ObjectiveTo characterize the quality differences among different germplasm and introduced varieties of Bupleurum scorzonerifolium roots（BSR）， and explore the underlying molecular mechanisms， providing a basis for high-quality production and quality control. MethodsWild BSR from Yulin（YLW） served as the quality reference， we conducted comparative analysis among YLW， locally domesticated wild germplasm in Yulin（YLC3）， Daqing germplasm introduced and cultivated in Yulin（YLDQC3）， and locally cultivated germplasm in Daqing（DQC3）. A combination of traditional pharmacognostic methods and modern multi-omics analyses was employed， including macroscopic traits（appearance， odor）， microscopic features（proportions of cork， phloem， xylem）， cell wall component contents（hemicellulose， cellulose， lignin）， carbohydrate contents（starch， water-soluble polysaccharides）， marker compound contents（ethanol-soluble extracts， total saponins， liposoluble extracts， and saikosaponins A， B₂， C， D）， metabolomics， and transcriptomics， in order to systematically characterize quality differences and investigate molecular mechanisms among these samples. ResultsMacroscopically， Yulin-produced BSR（YLW， YLC3， YLDQC3） exhibited significantly greater weight， length， and upper and middle diameters than Daqing-produced BSR（DQC3）. Odor-wise， YLW and YLC3 had a a fragrance taste， YLDQC3 had a rancid oil odor， and DQC3 had a sweet and fragrant taste. Microscopically， Yulin germplasm（YLW， YLC3） and Daqing germplasm（YLDQC3， DQC3） shared similar structural features， respectively. However， Yulin germplasm showed significantly higher proportions of cork and phloem， as well as stronger xylem vessel staining intensity compared to Daqing germplasm. Regarding various component contents， Yulin germplasm contained significantly higher levels of ethanol-soluble extracts， total saponins， and saikosaponins A， B₂， C， D， while Daqing germplasm had significantly higher levels of hemicellulose， starch， and liposoluble extracts. After introduction to Yulin， the Daqing germplasm（YLDQC3） showed increased starch， water-soluble polysaccharides and liposoluble extracts contents， decreased cell wall component content， but no significant difference in other component contents. Metabolomics revealed that saponins and terpenes accumulated significantly in Yulin germplasm， while alcohols and aldehydes accumulated predominantly in Daqing germplasm. Transcriptomics indicated similar gene expression patterns within the same germplasm but specificity between different germplasms. Integrative metabolomic-transcriptomic analysis identified 145 potential key genes associated with the saikosaponin biosynthesis pathway， including one acetyl-coenzyme A（CoA） acetyltransferase gene（ACAT）， one 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene（HMGS）， two hydroxymethylglutaryl-CoA（HMG-CoA） reductase genes（HMG）， one phosphomevalonate kinase gene（PMK）， one 1-deoxy-D-xylose-5-phosphate synthase gene（CLA）， one hydroxymethylbuten-1-aldol synthase gene（HDR）， two farnesyl pyrophosphate synthase genes（FPPS）， one squalene synthase gene（SQS）， one β-amyrin synthase gene（BAS）， 102 cytochrome P450（CYP450） gene family members， and 32 uridine diphosphate-glucuronosyltransferase（UGT） gene family members. ConclusionAmong the three cultivated types， YLC3 most closely resembles YLW in appearance， microscopic features， contents of major bioactive constituents， metabolomic and transcriptomic profiles. Yulin germplasm exhibits superior saponin synthesis capability compared to Daqing germplasm， and Yulin region is more suitable for the growth of B. scorzonerifolium. Based on these findings， it is recommended that artificial cultivation in northern Shaanxi and similar regions utilize the local Yulin germplasm source cultivated for at least three years.

ObjectiveBased on the traditional quality evaluation methods summarized in previous dynasties， this paper systematically contrasted the quality differences between wild Polygalae Radix（WPR） and cultivated Polygalae Radix（CPR） from the aspects of character， microscope and chemical composition by modern scientific and technological means， providing a basis for high-quality production and quality control. MethodsCPR and local WPR in Yulin city， Shaanxi province from 1 to 6 years were collected， and a systematic comparative analysis was conducted using traditional pharmacognosy research methods combined with modern multi-omics analysis techniques， including character traits（length， weight， diameter）， cross-sectional microscopic features（proportions of cork， phloem， xylem， etc）， cell wall component content（hemicellulose， cellulose， lignin）， extracts content（water-soluble extract and alcohol-soluble extract）， carbohydrate content（starch， water-soluble polysaccharides）， contents of total flavonoids， total saponins and specific marker compounds（3，6′-disinapoyl sucrose， polygalaxanthone Ⅲ， tenuifoliside A， tenuifoliside C， sibiricose A₅ and A₆） and other indexes. Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was employed to conduct comparative analysis of secondary metabolites in WPR and CPR， and multivariate statistical analysis such as principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were combined to screen the key differential components of them. ResultsIn terms of appearance， there were significant differences between WPR and CPR. The characteristics of WPR conformed to the "thick wrinkles on the epidermis" recorded in ancient books， featuring a wrinkled surface and grayish-brown appearance. However， CPR had a finer texture and a yellowish white appearance， with weight， length， and diameter increasing with longer cultivation periods. In terms of microscopy， WPR exhibited a thick cork layer with fissures in the phloem， whereas CPR had a thinner cork layer with uniformly arranged cork cells. Younger PR specimens showed numerous phloem fissures in cross-sections， while older specimens display progressively denser arrangements of phloem parenchyma cells. In terms of the contents of various major components， the contents of water-soluble extract， starch and total saponins in WPR were inversely proportional to the root diameter， while the contents of water-soluble extract， water-soluble polysaccharides and total saponins in CPR decreased with the increase of planting years. The content of xanthones in WPR was significantly higher than that of CPR， while the contents of other major components showed no significant change pattern. Among the six indicator components， the average content of sibiricose A₅ in WPR was significantly higher than that of CPR， followed by slightly higher content of tenuifoliside A. In CPR， the relative content of 3，6′-disinapoyl sucrose and tenuifoliside A was the highest. The former showed an increase in volatility with increasing cultivation years， while the latter showed a decrease in volatility. The results of differential compound analysis based on UPLC-Q-TOF-MS showed that there were significant differences in metabolites between WPR and CPR samples. Among them， the seven compounds with the largest differences among WPR samples of different thicknesses were polygalasaponins， and for CPR with different planting years， the main differential compounds were oligosaccharide esters. ConclusionThere are differences between WPR and CPR in character， microscopic structure and chemical composition， and some components are inversely proportional with the increase of diameter and cultivation duration due to the distribution characteristics. However， the longer the cultivation years of PR， the closer it is to the "thick wrinkles on the epidermis" of WPR， which has been respected by generations. It is suggested that this traditional character combined with modern component contents should be used as the index of artificial cultivation and quality control of PR.

ObjectiveTo investigate the functional role and underlying molecular mechanisms of fumarylacetoacetate hydrolase （FAH） in the progression of glioblastoma （GBM）. MethodsDifferential expression analysis was performed on the TCGA-GBM， GSE4290， and GSE116520 datasets. Weighted gene co-expression network analysis （WGCNA） was used to identify key modules， and Cox regression and risk modeling were used to screen prognostic genes. Immune infiltration analysis of prognostic genes was carried out by using single-cell RNA sequencing panels. The clinical expression signature of FAH in GBM was analyzed in the TCGA and HPA databases. The functional role of FAH was validated by in vitro and in vivo experiments， and pathway analysis was performed to explore the underlying mechanisms. ResultsA total of 152 overlapping genes were identified across the three GBM datasets （P<0.05）. WGCNA revealed that the turquoise module was most strongly associated with tumor purity， stromal score， immune score， and ESTIMATE score （P<0.001）. Compared with normal tissues， three prognostic genes （CTSD， FAH， and THBD） were upregulated in GBM and correlated with immune infiltration （P<0.05）. FAH mRNA and protein levels were elevated in GBM tissues relative to normal tissues， and its expression was significantly associated with age stratification and TP53 mutation （P<0.05）. CCK-8 assay results showed that， compared with the shNC group， the proliferative activity of GBM cells in the shFAH group was reduced （P<0.001）. Transwell migration and invasion assays demonstrated that， relative to the shNC group， the numbers of migrated and invaded cells in the shFAH group decreased （P<0.05）. Western blot analysis revealed that the protein expression levels of PI3K， p-AKT， and p-mTOR in the shFAH group decreased compared with those in the shNC group （P<0.05）. In vivo subcutaneous xenograft experiments further confirmed that tumor volume and weight significantly decreased in the shFAH group compared with the shNC group （P<0.001）. ConclusionFAH promotes GBM progression by activating the PI3K/AKT/mTOR signaling pathway and may serve as a potential therapeutic target for GBM.

BACKGROUND: Arsenic trioxide (ATO) is indicated as a broad-spectrum medicine for a variety of diseases, including cancer and cardiac disease. While the role of ATO in hepatic ischemia/reperfusion injury (HIRI) has not been reported. Thus, the purpose of this study was to identify the effects of ATO on HIRI. METHODS: In the present study, we established a 70% hepatic warm I/R injury and partial hepatectomy (30% resection) animal models in vivo and hepatocytes anoxia/reoxygenation (A/R) models in vitro with ATO pretreatment and further assessed liver function by histopathologic changes, enzyme-linked immunosorbent assay, cell counting kit-8, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Small interfering RNA (siRNA) for extracellular signal-regulated kinase (ERK) 1/2 was transfected to evaluate the role of ERK1/2 pathway during HIRI, followed by ATO pretreatment. The dynamic process of autophagic flux and numbers of autophagosomes were detected by green fluorescent protein-monomeric red fluorescent protein-LC3 (GFP-mRFP-LC3) staining and transmission electron microscopy. RESULTS: A low dose of ATO (0.75 μmol/L in vitro and 1 mg/kg in vivo ) significantly reduced tissue necrosis, inflammatory infiltration, and hepatocyte apoptosis during the process of hepatic I/R. Meanwhile, ATO obviously promoted the ability of cell proliferation and liver regeneration. Mechanistically, in vitro  studies have shown that nontoxic concentrations of ATO can activate both ERK and phosphoinositide 3-kinase-serine/threonine kinase (PI3K-AKT) pathways and further induce autophagy. The hepatoprotective mechanism of ATO, at least in part, relies on the effects of ATO on the activation of autophagy, which is ERK-dependent. CONCLUSION Low, non-toxic doses of ATO can activate ERK/PI3K-AKT pathways and induce ERK-dependent autophagy in hepatocytes, protecting liver against I/R injury and accelerating hepatocyte regeneration after partial hepatectomy.
Animals ; Arsenic Trioxide ; Autophagy/physiology* ; Reperfusion Injury/prevention & control* ; Mice ; Male ; Proto-Oncogene Proteins c-akt/physiology* ; Arsenicals/therapeutic use* ; Oxides/therapeutic use* ; Liver/metabolism* ; Extracellular Signal-Regulated MAP Kinases/metabolism* ; Mice, Inbred C57BL

BACKGROUND:The traditional method for treating infected bone defects is thorough debridement,filling with antibiotic-impregnated bone cement,and then autologous bone transplantation.However,there are problems such as insufficient antibiotic concentration and drug resistance,and limited bone mass.Therefore,it is of great clinical significance to seek dual-functional biomaterials that can locally release antibiotics at the site of infection and promote bone repair.OBJECTIVE:To design an injectable hydrogel composed of hyaluronic acid and oxidized dextran as a local delivery system for vancomycin to treat infection and promote bone regeneration.METHODS:(1)Vancomycin-loaded hyaluronic acid/oxidized dextran hydrogels were prepared to characterize the morphology,mechanical properties,and in vitro drug release of the hydrogels.(2)Hyaluronic acid/oxidized dextran hydrogels and vancomycin-loaded hyaluronic acid/oxidized dextran hydrogels were co-cultured with rabbit bone marrow mesenchymal stem cells.Cell activity and proliferation were detected by live-dead staining and CCK-8 assay.After osteogenic induction,alkaline phosphatase staining,alizarin red staining,RUNX2 immunofluorescence staining,and RT-qPCR detection(osteocalcin and bone morphogenetic protein 2 mRNA expression)were performed to evaluate the osteogenic differentiation of rabbit bone marrow mesenchymal stem cells.(3)The above two hydrogels were co-cultured with Escherichia coli(or Staphylococcus aureus)to detect the antibacterial ability of the hydrogels.(4)Thirty rabbits were selected to establish a 1.5 cm infected bone defect model in the middle of the left radius.Two weeks after modeling,they were randomly divided into three intervention groups:the blank group(n=10)did not receive any treatment;the control group(n=10)was injected with hyaluronic acid/oxidized dextran hydrogel at the bone defect site;the experimental group(n=10)was injected with vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel at the bone defect site.12 weeks after injection,the samples were collected for Micro-CT scanning,tissue morphology observation,RT-qPCR detection(tumor necrosis factor α,interleukin 6 mRNA expression),and immunohistochemical staining.RESULTS AND CONCLUSION:(1)Vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel had a good porous structure with a pore size between 100-300 μm,and had good mechanical properties and in vitro drug sustained release performance.(2)Live-dead staining and CCK-8 assay results confirmed that vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel had good biocompatibility.Alkaline phosphatase staining,alizarin red staining,RUNX2 immunofluorescence staining,and RT-qPCR test results showed that both hydrogels could promote osteogenic differentiation of rabbit bone marrow mesenchymal stem cells.(3)Compared with hyaluronic acid/oxidized dextran hydrogel,vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel could significantly inhibit the growth of Escherichia coli and Staphylococcus aureus.(4)Micro-CT scanning results showed that the bone volume fraction and bone density of new bone in the bone defect area of the experimental group were higher than those of the blank group and the control group(P＜0.05).The bone tissue morphology observation results showed that the experimental group had better repair effect of bone defects compared with the blank group and control group.The expression of tumor necrosis factor α and interleukin 6 mRNA in the bone defect site in the experimental group was lower than that in the blank group and control group(P＜0.05).Immunohistochemical staining showed that the protein expressions of osteocalcin and RUNX2 at the site of bone defects in the experimental group and control group were higher than those in the blank group(P＜0.05).These findings indicate that vancomycin-loaded hyaluronic acid/oxidized dextran hydrogel can effectively promote bone regeneration under infection.

Objective To explore the function of electroacupuncture(EA)on body mass,fasting blood glucose,heat pain threshold,and transient receptor potential vanilloid 4(TRPV4)in the dorsal root ganglia(DRG)of rats with diabetic neuropathic pain(DNP).Methods A DNP rat model was formed by intraperitoneally injecting the animals with STZ.From days 15 to 21,bilateral Zusanli and Kunlun points of the DNP rat model were treated with electroacupuncture once daily for 30 min.We then measured their body mass,fasting blood glucose,and heat pain threshold.The co-expression of TRPV4 and NeuN in the rat L4～L6 DRG was detected by immunofluorescence.The effects of the TRPV4 agonist GSK1016790A on body mass,fasting blood glucose,and the heat pain threshold of DNP rats treated with electroacupuncture were detected.Results After the 7th day,body mass was significantly decreased(P<0.01)and fasting glucose was significantly increased(P<0.01)in the model group compared with the normal group.After the 21st day,compared with the model group,heat pain threshold of the model+electroacupuncture group was significantly higher(P<0.01);the results of co-expression of TRPV4 and NeuN immunofluorescence on rat L4～L6 DRG showed that:the expression of positive cells in the model group was significantly higher(P<0.01)than that in the normal group,the co-expression of TRPV4 and NeuN positive cells in L4～L6 DRG of rats in the model+electroacupuncture group was significantly lower(P<0.01)than that in the model group.The TRPV4 agonist GSK1016790A can reverse the downregulation of thermal pain threshold induced by electroacupuncture in DNP rats(P<0.01).Conclusion Electroacupuncture alleviated the DNP induced by STZ,and its mechanism may involve the inhibition of TRPV4 protein expression in the DRG.

Objective To analyze the changes of serum inhibin B(INHB),anti-Müllerian hormone(AMH)and sex hormone levels in patients with infertility caused by polycystic ovary syndrome(PCOS).Methods A total of 302 patients with infertility caused by PCOS admitted to the hospital from June 2021 to June 2023 were selected as the experimental group,and 300 healthy women of childbearing age with normal menstrual cycle were selected as the control group.The levels of serum INHB,AMH and sex hormone indexes[serum luteinizing hormone(LH),follicle stimulating hormone(FSH),prolactin(PRL),progesterone(P),testoster-one(T),estradiol(E2)]and homeostasis model assessment of insulin resistance(HOMA-IR)were observed and compared between the two groups.Results The serum levels of AMH,INHB and HOMA-IR in test group were significantly higher than those in control group,with statistical significance(P＜0.05).Serum levels of LH/FSH,PRL,T and P in experimental groups were significantly higher than those in control group,and serum E2 levels were significantly lower than those in control group,with statistical significance(P＜0.05).With the passage of treatment time,serum AMH,INHB,LH/FSH,PRL,T and P levels gradually decreased,and E2 levels gradually increased(P＜0.05).The area under the curve(AUC)of AMH and INHB combined detection in the diagnosis of infertility caused by PCOS was 0.905,which was higher than the AUC of AMH or INHB detection alone(P＜0.05).Serum AMH and INHB levels were positively correlated with FSH,LH,T and HOMA-IR,PRL,P(P＜0.05),and negatively correlated with serum E2 levels(P＜0.05).Conclusion The levels of AMH and INHB in serum of infertility patients caused by PCOS are significantly increased,and AMH and INHB are correlated with sex hormone levels.Detection of serum AMH,INHB and sex hormone levels is helpful for the diagnosis of infertility caused by PCOS.

Objective:To explore the impact of the interaction between alcohol consumption and overweight/obesity on the risk of hypertension.Methods:It was a cross-sectional study, and convenient sampling was used to enroll physical examination participants aged 18-60 years from the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, the Affiliated Hospital of Southwest Medical University and Luzhou People′s Hospital from June to November in 2020. All the participants were given questionnaire survey, physical examination and biochemical tests. A total of 5 000 questionnaires were distributed in the study, and 4 878 questionnaires were collected, of which 4 397 (90.14%) were valid. According to the diagnostic criteria for hypertension, the study participants were divided into hypertension group (1 128 cases) and non-hypertension group (3 269 cases), and t-test and chi-square test were used to compare the differences in gender, age, and other data between the two groups; and multivariate logistic regression analysis was used to analyze the association of alcohol consumption, overweight/obesity with the risk of hypertension and the interaction between alcohol consumption and overweight/obesity on the risk of hypertension, and relative excess risk of interaction, attributable proportion of interaction and the synergy index were used to evaluate the impact of the interaction between alcohol consumption and overweight/obesity on the risk of hypertension. Results:Among the 4 397 individuals included in the analysis, 3 116 were male and 1 281 were female, with a mean age of (42.42±8.83) years. The detection rate of hypertension was 25.7% (1 128/4 397). The risk of hypertension in overweight/obese individuals was 2.566 times ( OR=2.566, 95% CI: 2.167-3.038) higher than that of non-overweight/obese individuals, and the risk of hypertension in alcohol consumption individuals was 1.486 times ( OR=1.486, 95% CI: 1.250-1.766) higher than that of non-drinkers. The risk of hypertension in drinking+non-overweight/obesity group, non-drinking+overweight/obesity group, and drinking+overweight/obesity group was 1.468 times ( OR=1.468, 95% CI: 1.112-1.936), 2.538 times ( OR=2.538, 95% CI: 1.968-3.272), and 3.796 times ( OR=3.796, 95% CI: 2.963-4.863) higher than that of non-drinking+non-overweight/obesity group, respectively (all P<0.05). Alcohol consumption and overweight/obesity had an additive interaction effect on the risk of hypertension, and the relative excess risk of interaction, attributable proportion of interaction and the synergy index was 0.791 (95% CI: 0.158-1.424), 0.208 (95% CI: 0.049-0.368), 1.394 (95% CI: 1.030-1.888), respectively. There was no significant multiplicative interaction between alcohol consumption and overweight/obesity on the risk of hypertension ( P>0.05). Conclusions:Alcohol consumption and overweight/obesity are both associated higher risk of hypertension. In addition, there is an additive interaction between alcohol consumption and overweight/obesity on the risk of hypertension.

Objective:To investigate the relationship between protein tyrosine phosphatase non-receptor type 1 (PTPN1) gene polymorphism and the risk of gestational diabetes mellitus (GDM).Methods:In this case-control study, 4 835 pregnant women who delivered from March, 2012 to July, 2014 in the Department of Gynecology and Obstetrics at the First Hospital of Shanxi Medical University were consecutively enrolled. Among them, 789 cases were diagnosed with GDM. A simple random sampling method was used to select 334 pregnant women with GDM as the case group, and 334 healthy pregnant women matched by maternal age, gestation time and residence were set as control. The DNA genotyping was performed in the subjects, and those with genotyping deletions10% were excluded; and finally, 322 and 317 subjects were included in case and control group, respectively. Under the codominant, dominant, recessive, and allelic genetic models, the unconditional logistic regression model was used to check the relationship between 13 candidate single nucleotide polymorphism (snp) loci in PTPN1 gene and the risk of GDM. The Haploview was used to analyze the relationship between haplotypes and risk of GDM, and multiple comparisons were adjusted with the false discovery rate (FDR) method.Results:The age of the 639 pregnant women analyzed in this study was (30.28±4.32) years. The proportions of pre-pregnancy body mass index (BMI)≥24.0 kg/m 2 and having a family history of diabetes were significantly higher in the GDM group compared to those in the control group (29.19% vs 16.72% and 13.04% vs 6.31%, respectively, both P0.05). The rs6096644 locus was positively associated with increased risk of GDM in co-dominant (GG vs AA, OR=2.76, 95% CI: 1.18-6.44) and recessive (GG vs AA+AG, OR=2.78, 95% CI: 1.20-6.46) genetic models (all q0.2). The rs6096655 locus was positively associated with increased risk of GDM in codominant (AA vs GG, OR=5.90, 95% CI: 1.27-27.36) and recessive (AA vs GG+GA, OR=5.50, 95% CI: 1.19-25.38) and alleles (A vs G, OR=1.51, 95% CI: 1.09-2.08) genetic models (all q0.2). The rs6013317 locus was associated with an increased risk of GDM in the allele (A vs G, OR=1.74, 95% CI: 1.15-2.63) genetic model (all q0.2). The GAGG haplotype and GGAG haplotype in haplotype block 1 (rs4811262, rs6096646, rs6096655, rs6013317), and the GGGA haplotype in haplotype block 2 (rs6068018, rs6123105, rs6013324, rs2869621) of the PTPN1 gene were all positively associated with an increased risk of GDM (all P0.05). Conclusion:PTPN1 gene polymorphisms may associated with risk of GDM, moreover, complex haplotype structures within the gene influence the risk of GDM.

Objective:To evaluate the application effect of improved problem-based learning (PBL) based on 5W2H thinking in the course of "massage therapy".Methods:The undergraduate students in Class 1 and Class 2 of Acupuncture and Massage in the first semester of the 2023-2024 academic year at Heilongjiang University of Chinese Medicine were selected as the research subjects. The "massage therapy" was taught in Class 1 with the traditional method (control group, n=66) and in Class 2 with the method of improved PBL with 5W2H thinking (observation group, n=68). The two groups of students were compared in terms of theoretical knowledge of massage therapy, the assessment of massage skills, comprehensive ability, innovation ability, and practical ability, as well as the procedural evaluation indicators of massage. SPSS 25.0 was used for normality test and t test χ2 test. Results:After the completion of the study, the observation group showed higher scores in theoretical knowledge (90.56±11.73), massage skill proficiency [(87.22±11.57) vs. (78.23±9.45)], massage skill demonstration [(84.56±11.20) vs. (74.69±9.13)], mannequin usage [(82.98±10.75) vs. (72.66±8.89)], and massage skill mechanics [(86.38±11.45) vs. (73.35±8.94)] compared to the control group. The observation group also outperformed the control group in medical humanities [(85.53±10.64) vs. (64.86±7.33)], teacher-student and student-student interaction [(85.35±8.67) vs. (80.15±7.46)], clinical thinking and operation [(90.05±12.13) vs. (74.26±8.94)], and team coordination and cooperation [(85.60±10.06) vs. (81.53±9.33)]. In addition, the observation group scored higher than the control group in learning participation rate [(18.20±3.75) vs. (13.15±2.93)], error correction rate [(27.56±5.85) vs. (22.11±4.62)], operational stability [(48.43±7.79) vs. (42.06±6.60)], and total score [(94.19±7.56) vs. (77.32±6.78)].Conclusions:The application of improved PBL based on 5W2H thinking in the course of "massage therapy" has significant clinical implications. It can not only improve the teaching quality and effect, but also cultivate students' clinical thinking, teamwork, learning interest, and innovation ability, making it suitable for further promotion and application.