Based on a review of classical literature combined with modern anatomical and biomechanical research， this paper proposes the "four intersections" of channel sinew diseases from the perspective of structural interfaces， which involves sinew-bone， sinew-sinew， sinew-joint， and sinew-vessel intersections， and analyzes the corresponding pathological characteristics. It is believed that the dynamic evolution of channel sinew diseases can be summarized as "starting with mechanical imbalancew， with interface transmission as the pivot， compensation continuation as the variation， and neurological adaptation as the consolidation". Furthermore， the pathological changes at the "four intersections" structural interfaces correspond respectively to clinical manifestations of enthesial pain， referred pain， joint dysfunction and sensory abnormalities. Understanding channel sinew diseases based on the "four intersections" structural interface is conducive to deepening the understanding of the pathogenesis， thereby providing a reference for clinical identification and precise treatment.

Based on the close structural and functional associations of the Shaoyang （少阳） channel and related zang-fu （脏腑） organs with the brain and the pharynx， this paper proposes that the pathogenesis of post-stroke dysphagia involves constraint and stagnation of the pivot mechanism， binding of phlegm and stasis， frenetic stirring of ministerial fire， and incoordination of the spirit mechanism， ultimately leading to loss of pharyngeal function. An acupuncture therapy centered on regulating the pivot and unblocking the orifice is established. Acupoints of the Shaoyang channel， including Fengchi （GB 20）， Wangu （GB 12）， Yifeng （SJ 17）， and Waiguan （SJ 5）， are selected to regulate the pivot and treat the root； acupoints of the Du （督） and Ren （任） channels， including Fengfu （GV 16）， Lianquan （CV 23）， and Tiantu （CV 22）， are used to unblock the orifice and treat the branch. In addition， staged treatment can be applied according to syndrome differentiation. In the acute stage with predominant phlegm turbidity， Fenglong （ST 40）， Yinlingquan （SP 9）， Jinjin （EX-HN 12）， and Yuye （EX-HN 13） are added to resolve phlegm and facilitate orifice function. During the recovery stage with excess ministerial fire scorching the pharynx， Zulinqi （GB 41） and Zhaohai （KI 6） are added to drain fire and nourish yin； when ministerial fire disturbs the spirit， Tongli （HT 5） and Neiguan （PC 6） are added to calm the spirit and the heart. During the chronic stage with blood deficiency and collateral stasis， Zusanli （ST 36） and Sanyinjiao （SP 6） are added to nourish blood and unblock collaterals. In clinical practice， the angle and depth of needling are adjusted based on the stage of dysphagia， with the combination of elongated needles and medicinal moxibustion are used to enhance the therapeutic effect.

OBJECTIVE To screen a single matrix， prepare dissolving microneedles of Pterocarya hupehensis total flavonoids（PHTF-MN）， and characterize it while evaluating its analgesic effects.METHODS Using microneedle morphology and puncture rate as evaluation indicators， the matrix was screened from polyvinyl alcohol （PVA）， polyvinylpyrrolidone K30 （PVP K30）， PVP K90， and sodium hyaluronate with relative molecular weights of 15 000 and 60 000 （HA 1.5w and HA 6w ）. PHTF-MN was prepared by the layer-by-layer casting method with the selected matrix， and the appearance and morphology， total flavonoid content， mechanical properties， puncture performance， dissolution properties， and drug release behavior of the obtained PHTF-MN were characterized. Using male BALB/c mice as test subjects and aspirin （ASA） as a positive control， the analgesic effects of the microneedles were evaluated using a formalin-induced pain model.RESULTS Blank microneedles prepared with 30% HA 1.5w exhibited optimal surface flatness and the membrane puncture rate （through 4 layers of parafilm membrane） reaching 78%. The PHTF-MNs （with PHTF contents of 10， 20， 30， 40， and 50 mg/g in the matrix） exhibited uniform arrays and intact tips， with RSDs for total flavonoid contents all below 7% （ n ＝8）. The single-needle puncture forces were 1.46 and 1.07 N （with PHTF contents of 30 and 50 mg/g in the matrix， respectively）. The skin penetration rate approached 100%， with visible puncture marks in the stratum corneum and drug residue （with a PHTF content of 50 mg/g in the matrix）. After 30 min of fixation， the needles had almost completely dissolved， and the skin retention rates all exceeded 90% （with PHTF contents of 10， 30， and 50 mg/g in the matrix， respectively）. The cumulative release within 8 h was （134.64±2.03） μg/cm 2 ， and the fit to the first-order kinetic equation （ R 2 ＝0.950 1） was highest （with a PHTF content of 50 mg/g in the matrix）. Low- and high-dose PHTF-MN （containing 85.26 and 145.03 μg of total flavonoids per patch， respectively； 1 patch/day） significantly shortened the cumulative time of phase Ⅱ pain response in mice （ P ＜0.05）， with pain inhibition rates （43.67% and 59.78%） comparable to those of ASA （53.70%）.CONCLUSIONS PHTF-MN using HA 1.5w as a single matrix was successfully prepared. This formulation features a simplified preparation process， uniform drug loading， favorable mechanical properties， resistance to fracture， and rapid dissolution. Its drug release behavior conforms to a first-order kinetic model， and its analgesic effect is comparable to that of ASA.

"Sinew prescription correspondence" is the principle of selecting prescriptions for channel sinew diseases. On the basis of the theory of syndrome differentiation and treatment， the pulse manifestation corresponds to the channel sinew syndrome， which can improve the flexibility and standardization of clinical prescriptions. From the perspective of systematic dialectical sphygmology， this paper explains the dialectical relationship between channel sinew theory and pulse body elements， pulse wall elements， pulse elements and blood flow elements， and clarifies the internal relationship between pulse manifestation and prescriptions at the level of channel sinew disease. The prescription is derived from the method， while the method is established with the syndrome， and the prescription is unified by the method. According to the theory of "sinew prescription correspondence"， the treatment ideas of channel sinew diseases were analyzed from the perspective of channel sinew distribution， functional characteristics and structural changes. On this basis， the diagnosis of channel sinew disease and the application of prescriptions are expanded， and the research on the internal treatment and diagnosis mode of "pulse manifestation-channel sinew-zang fu （脏腑）" is prospected， so as to expand the differentiation and treatment methods of channel sinew theory.

ObjectiveTo elucidate the underlying mechanism through which Zhuluan decoction suppresses excessive autophagy in human ovarian granulosa cells （KGN） and ameliorates premature ovarian insufficiency （POI） via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsThe optimal concentration of cyclophosphamide for inducing a POI model in KGN cells was identified via the cell counting kit-8 （CCK-8） assay. Subsequently， the impacts of varying concentrations of Zhuluan decoction-containing serum on the viability of the KGN cell model were assessed. After the optimal drug concentration was determined， KGN cells were categorized into the following groups： blank control （20% blank serum）， model （20% blank serum + 5 μmol·L^-1 cyclophosphamide）， Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide）， autophagy inhibitor （20% blank serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， autophagy inhibitor + Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， and estradiol valerate （20% estradiol valerate-containing serum + 5 μmol·L^-1 cyclophosphamide）. Following 48 hours of incubation， flow cytometry was utilized to measure the apoptosis rate of KGN cells in each group. Western blotting was employed to quantify the protein levels of PI3K， phosphorylated （p）-Akt， Akt， p-mTOR， and mTOR， along with the expression levels of autophagy-related proteins such as Beclin1， autophagy-related 5 homolog （ATG5）， and microtubule-associated protein 1 light chain 3 （LC3）， in each group. Additionally， monodansylcadaverine （MDC） staining was performed to evaluate the extent of autophagy in each group. ResultsIncubation of KGN cells with 5 μmol·L^-1 cyclophosphamide for 48 h successfully established a POI model， marked by a significant inhibition of KGN cell proliferation. Notably， the inhibitory effect of cyclophosphamide on KGN cell proliferation exhibited a positive correlation with its concentration. Zhuluan decoction-containing serum at 20% and 30% promoted cell proliferation and mitigated the inhibitory effect of cyclophosphamide on KGN cell proliferation， with comparable therapeutic efficacy observed at both concentrations. Compared with the blank control group， the model group displayed an elevated apoptosis rate （P<0.01）， reduced protein levels of PI3K， p-Akt， and p-mTOR （P<0.01）， increased protein levels of Beclin1， LC3， and ATG5 （P<0.01）， no significant alterations in the protein levels of Akt and mTOR， and an enhanced MDC autophagy fluorescence intensity （P<0.01）. In comparison to that the model group， the apoptosis rates in the blank control group， model group， Zhuluan decoction-containing serum group， autophagy inhibitor group， autophagy inhibitor + Zhuluan decoction-containing serum group， and estradiol valerate group all reduced （P<0.05， P<0.01）， with the most pronounced reduction observed in the autophagy inhibitor + Zhuluan decoction-containing serum group. The protein levels of PI3K， p-Akt， and p-mTOR were higher in other groups than in the model group （P<0.05， P<0.01）， being the highest in the autophagy inhibitor + Zhuluan decoctio-containing serum group （P<0.01）. The protein levels of Beclin1 and ATG5 were lower in other groups than in the model group （P<0.05， P<0.01）. The expression level of LC3 declined in the Zhuluan decoction-containing serum group and the estradiol valerate group （P<0.05， P<0.01）， while it decreased without statistical significance in the autophagy inhibitor group and the autophagy inhibitor + Zhuluan decoction-containing serum group. ConclusionZhuluan decoction may activate the PI3K/Akt/mTOR pathway to inhibit excessive autophagy and counteract the detrimental effects of cyclophosphamide on the KGN cell model， thus managing POI.

ObjectiveTo elucidate the underlying mechanism through which Zhuluan decoction suppresses excessive autophagy in human ovarian granulosa cells （KGN） and ameliorates premature ovarian insufficiency （POI） via the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsThe optimal concentration of cyclophosphamide for inducing a POI model in KGN cells was identified via the cell counting kit-8 （CCK-8） assay. Subsequently， the impacts of varying concentrations of Zhuluan decoction-containing serum on the viability of the KGN cell model were assessed. After the optimal drug concentration was determined， KGN cells were categorized into the following groups： blank control （20% blank serum）， model （20% blank serum + 5 μmol·L^-1 cyclophosphamide）， Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide）， autophagy inhibitor （20% blank serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， autophagy inhibitor + Zhuluan decoction-containing serum （20% Zhuluan decoction-containing serum + 5 μmol·L^-1 cyclophosphamide + 20 μmol·L^-1 chloroquine phosphate）， and estradiol valerate （20% estradiol valerate-containing serum + 5 μmol·L^-1 cyclophosphamide）. Following 48 hours of incubation， flow cytometry was utilized to measure the apoptosis rate of KGN cells in each group. Western blotting was employed to quantify the protein levels of PI3K， phosphorylated （p）-Akt， Akt， p-mTOR， and mTOR， along with the expression levels of autophagy-related proteins such as Beclin1， autophagy-related 5 homolog （ATG5）， and microtubule-associated protein 1 light chain 3 （LC3）， in each group. Additionally， monodansylcadaverine （MDC） staining was performed to evaluate the extent of autophagy in each group. ResultsIncubation of KGN cells with 5 μmol·L^-1 cyclophosphamide for 48 h successfully established a POI model， marked by a significant inhibition of KGN cell proliferation. Notably， the inhibitory effect of cyclophosphamide on KGN cell proliferation exhibited a positive correlation with its concentration. Zhuluan decoction-containing serum at 20% and 30% promoted cell proliferation and mitigated the inhibitory effect of cyclophosphamide on KGN cell proliferation， with comparable therapeutic efficacy observed at both concentrations. Compared with the blank control group， the model group displayed an elevated apoptosis rate （P<0.01）， reduced protein levels of PI3K， p-Akt， and p-mTOR （P<0.01）， increased protein levels of Beclin1， LC3， and ATG5 （P<0.01）， no significant alterations in the protein levels of Akt and mTOR， and an enhanced MDC autophagy fluorescence intensity （P<0.01）. In comparison to that the model group， the apoptosis rates in the blank control group， model group， Zhuluan decoction-containing serum group， autophagy inhibitor group， autophagy inhibitor + Zhuluan decoction-containing serum group， and estradiol valerate group all reduced （P<0.05， P<0.01）， with the most pronounced reduction observed in the autophagy inhibitor + Zhuluan decoction-containing serum group. The protein levels of PI3K， p-Akt， and p-mTOR were higher in other groups than in the model group （P<0.05， P<0.01）， being the highest in the autophagy inhibitor + Zhuluan decoctio-containing serum group （P<0.01）. The protein levels of Beclin1 and ATG5 were lower in other groups than in the model group （P<0.05， P<0.01）. The expression level of LC3 declined in the Zhuluan decoction-containing serum group and the estradiol valerate group （P<0.05， P<0.01）， while it decreased without statistical significance in the autophagy inhibitor group and the autophagy inhibitor + Zhuluan decoction-containing serum group. ConclusionZhuluan decoction may activate the PI3K/Akt/mTOR pathway to inhibit excessive autophagy and counteract the detrimental effects of cyclophosphamide on the KGN cell model， thus managing POI.

ObjectiveThis study aimed to evaluate the effectiveness of various statistical methods based on the complementary sensitivity analysis framework recommended by the European Medicines Agency (EMA) in Alzheimer’s disease clinical trials, providing a reference for statistical practice that meets international regulatory standards for similar studies. MethodsBased on longitudinal data from a phase Ⅲ clinical trial for the treatment of Alzheimer’s disease, the change from baseline in Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) score from baseline at week 26 (with a decrease indicating cognitive improvement) was used as the primary outcome measure. This study employed a mixed-effects model with repeated measures (MMRM) under the assumption of missing at random (MAR) as the primary analysis, while using critical point analysis, rank-based analysis, and slope-based analysis for sensitivity verification. A systematic comparison of the efficacy estimation results from the four analytical methods was conducted to compare the consistency and robustness differences in research conclusions across different analytical strategies. ResultsThe primary analysis results from the MMRM showed that the improvement in ADAS-Cog score from baseline at week 26 in the treatment group was superior to that in the placebo group (LSMD=-3.22, 95%CI: -4.55 to -1.88, P<0.001). Both rank-based analyses and slope-based analyses indicated significant differences between groups (both P<0.001). Critical point analyses revealed that the treatment effect was negative under most reasonable missing hypothesis scenarios, with the effect value only potentially turning positive in extreme non-random missing scenarios. There was no overall reversal of the efficacy conclusion. ConclusionThe combination of MMRM, critical point analysis, rank-based analysis, and slope-based analysis can effectively address the challenge of missing data in clinical trials for progressive diseases. This strategy fully complies with international regulatory standards, verifies efficacy conclusions from multiple perspectives, and enhances the credibility and scientific validity of clinical trial results. It can provide a practical reference for statistical analysis of missing data in similar clinical trials for progressive diseases.

BACKGROUND:Growth factor is the key effect molecule that plays a role in platelet-rich plasma in clinical treatment.There are differences in the concentration of growth factor after different activators activate platelet-rich plasma,which is an important factor affecting clinical efficacy. OBJECTIVE:To analyze the influence of different activators on the mass concentration of growth factors in platelet-rich plasma. METHODS:Totally 12 healthy volunteers were recruited to collect EDTA-K2 anticoagulant venous blood.Secondary centrifugation was used to prepare platelet-rich plasma.The difference in mass concentrations of growth factors was compared between venous blood and platelet-rich plasma.The platelet-rich plasma was mixed with four activators(normal saline,thrombin,calcium gluconate,calcium gluconate+thrombin)according to the volume ratio of 10:1,and incubated in a constant temperature water bath at 37 °C for 30 minutes.After centrifugation,the supernatant was extracted and the mass concentration of growth factor was detected.The bacterial growth in supernatant was measured by blood agar plate.Pearson correlation was used to analyze the correlation between different activators and the mass concentration of growth factor in platelet-rich plasma,and the correlation between the value of thrombocytometer and the mass concentration of growth factors in platelet-rich plasma. RESULTS AND CONCLUSION:(1)The mass concentrations of platelet-derived growth factor-BB,platelet-derived growth factor-AB,vascular endothelial growth factor,and epidermal growth factor in platelet-rich plasma were 8.7,22.2,2.3,and 2.8 times of those in venous blood,respectively(P<0.05).(2)Compared with normal saline group,the mass concentrations of platelet-derived growth factor BB,platelet-derived growth factor AB,vascular endothelial growth factor,and epidermal growth factor were increased in the thrombin group,calcium gluconate group,and calcium gluconate+thrombin group(P<0.05).The mass concentration of platelet-derived growth factor BB in the thrombin group and calcium gluconate group was higher than that in the calcium gluconate+thrombin group(P<0.05),and the mass concentration of platelet-derived growth factor AB in the thrombin group was higher than that in the calcium gluconate group and calcium gluconate+thrombin group(P<0.05).Epidermal growth factor mass concentration in the thrombin group was lower than that in the calcium gluconate group and calcium gluconate+thrombin group(P<0.05).(3)The results of blood agar plate test showed no bacterial growth in the supernatant of the four groups.(4)Pearson correlation analysis showed that the mass concentration of platelet-derived growth factor BB in platelet-rich plasma was strongly positively correlated with thrombin(r=0.683,P<0.05),and the mass concentration of vascular endothelial growth factor was strongly positively correlated with thrombin,calcium gluconate,calcium gluconate+thrombin stimulant(r=0.730,0.789,0.686,P<0.05).There was no correlation between the value of thrombocytometer and the mass concentration of four kinds of growth factors(P>0.05).(5)The results suggest that different activators have an impact on the concentration of growth factors in platelet-rich plasma.It is suggested to choose different activators to improve clinical efficacy according to different growth factor mass concentrations and treatment needs.

Objective:To investigate the effects and mechanism of Jianpi Yangzheng Xiaozheng Prescription in lung pre-metastatic niche formation of gastric cancer by regulating the content of programmed death receptor ligand 1 (PD-L1) in gastric cancer exosomes.Methods:Totally 30 615 mice were divided into normal group, model group, Jianpi Yangzheng Xiaozheng Prescription low- and high-dosage groups, and exosome inhibitor group according to random number table method, with 6 mice in each group. The mouse model of lung pre-metastatic niche formation of gastric cancer was established by tail vein injection of MFC cells. After 12 days of administration, the lung metastasis under the intervention of Jianpi Yangzheng Xiaozheng Prescription was evaluated by observing the infiltration of tumor into lung tissue, weighing lung weight and hematoxylin-eosin (HE) staining of lung tissue. Mouse serum exosomes were extracted by ExoQuick kit and identified by transmission electron microscopy and nanoparticle tracking analysis (NTA). The content of PD-L1 in serum exosomes and the expression levels of serum transforming growth factor-β (TGF-β), interleukin-10 (IL-10) and interleukin-6 (IL-6) were detected by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to detect the phenotype of myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) and the expression of PD-L1 in MDSC and TAM.Results:Compared with the model group, after the intervention of Jianpi Yangzheng Xiaozheng Prescription, the lung metastases of mice were reduced ( P<0.05), and the weight of metastatic tumors decreased ( P<0.05). PD-L1 in serum exosomes and the proportion of MDSC and M2 TAM in lung tissue microenvironment decreased, as well as the expression of PD-L1 on MDSC and TAM decreased ( P<0.05). The serum levels of IL-10 and IL-6 significantly decreased ( P<0.05). Conclusion:Jianpi Yangzheng Xiaozheng Prescription can reduce the proportion of MDSC and M2 TAM and the expression level of PD-L1 in the microenvironment of lung tissue before metastasis by inhibiting the transmission of gastric cancer exosome PD-L1 to MDSC and TAM, and reduce the contents of inflammatory factorsIL-10 and IL-6, so as to play a role in improving the microenvironment before lung metastasis of gastric cancer.

Objective To investigate the relationship between serum trimethylamine oxide(TMAO),endo-thelial cell specific molecule 1(Endocan)and cardiac function and pregnancy outcome in patients with hyper-tensive disorders of pregnancy(HDP).Methods A total of 182 patients with HDP admitted to Handan Ma-ternal and Child Health Hospital from January 2021 to June 2023(HDP group)and 98 healthy pregnant women admitted to this hospital during the same period(control group)were selected as research subjects.Serum TMAO,Endocan and left ventricular cardiac function indexes[left ventricular ejection fraction(LVEF),left ventricular end-diastolic volume(LVEDV)and left ventricular end-systolic volume(LVESV)]were compared between the two groups.According to pregnancy outcome,HDP patients were divided into poor outcome group(78 cases)and good outcome group(104 cases).Spearman correlation analysis was used to analyze the correlation between serum TMAO and Endocan and cardiac function indexes in HDP patients,and multi-factor Logistic regression was used to analyze the influencing factors of adverse pregnancy outcomes in HDP patients.The predictive value of serum TMAO and Endocan for adverse pregnancy outcomes in HDP patients was analyzed by receiver operating characteristic(ROC)curve.Results Compared with control group,serum TMAO,Endocan,LVEDV and LVESV were increased in HDP group,and LVEF was decreased(P＜0.05).Serum TMAO and Endocan in HDP patients were negatively correlated with LVEF(P＜0.05),and positively correlated with LVEDV and LVESV(P＜0.05).The incidence of adverse pregnancy outcomes in 182 HDP patients was 42.86％(78/182).Preeclampsia(PE),severe preeclampsia(SPE),24 h urine protein increase,LVEDV increase,LVESV increase,TMAO increase,Endocan increase were independent risk factors for adverse pregnancy outcomes in HDP patients,and LVEF increase was protective factor(P＜0.05).The area under the curve of serum TMAO combined with Endocan in predicting adverse pregnancy outcomes in HDP patients was 0.880,which was greater than 0.793 and 0.788 predicted by serum TMAO and Endocan a-lone.Conclusion The increase of serum TMAO and Endocan levels in HDP patients are relate to the decrease of cardiac function and adverse pregnancy outcomes,and the combined detection of the two has high predictive value for adverse pregnancy outcomes in HDP patients.