1.Quercetin Ameliorates Gouty Arthritis in Rats via ROS/NLRP3/IL-1β Signaling Pathway
Baowei FENG ; Yan WANG ; Chang LI ; Yujing ZHANG ; Dingxing FAN ; Xin LI
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):145-153
ObjectiveTo investigate the effect of quercetin on acute gouty arthritis (GA) in rats by inhibiting the reactive oxygen species (ROS)/NOD-like receptor protein 3 (NLRP3)/interleukin-1β (IL-1β) signaling pathway. MethodsSixty SPF-grade male SD rats were randomized into normal, model, colchicine (0.3 mg·kg-1), and low-, medium-, and high-dose (25, 50, 100 mg·kg-1, respectively) quercetin groups (n=10). The rats in the dosing groups were administrated with the corresponding drugs (10 mL·kg-1) by gavage once a day for one week. An equal volume of normal saline was given by gavage to rats in normal and model groups. One hour after drug administration on day 5, an acute GA model was established in other groups except the control group via intra-articular injection of monosodium urate (MSU) suspension into the right posterior ankle joint cavity. The joint swelling and gait were scored at the time points of 6, 12, 24, 48 h after modeling. Histopathological alterations in the ankle joint tissue from each group were assessed by hematoxylin-eosin (HE) staining. Malondialdehyde (MDA), xanthine oxidase (XOD), and total superoxide dismutase (T-SOD) assay kits were used to assess the levels of MDA, XOD, and T-SOD in the serum. The levels of tumor interleukin-6 (IL-6), necrosis factor-α (TNF-α), and IL-1β in the rat serum, as well as ROS in the ankle joint tissue, were measured by enzyme-linked immunosorbent assay (ELISA). Western blot was performed to determine the protein levels of NLRP3, thioredoxin-interacting protein (TXNIP), apoptosis-associated speck-like protein containing a CARD domain (ASC), precursor cysteinyl aspartate-specific proteinase-1 (pro-Caspase-1), cleaved Caspase-1 (Caspase-1 p20), and IL-1β in the ankle joint tissue. Real-time PCR was employed to assess the mRNA levels of TXNIP, NLRP3, ASC, IL-1β, and TNF-α in the ankle joint tissue. ResultsCompared with the normal group, the model group exhibited decreased spontaneous activity, mental fatigue, increased ankle joint swelling and gait scores (P<0.01), aggravated synovial tissue edema and inflammatory cell infiltration (P<0.01), elevated levels of XOD, MDA, TNF-α, IL-1β, and IL-6 in the serum and ROS in the joint tissue (P<0.01), a declined level of T-SOD (P<0.01), up-regulated protein levels of NLRP3, TXNIP, ASC, pro-Caspase-1, Caspase-1 p20, and IL-1β in the ankle joint tissue (P<0.01), and up-regulated mRNA levels of NLRP3, TXNIP, ASC, IL-1β, and TNF-α in the ankle joint tissue (P<0.01). Compared with the model group, the medium- and high-dose quercetin groups showed improved general conditions, decreased gait scores (P<0.05, P<0.01), reduced joint swelling (P<0.01), alleviated synovial tissue edema and inflammatory cell infiltration (P<0.05, P<0.01), lowered levels of XOD, MDA, TNF-α, IL-1β, and IL-6 in the serum and ROS in the joint tissue (P<0.01), increased levels of T-SOD (P<0.01), down-regulated protein levels of TXNIP, NLRP3, ASC, pro-Caspase-1, Caspase-1 p20, and IL-1β in the ankle joint tissue (P<0.05, P<0.01), and down-regulated mRNA levels of TXNIP, NLRP3, ASC, IL-1β, and TNF-α in the ankle joint tissue (P<0.01). Low-dose quercetin also ameliorated some of the above parameters (P<0.05, P<0.01). ConclusionQuercetin exerts anti-GA effects by blocking the ROS/NLRP3/IL-1β signaling pathway, downregulating NLRP3 inflammasome activation, and inhibiting the production of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6.
2.Analysis of the application status of prescription pre-review systems in Yunnan province
Fan XU ; Wenjie YIN ; Kejia LI ; Zhengfu LI ; Jie CHEN ; Meixian WU ; Ruixiang CHEN ; Songmei LI ; Guowen ZHANG ; Te LI
China Pharmacy 2026;37(1):6-10
OBJECTIVE To investigate the application status of prescription pre-review systems in healthcare institutions of Yunnan province, evaluate their system functions and management capabilities, and provide a practical basis for promoting rational drug use. METHODS A questionnaire survey was conducted among public healthcare institutions at or above the secondary level in Yunnan province to investigate the deployment status of the systems. A capability maturity assessment framework was constructed, encompassing 6 dimensions and 39 indicators, including real-time prescription review, prescription correlation review, rule setting, evidence-based information support, prescription authority management, and system operation management. This framework was then used to evaluate the institutions that had implemented the pre-review systems. RESULTS A total of 100 valid questionnaires were collected, with 37 institutions having adopted prescription pre-review systems, mainly tertiary hospitals. The system predominantly adopted a modular architecture and was embedded into the hospital information system through application programming interfaces and middleware, providing certain capabilities for real-time prescription risk identification. Evaluation results indicated that basic functions such as reviewing indications, contraindications, and drug compatibility performed well, while deficiencies remained in functions related to parenteral nutrition prescription, review of drug dosage for specific diseases, individual patient characteristic recognition, and rule setting. Moreover, the construction of review centers and establishment of management systems were also not well-developed. CONCLUSIONS The overall application rate of prescription pre-review systems in Yunnan province remains low. System functions and management mechanisms require further improvement. It is recommended to enhance information infrastructure in lower-level institutions and explore regionally unified review models to promote standardized and intelligent development of prescription review practices.
3.Clinical research on corneal epithelium remodeling after small incision lenticule extraction and femtosecond laser-assisted in situ keratomileusis
Yangjing ZHANG ; Liwei MA ; Fan ZHANG ; Chunmei KE ; Ruifu WANG ; Lipye ZU
International Eye Science 2025;25(1):37-41
AIM: To compare the changes in corneal epithelial thickness(CET)after small incision lenticule extraction(SMILE)and femtosecond laser-assisted in situ keratomileusis(FS-LASIK).METHODS: A total of 187 patients(187 eyes)who underwent either SMILE or FS-LASIK at Urumqi Aier Eye Hospital between December 2022 and November 2023 were collected. The patients were divided into SMILE group and FS-LASIK group according to surgical methods. The CET of the patients was measured by optical coherence tomography(OCT)system before surgery and at 1 wk, 1, 3, and 6 mo postoperatively.RESULTS: Changes in corneal epithelial thickness(△CET)in the central, paracentral, and mid-peripheral regions were compared at 6 mo postoperatively. The SMILE group was characterized by the most significant thickening in the central area and the least thickening in the mid-peripheral area; while the FS-LASIK group was characterized by the most significant thickening in the paracentral area and the least thickening in the mid-peripheral region. At 1 wk, 1, 3, and 6 mo postoperatively, within the 0-7 mm corneal area, the △CET for both the SMILE and FS-LASIK groups was correlated with the preoperative spherical equivalent.CONCLUSION: Within 6 mo postoperatively, both SMILE and FS-LASIK showed a similar trend in epithelial thickening but with distinct characteristics. The change in corneal epithelial thickness for both procedures was positively correlated with the preoperative diopter.
4.Exploration on Mechanism of Topical Treatment of Allergic Contact Dermatitis in Mice with Portulacae Herba Based on Nrf2/HO-1/NF-κB Signaling Pathway
Xiaoxue WANG ; Guanwei FAN ; Xiang PU ; Zhongzhao ZHANG ; Xia CHEN ; Ying TANG ; Nana WU ; Jiangli LUO ; Xiangyan KONG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):115-123
ObjectiveTo investigate the mechanism of topical treatment of allergic contact dermatitis (ACD) mice with Portulacae Herba based on the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/nuclear factor-κB (NF-κB) signaling pathway. MethodsA total of 70 6-week-old specific pathogen free (SPF) female Kunming mice were adaptively fed for 1 week and randomly divided into blank group, model group, compound dexamethasone acetate cream group (2.075×10-2 g·g-1), blank matrix cream group, low-dose Portulacae Herba cream group (0.1 g·g-1), high-dose Portulacae Herba cream group (0.2 g·g-1), and Portulacae Herba + inhibitor group (0.2 g·g-1 + 30 mg·kg-1 ML385), with 10 mice in each group. One day before the experiment, the mice were shaved on the neck and back. Except for the blank group, the mice in the other groups were treated with 2,4-dinitrochlorobenzene (DNCB) to establish an ACD model. After respective administration, the skin lesion of the mice was scored, and the histopathological changes of the skin were stained with hematoxylin-eosin (HE). Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), reactive oxygen species (ROS), superoxide dismutase (SOD) activity, and malondialdehyde (MDA) in serum of mice. The expression of Nrf2/HO-1/NF-κB signaling pathway-related proteins in mouse skin tissue was detected by immunohistochemistry (IHC), Western blot, and real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). ResultsCompared with the blank group, the mice in the model group had an increased skin lesion score (P<0.01), severe pathological damage to skin tissue, increased content of IL-1β, IL-6, ROS, and MDA in their serum (P<0.01), and decreased content of SOD (P<0.01). In addition, the mRNA and protein expression levels of Nrf2, HO-1, and nuclear factor-κB inhibitor α (IκBα) in skin tissue were up-regulated (P<0.01), while the protein expression levels of phosphorylated (p)-IκBα and p-NF-κB p65 and the mRNA expression of NF-κB p65 were down-regulated (P<0.01). Compared with the model group and the blank matrix cream group, the mice treated with Portulacae Herba had a decreased skin lesion score (P<0.01), reduced pathological damage to skin tissue, decreased content of IL-1β, IL-6, ROS, and MDA in their serum (P<0.01), and increased content of SOD (P<0.01). Additionally, the mRNA and protein expression levels of Nrf2, HO-1, and IκBα in skin tissue were down-regulated (P<0.05,P<0.01), and the protein expression levels of p-IκBα and p-NF-κB p65 and the mRNA expression of NF-κB p65 were up-regulated (P<0.05,P<0.01). Compared with the Portulacae Herba + inhibitor group, the high-dose Portulacae Herba cream group had a decreased skin lesion score (P<0.01), alleviated pathological damage to skin tissue, decreased content of IL-1β, IL-6, ROS, and MDA in the serum of mice (P<0.05,P<0.01), and increased content of SOD (P<0.01). The protein expression levels of Nrf2, HO-1, and IκBα and the mRNA expression of Nrf2 and HO-1 in skin tissue were up-regulated (P<0.05,P<0.01), and the protein expression levels of p-IκBα and p-NF-κB p65 and the mRNA expression of NF-κB p65 were down-regulated (P<0.05). ConclusionPortulacae Herba can improve DNCB-induced ACD skin damage in mice by regulating the Nrf2/HO-1/NF-κB signaling pathway.
5.Fufang Kangjiaolv Capsules Treat Anxiety in Rat Model of Chronic Restraint Stress via Microbiota-gut-brain Axis
Wenxin FAN ; Tingyue JIANG ; Yu WANG ; Ge ZHANG ; Yifan LU ; Mengmeng LIU ; Jiayuan LI ; Renzhi MA ; Jinli SHI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(4):95-107
ObjectiveTo observe the intervention effect of Fufang Kangjiaolv capsules on anxiety-like behaviors in the rat model of chronic restraint stress (CRS) and explore the mechanism underlying the anti-anxiety effect via the microbiota-gut-brain axis. MethodsRats were assigned into blank, model, positive drug (diazepam, 1 mg·kg-1), and low-, medium-, and high-dose (0.75, 1.5, 3 g·kg-1, respectively) Fufang Kangjiaolv capsules groups. After 14 days of administration, the elevated plus maze test, open field test, light and dark box test, and marble burying test were performed. Hematoxylin-eosin staining was employed to observe the pathological changes in the hippocampus and colon of rats, and Nissl staining was conducted to observe the damage of hippocampal neurons. The gut microbiota was analyzed by 16S rRNA gene sequencing. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was employed to determine the mRNA levels of zonula occludens-1 (ZO-1) and occludin in the colon of rats. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the colon, serum, and hippocampus were determined by enzyme-linked immunosorbent assay. Western blot was employed to determine the protein levels of ZO-1, occludin, nuclear factor-κB p65 (NF-κB p65) in the colon tissue and NF-κB p65 and brain-derived neurotrophic factor (BDNF) in the hippocampal tissue. ResultsCompared with the blank group, the model group showed reductions in the time and frequency ratio of rats entering the elevated plus maze, the time and frequency of rats entering the central area of the open field, the time of entering the open box, the times of passing through the light and dark box, and the number of unburied beads (P<0.05, P<0.01). Compared with the model group, Fufang Kangjiaolv capsules ameliorated the anxiety of the model rats to varying degrees, and the high-dose group had the best effect, with increases in the proportions of time and frequency of rats entering the open arm in the elevated plus maze (P<0.05), the number of rats entering the central area in the open field (P<0.05), the time of entering the open box, the times of passing through the light and dark boxes, and the number of unburied beads (P<0.01). Moreover, the high-dose group showed alleviated pathological damage of hippocampal neurons and colon. The results of 16S rRNA gene sequencing showed that the model group had increased relative abundance of Firmicutes, Deferribacterota, Romboutsia, and Phascolarctobacterium, while it had decreased relative abundance of Bavcteroidota and Lactobacillus. The drug administration groups showed increased relative abundance of Bavcteroidota, Bacteroides, norank f norank o Clostridia UCG-014, and Blautia and decreased relative abundance of Firmicutes and Deferribacterota. Compared with the blank group, the model group showed down-regulated protein and mRNA levels of ZO-1 and occludin in the colon (P<0.01), elevated levels of TNF-α, IL-6, and IL-β in the colon, serum, and hippocampus (P<0.01), up-regulated protein level of NF-κB p65 in the colon and hippocampus (P<0.01), and down-regulated protein level of BDNF in the hippocampus (P<0.05). Compared with the model group, high-dose Fufang Kangjiaolv capsules up-regulated the mRNA levels of ZO-1 and occludin in the colon (P<0.01), lowered the levels of TNF-α, IL-6, and IL-β in the colon, serum, and hippocampus (P<0.01), up-regulated the protein levels of ZO-1 (P<0.01) and occludin (P<0.05) in the colon, down-regulated the protein level of NF-κB p65 in the colon and hippocampus (P<0.05), and up-regulated the protein level of BDNF in the hippocampus. ConclusionFufang Kangjiaolv capsules can reduce the anxiety-like behaviors in the rat model of CRS by regulating the gut microbiota disturbance, up-regulating the expression of tight junction proteins in the colon, repairing intestinal mucosal mechanical barrier, and down-regulating NF-κB/BDNF signaling pathway, thereby reducing peripheral and central inflammation. This study proves the hypothesis that Fufang Kangjiaolv capsules play an anti-anxiety role via the microbiota-gut-brain axis, providing a new idea for further research.
6.Expert Consensus on Clinical Application of Qinbaohong Zhike Oral Liquid in Treatment of Acute Bronchitis and Acute Attack of Chronic Bronchitis
Jian LIU ; Hongchun ZHANG ; Chengxiang WANG ; Hongsheng CUI ; Xia CUI ; Shunan ZHANG ; Daowen YANG ; Cuiling FENG ; Yubo GUO ; Zengtao SUN ; Huiyong ZHANG ; Guangxi LI ; Qing MIAO ; Sumei WANG ; Liqing SHI ; Hongjun YANG ; Ting LIU ; Fangbo ZHANG ; Sheng CHEN ; Wei CHEN ; Hai WANG ; Lin LIN ; Nini QU ; Lei WU ; Dengshan WU ; Yafeng LIU ; Wenyan ZHANG ; Yueying ZHANG ; Yongfen FAN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(4):182-188
The Expert Consensus on Clinical Application of Qinbaohong Zhike Oral Liquid in Treatment of Acute Bronchitis and Acute Attack of Chronic Bronchitis (GS/CACM 337-2023) was released by the China Association of Chinese Medicine on December 13th, 2023. This expert consensus was developed by experts in methodology, pharmacy, and Chinese medicine in strict accordance with the development requirements of the China Association of Chinese Medicine (CACM) and based on the latest medical evidence and the clinical medication experience of well-known experts in the fields of respiratory medicine (pulmonary diseases) and pediatrics. This expert consensus defines the application of Qinbaohong Zhike oral liquid in the treatment of cough and excessive sputum caused by phlegm-heat obstructing lung, acute bronchitis, and acute attack of chronic bronchitis from the aspects of applicable populations, efficacy evaluation, usage, dosage, drug combination, and safety. It is expected to guide the rational drug use in medical and health institutions, give full play to the unique value of Qinbaohong Zhike oral liquid, and vigorously promote the inheritance and innovation of Chinese patent medicines.
7.Hypoglycemic Effect and Mechanism of ICK Pattern Peptides
Lin-Fang CHEN ; Jia-Fan ZHANG ; Ye-Ning GUO ; Hui-Zhong HUANG ; Kang-Hong HU ; Chen-Guang YAO
Progress in Biochemistry and Biophysics 2025;52(1):50-60
Diabetes is a very complex endocrine disease whose common feature is the increase in blood glucose concentration. Persistent hyperglycemia can lead to blindness, kidney and heart disease, neurodegeneration, and many other serious complications that have a significant impact on human health and quality of life. The number of people with diabetes is increasing yearly. The global diabetes prevalence in 20-79 year olds in 2021 was estimated to be 10.5% (536.6 million), and it will rise to 12.2% (783.2 million) in 2045. The main modes of intervention for diabetes include medication, dietary management, and exercise conditioning. Medication is the mainstay of treatment. Marketed diabetes drugs such as metformin and insulin, as well as GLP-1 receptor agonists, are effective in controlling blood sugar levels to some extent, but the preventive and therapeutic effects are still unsatisfactory. Peptide drugs have many advantages such as low toxicity, high target specificity, and good biocompatibility, which opens up new avenues for the treatment of diabetes and other diseases. Currently, insulin and its analogs are by far the main life-saving drugs in clinical diabetes treatment, enabling effective control of blood glucose levels, but the risk of hypoglycemia is relatively high and treatment is limited by the route of delivery. New and oral anti-diabetic drugs have always been a market demand and research hotspot. Inhibitor cystine knot (ICK) peptides are a class of multifunctional cyclic peptides. In structure, they contain three conserved disulfide bonds (C3-C20, C7-C22, and C15-C32) form a compact “knot” structure, which can resist degradation of digestive protease. Recent studies have shown that ICK peptides derived from legume, such as PA1b, Aglycin, Vglycin, Iglycin, Dglycin, and aM1, exhibit excellent regulatory activities on glucose and lipid metabolism at the cellular and animal levels. Mechanistically, ICK peptides promote glucose utilization by muscle and liver through activation of IR/AKT signaling pathway, which also improves insulin resistance. They can repair the damaged pancrease through activation of PI3K/AKT/Erk signaling pathway, thus lowering blood glucose. The biostability and hypoglycemic efficacy of the ICK peptides meet the requirements for commercialization of oral drugs, and in theory, they can be developed into natural oral anti-diabetes peptide drugs. In this review, the structural properties, activity and mechanism of ICK pattern peptides in regulating glucose and lipid metabolism were summaried, which provided a reference for the development of new oral peptides for diabetes.
8.Mechanisms of Intestinal Microecology in Hyperuricemia and Traditional Chinese Medicine Intervention:A Review
Mingyuan FAN ; Jiuzhu YUAN ; Hongyan XIE ; Sai ZHANG ; Qiyuan YAO ; Luqi HE ; Qingqing FU ; Hong GAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(5):329-338
In recent years, hyperuricemia (HUA) has shown a rapidly increasing incidence and tends to occur in increasingly young people, with a wide range of cardiac, renal, joint, and cancerous hazards and all-cause mortality associations. Western medicine treatment has limitations such as large liver and kidney damage, medication restriction, and easy recurrence. The intestine is the major extra-renal excretion pathway for uric acid (UA), and the intestinal microecology can be regulated to promote UA degradation. It offers great potential to develop UA-lowering strategies that target the intestinal microecology, which are promising to provide safer and more effective therapeutic approaches. Traditional Chinese medicine (TCM) can treat HUA via multiple targets and multiple pathways from a holistic view, with low toxicity and side effects. Studies have shown that intestinal microecology is a crucial target for TCM in the treatment of HUA. However, its specific mechanism of action has not been fully elucidated. Focusing on the key role of intestinal microecology in HUA, this review explores the relationship between intestinal microecology and HUA in terms of intestinal flora, intestinal metabolites, intestinal UA transporters, and intestinal barriers. Furthermore, we summarize the research progress in TCM treatment of HUA by targeting the intestinal microecology, with the aim of providing references for the development of TCM intervention strategies for HUA and the direction of future research.
9.Interpretation of perioperative immunotherapy for lung cancer in 2024 WCLC/ESMO
Jiahe LI ; Xiaopeng REN ; Jiayu LU ; Chenyuan ZHANG ; Ruitao FAN ; Xuxu ZHANG ; Xinyao XU ; Guizhen LI ; Jipeng ZHANG ; Wei LI ; Qiang LU
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2025;32(03):300-307
The 2024 World Conference on Lung Cancer (WCLC) and the European Society for Medical Oncology (ESMO) Annual Meeting, two of the most prestigious events in oncology, have concluded sequentially. As the most authoritative annual gatherings in lung cancer and the entire oncology field, the WCLC and ESMO conferences brought together top oncology experts and scientists from around the world to share, discuss, and publish the latest cutting-edge advancements in oncology. In both conferences, lung cancer immunotherapy remained a hot topic of considerable interest. This article aims to summarize and discuss the important research progress on perioperative immunotherapy for non-small cell lung cancer reported at the two conferences.
10.Construction of lentiviral vectors for solute carrier family 1 member 5 overexpression and knockdown and stably transfected RAW264.7 cell line
Daxin GUO ; Susu FAN ; Zhendong ZHU ; Jianhong HOU ; Xuan ZHANG
Chinese Journal of Tissue Engineering Research 2025;29(7):1414-1421
BACKGROUND:Solute carrier family 1 member 5(SLC1A5)plays a potential role in a variety of diseases,but the exact mechanism of action is unclear.The construction of stable SLC1A5 overexpression and knockdown cell models can provide a powerful experimental tool for in-depth study of the exact role and mechanism of SLC1A5 in diseases and the discovery of potential therapeutic targets. OBJECTIVE:To construct lentiviral vectors for overexpression and knockdown of mouse SLC1A5 and establish stable transfected RAW264.7 cell lines,so as to provide an experimental foundation for further investigation of the role of SLC1A5 in inflammation. METHODS:Primers were designed and synthesized based on the SLC1A5 gene sequence,and the gene segment was amplified using polymerase chain reaction.Subsequently,the target gene segment was directionally inserted into the GV492 vector plasmid,which had been digested with AgeI/NheI enzymes,to construct recombinant lentiviral plasmids.Positive clones were further selected,and their sequences were confirmed.The pHelper1.0 plasmid vector and pHelper2.0 plasmid vector,along with the target plasmid vector,was co-cultured with 293T cells for transfection,resulting in the production and titration of lentiviral stocks.Furthermore,RAW264.7 cells were cultured in vitro,and the working concentration of puromycin was determined.Lentiviruses were separately co-cultured with RAW264.7 cells,and transfection efficiency was determined by measuring fluorescence intensity.Stable transfected cells were selected using puromycin,and real-time fluorescence quantitative PCR and western blot assay were employed to assess the gene and protein expression levels of SLC1A5 in stably transfected cell lines. RESULTS AND CONCLUSION:(1)Sequencing results indicated a perfect match between the sequencing and target sequences,confirming the successful construction of recombinant lentiviral vectors.(2)The titer for the overexpression SLC1A5 lentivirus was 1×109 TU/mL,while the titer for the knockdown SLC1A5 lentivirus was 3×109 TU/mL.(3)The working concentration of puromycin for RAW264.7 cells was determined to be 3 μg/mL.(4)The optimal conditions for transfecting RAW264.7 cells with overexpression/knockdown expression of SLC1A5 lentivirus involved the use of HiTransG P transfection enhancer with a multiplicity of infection value of 50.(5)A significant upregulation of the gene and protein expression levels of SLC1A5 was detected in cell lines stably overexpressing SLC1A5,while gene and protein expression levels of SLC1A5 were significantly decreased in the knockdown stable cell lines.These findings indicate that lentiviral vectors for mouse SLC1A5 overexpression and knockdown have been successfully constructed and a stably transfected RAW264.7 cell line has been obtained.

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