-kingdom gene transfer of Chinese medicine,345 overlapping target genes were obtained;GO functional enrichment analysis revealed 16 biological processes,7 cellular components,and 2 molecular functions among the top 25 enriched functions,while KEGG pathway analysis mainly focused on the transforming growth factor-βsignaling pathway,neurotrophin signaling pathway,which are closely related to neural repair and functional recovery processes such as glial scar formation and synaptic plasticity after cerebral ischemia.Conclusion Qixian Tongluo Formula can significantly improve the functional independence level of patients with kidney deficiency and blood stasis syndrome in the recovery period of paralysis after cerebral infarction,offering a safe and effective treatment option for these patients;There were a large number of miRNAs in Qixian Tongluo decoction,some of which could cross-kingdom transferred into the human blood circulation,and promote the recovery of motor function in patients with cerebral infarction through multi-target,multi link and multi pathway gene network regulation.This study provides a new idea for subsequent clinical and basic research.

Colorectal cancer(CRC)is a common malignancy of the digestive system,with increasing morbidity and mortality worldwide.The standard treatment for CRC has,in the past,been surgery,chemotherapy,and radiotherapy.However,these treatments have significant side effects and are particularly challenging for patients with advanced metastatic CRC(mCRC).Therefore,it is crucial to explore alternative and more effective treatments.In recent years,with the development of molecular technology,numerous biomarkers have been identified,and targeted therapy and immunotherapy have achieved surprisingly positive results in the treatment of mCRC.Patients receiving targeted ther-apy and immunotherapy have an improved prognosis and quality of life.On this basis,this article reviews the latest research progress in tar-geted therapy and immunotherapy for mCRC,providing a reference for precision treatment of mCRC.

The objective of this study was to develop a rapid and precise detection technique for por-cine Senecavirus A(SVA)employing reverse transcription recombinase polymerase amplification(RT-RAA)in conjunction with CRISPR Cas13a technology.Additionally,the study aimed to opti-mize the assay's reaction conditions to enhance amplification efficiency.Eight RT-RAA primer sets were designed based on the conserved gene sequence of porcine SVA,and a series of reaction condi-tions were evaluated to refine the RT-RAA reaction system.Subsequently,CRISPR-derived RNA(crRNA)sequences were developed and selected to construct the RT-RAA-CRISPR reaction sys-tem.The method's specificity was determined by examining six prevalent porcine pathogenic nucleic acids,while its sensitivity was assessed using SVA cRNA standards quantified by digital PCR.The method's stability and the consistency of clinical sample analysis were also evaluated.The findings revealed that the optimized RT-RAA and CRISPR reaction systems exhibited the highest amplifi-cation efficiency at a reaction temperature of 37 ℃.Among the eight crRNAs,five were identified as exhibiting the strongest detection signals.The formulated RT-RAA-CRISPR Cas13a method demonstrated exceptional specificity,showing no cross-reactivity with other common porcine disea-ses,including ASFV,PRRSV,PEDV,PCV2,CSFV,and PRV.The method achieved high sensitivi-ty,detecting as low as 0.86 copies/μL of SVA,and exhibited stable fluorescence output,robust re-producibility,and the ability to complete clinical sample analysis within 50 minutes.Consistency e-valuation with six positive and 58 negative samples indicated 100％agreement in outcomes.These results substantiate that the study successfully established a rapid and specific RT-RAA-CRISPR Cas13a detection method for the on-site identification of porcine Senecavirus A,demonstrating high specificity and sensitivity,and holds promise for application in SVA monitoring and control initia-tives.