ObjectiveThis paper aims to investigate the effects and mechanism of Mimenghua prescription in modulating the mitogen-activated protein kinase kinase （MEK）/rat sarcoma viral oncogene homolog （Ras）/rapidly accelerated fibrosarcoma kinase （Raf）/extracellular signal-regulated kinase （ERK） signaling pathway to inhibit inflammatory responses in a dry eye animal model. MethodsA total of 60 C57BL/6J mice （eight weeks old， half male and half female） were used in the experiment. Ten mice were randomly selected as the blank control group， while the remaining 50 were exposed to a controlled dry system and received instillation of 0.2% benzalkonium chloride （BAC） into the eyes for four weeks to establish a dry eye mouse model. After successful modeling， the mice were randomly divided into five groups： Model group， sodium hyaluronate group， and Mimenghua prescription groups with low dose （4.83 g·kg^-1）， medium dose （9.67 g·kg^-1）， and high dose （19.34 g·kg^-1）. The mice in the model group received an equal volume of normal saline via gavage for four weeks. The mice in the sodium hyaluronate group received instillation of sodium hyaluronate eye drops twice daily for 14 consecutive days. The tear secretion volume， tear film break-up time （TBUT）， and corneal fluorescein staining were evaluated once every two weeks. After four weeks of administration， mice were euthanized， and their lacrimal gland tissues and corneas were harvested. Hematoxylin-eosin （HE） staining was used to assess histopathological morphology. Western blot was performed to detect the protein expression levels of MEK， Ras， Raf， and ERK. Enzyme-linked immunosorbent assay （ELISA） was used to measure the contents and expressions of MEK， Ras， Raf， ERK， and interleukin （IL）-1β in lacrimal gland and corneal tissues of the mice in each group. Quantitative real-time polymerase chain reaction （Real-time PCR） was employed to determine mRNA expression levels of MEK， Ras， Raf， and ERK. ResultsThe Mimenghua prescription groups and the sodium hyaluronate group exhibited significantly increased tear secretion volume （P<0.05） and prolonged TBUT （P<0.05） after treatment. Ocular surface damage of mice was visibly recovered. Western blot results indicated that protein expression levels of MEK， Ras， Raf， and ERK in the lacrimal gland and corneal tissues were significantly downregulated in the sodium hyaluronate group and Mimenghua prescription group with high dose （P<0.05）. ELISA results showed that IL-1β levels were highest in the model group but significantly reduced in the sodium hyaluronate group and Mimenghua prescription groups （P<0.05）. Both ELISA and Real-time PCR results demonstrated that the expression levels of MEK， Ras， Raf， and ERK in the lacrimal glands and corneal tissues were significantly elevated in the model group （P<0.05）， but markedly downregulated in the sodium hyaluronate group and Mimenghua prescription groups （P<0.05）， suggesting that Mimenghua prescription can decrease the expressions of MEK， Ras， Raf， and ERK in the lacrimal glands and corneal tissues. ConclusionMimenghua prescription can reduce inflammatory responses， increase tear secretion， prolong TBUT， and promote corneal recovery by inhibiting the MEK， Ras， Raf， and ERK signaling pathways in lacrimal gland and corneal tissues.

Aim To investigate the effects of dimethyl-arginine dimethylamino hydrolase 1(DDAH1)on high glucose-induced mitochondrial dysfunction and mitoph-agy in vascular endothelial cells.Methods JC-1 stai-ning was used to detect mitochondrial membrane poten-tial.DCFH-DA fluorescent probe was employed to measure reactive oxygen species(ROS)levels.Ho-echst staining was used to assess cell apoptosis.Real-time PCR was conducted to detect DDAH1 mRNA lev-els.Western blot was performed to analyze the expres-sion of DDAH1,LC3-Ⅰ and LC3-Ⅱ proteins.Mitochon-drial probe Mitotracker and autophagosome marker pro-tein LC3 were used in cell immunofluorescence co-lo-calization to assess mitochondrial autophagy,and high-performance liquid chromatography was utilized to measure the levels of asymmetric dimethylarginine(ADMA)in cell supernatant.Results High glucose treatment for 48 h significantly reduced mitochondrial membrane potential,increased ROS production,and promoted apoptosis in human umbilical vein endothelial cells(HUVECs).High glucose downregulated the ex-pression of LC3-Ⅱ/LC3-Ⅰ proteins,reduced the co-lo-calization of Mitotracker and LC3,and inhibited mito-chondrial autophagy.Autophagy inhibitors 3-MA or CQ exacerbated high glucose-induced mitochondrial dam-age and apoptosis in HUVECs,while autophagy activa-tor RAPA alleviated these effects.High glucose signifi-cantly downregulated DDAH1 protein expression in HUVECs and increased ADMA levels in cell superna-tant.DDAH1 siRNA inhibited mitochondrial autoph-agy,reduced mitochondrial membrane potential,and promoted apoptosis,whereas DDAH1 overexpression enhanced mitochondrial autophagy and alleviated high glucose-induced apoptosis in HUVECs.Conclusion High glucose-induced endothelial mitochondrial dys-function is associated with the suppression of DDAH1 expression,the increase of ADMA levels,and thereduc-tion of mitochondrial autophagy.

Aim To explore the mechanism of the syn-ergistic effect of the ferroptosis inducer erastin com-bined with shikonin in promoting ferroptosis in human hypertrophic scar fibroblasts(HSFBs).Methods Hypertrophic scar tissues provided by the General Hos-pital of Ningxia Medical University were collected,and HSFBs were extracted.HSFBs were identified by HE staining and immunofluorescence.The inhibitory rates of Era and SHK on HSFBs at different concentrations were detected by CCK-8 assay,and the IC50 value was calculated.CompuSyn software was used to calculate the co-use index(CI).Control group,Erastin(Era)group,shikonin(SHK)group and Era+SHK group were set up,and the number and morphological chan-ges of cells were observed after 24 hours of interven-tion.The ability of cell migration and invasion was de-tected by scratch test and Transwell test.The changes of malondialdehyde(MDA),total iron ion and reactive oxygen species(ROS)were detected by corresponding biochemical kits.The expressions of collagen I,α-SMA and GOT1,SLC7A11,GPX4 and FTH1 were detected by Western blot.Results The IC50 value of Era and SHK of primary HSFBs was 2.22 μmol·L-1 and 3.94μmol·L-1 respectively,which was used as the single drug concentration for subsequent experiments.The CompuSyn software was employed to calculate the CI value when the two drugs were used in combination,and the concentrations corresponding to CI=0.39597(Era:1.2 μmol·L-1+SHK:1.5 μmol·L-1)were selected as subsequent combination concentrations(Because when CI was equal to 0.395 97,the concen-tration of each drug was lower than the concentration of single drug,and the inhibition rate of combined drug was greater than 50％).Compared with the monother-apy group,the number of HSFBs in the SHK+Era group was significantly reduced,cell membrane showed breakage and vesiculation,cell wrinkling became smal-ler,and cytoplasm was concentrated.The migration and invasion ability of HSFBs in the SHK+Era group were obviously weakened(P＜0.05),and the expres-sion of fibrosis-related proteins collagen Ⅰ and α-SMA was reduced(P＜0.05);the contents of MDA,total i-ron ions,and ROS in HSFBs of the SHK+Era group increased(P＜0.05),and the protein expression lev-els of SLC7A11,GOT1,GPX4,and FTH1 further de-creased(P＜0.05).Conclusions Erastin in combi-nation with shikonin can synergistically inhibit the pro-liferation,migration and fibrosis levels of HSFBs.The mechanism may be that erastin enhances the inhibition of shikotin on GOT1,increases the levels of cellular i-ron ions,ROS,and lipid peroxides,thereby promoting ferroptosis in HSFBs.

Objective:To observe the effects of moxibustion at Shenshu(BL23)and Zusanli(ST36)on Toll-like receptor 4(TLR4)-myeloid differentiation factor 88(Myd88)signaling pathway-mediated inflammatory factors in the synovial tissue of ankle joints of rats with rheumatoid arthritis(RA),and to explore the molecular and biological mechanisms underlying the anti-inflammatory and analgesic effects.Methods:A total of 24 male Sprague-Dawley(SD)rats were randomly divided into a normal group,a model group,and a moxibustion group,with 8 rats in each group.The RA model was established with exposure to wind,cold,and damp environmental factors,along with Freund's complete adjuvant.After three days of modeling,mild moxibustion was applied to bilateral Shenshu(BL23)and Zusanli(ST36)in the moxibustion group using moxa sticks of 0.9 cm in diameter for 30 min each time,once a day for 14 d.Structural changes in the synovial tissue and cells were then observed using hematoxylin-eosin staining and transmission electron microscopy,while immunohistochemistry analysis was used to detect tumor necrosis factor(TNF)-α,interleukin(IL)-17,IL-1β,and IL-6 levels.Moreover,the protein expression levels of Myd88,TLR4,and transient potential receptor vanilloid type 1(TRPV1)in the synovial tissue were detected using Western blotting,while their mRNA expression levels were detected using reverse transcription-polymerase chain reaction.Finally,the levels of IL-1β,IL-2,IL-6,IL-17A,and TNF-α in rat serum were detected using enzyme-linked immunosorbent assay.Results:Compared to the normal group,the model group exhibited notable pathological synovial tissue damage,along with significantly higher IL-1β,IL-6,and TNF-α levels(P<0.01)and a slightly higher IL-17 content(P>0.05).Furthermore,the Myd88,TLR4,and TRPV1 protein and mRNA expression levels and serum IL-1β,IL-2,IL-6,IL-17A,and TNF-α levels were all significantly higher in the model group than in the normal group(P<0.01).Compared to the model group,the moxibustion group exhibited a lower degree of synovial tissue pathological damage,along with significantly lower IL-1β,IL-6,and TNF-α levels(P<0.05 or P<0.01)and a lower IL-17 content without statistical significance(P>0.05).Moreover,the Myd88,TLR4,and TRPV1 protein and mRNA expression levels,and serum IL-1β,IL-2,IL-6,IL-17A,and TNF-α levels were all significantly lower in the moxibustion group than in the model group(P<0.01 or P<0.05).Conclusion:Mild moxibustion at Shenshu(BL23)and Zusanli(ST36)can effectively inhibit TLR4-MyD88 signaling pathway-mediated inflammatory factor expression in the synovial tissue of ankle joints of RA rats.Furthermore,the effect of moxibustion on synovial tissue inflammation in RA rats may be attributed to TRPV1 channel activation.

Lung cancer poses a serious threat to global public health security.Chemotherapy,as the main strategy for cancer treatment,faces challenges such as high toxicity and drug resistance.Anticancer peptides have the potential of being developed into new anticancer drugs due to their advantages of broad-spectrum anticancer activity,rapid action,and difficulty in generating drug resistance,but they also face shortcomings such as weak activity and strong toxic side effects.The weakly acidic microenvironment of tumors(pH 6.5-6.8)provides a good idea for the design of anticancer peptides of high-efficiency and low-toxicity.Previously,we designed the acid-sensitive antibacterial peptide pHly-1 using the wolf spider(Lycosa singoriensis)toxin Lycosin-Ⅰ as a template.In this study,we found that pHly-1 also had acid-sensitive anticancer activity.Further alanine scanning analysis of pHly-1 was carried out,and we ob-tained a mutant pHTP-2 with better acid sensitivity,whose IC50(half maximal inhibitory concentration)against A549 cells was 15.68 μmol/L at pH 6.6 and was greater than 100 μmol/L at pH 7.4.At pH 6.6,pHTP-2 could act on various lung cancer cell lines and induce the death of A549 cells by rapid ly-sis;at pH 7.4,500 μmol/L pHTP-2 had weak toxicity to red blood cells(the hemolysis rate was ap-proximately 38％)and primary myocardial cells(the inhibition rate was 49.7％,with P＜0.05).Analy-sis of its charge,particle size,morphology,and secondary structure showed that at pH 6.6,the histidine in the sequence of pHTP-2 was protonated,increasing the positive charge(P＜0.01),decreasing the hy-drated particle size(P＜0.05)and forming an α-helical structure to induce membrane lysis of A549 cells.At pH 7.4,it was deprotonated,the positive charge decreases,a β-sheet structure was formed and self-aggregation occurred,limiting its effect on the A549 cell membrane and showing weak activity.In summary,pHTP-2 could respond to the weakly acidic microenvironment of tumors to exert selective cyto-toxic activity,effectively overcoming the shortcomings of anticancer peptides such as low efficiency and high toxicity.Our findings suggest that it is a high-quality lead molecule for anticancer drugs.

Lung cancer poses a serious threat to global public health security.Chemotherapy,as the main strategy for cancer treatment,faces challenges such as high toxicity and drug resistance.Anticancer peptides have the potential of being developed into new anticancer drugs due to their advantages of broad-spectrum anticancer activity,rapid action,and difficulty in generating drug resistance,but they also face shortcomings such as weak activity and strong toxic side effects.The weakly acidic microenvironment of tumors(pH 6.5-6.8)provides a good idea for the design of anticancer peptides of high-efficiency and low-toxicity.Previously,we designed the acid-sensitive antibacterial peptide pHly-1 using the wolf spider(Lycosa singoriensis)toxin Lycosin-Ⅰ as a template.In this study,we found that pHly-1 also had acid-sensitive anticancer activity.Further alanine scanning analysis of pHly-1 was carried out,and we ob-tained a mutant pHTP-2 with better acid sensitivity,whose IC50(half maximal inhibitory concentration)against A549 cells was 15.68 μmol/L at pH 6.6 and was greater than 100 μmol/L at pH 7.4.At pH 6.6,pHTP-2 could act on various lung cancer cell lines and induce the death of A549 cells by rapid ly-sis;at pH 7.4,500 μmol/L pHTP-2 had weak toxicity to red blood cells(the hemolysis rate was ap-proximately 38％)and primary myocardial cells(the inhibition rate was 49.7％,with P＜0.05).Analy-sis of its charge,particle size,morphology,and secondary structure showed that at pH 6.6,the histidine in the sequence of pHTP-2 was protonated,increasing the positive charge(P＜0.01),decreasing the hy-drated particle size(P＜0.05)and forming an α-helical structure to induce membrane lysis of A549 cells.At pH 7.4,it was deprotonated,the positive charge decreases,a β-sheet structure was formed and self-aggregation occurred,limiting its effect on the A549 cell membrane and showing weak activity.In summary,pHTP-2 could respond to the weakly acidic microenvironment of tumors to exert selective cyto-toxic activity,effectively overcoming the shortcomings of anticancer peptides such as low efficiency and high toxicity.Our findings suggest that it is a high-quality lead molecule for anticancer drugs.

Objective:To investigate lymph node metastasis(LNM)in the prostatic anterior fat pad(PAFP)of PCa patients after robot-assisted radical prostatectomy(RARP),and analyze the clinicopathological features and prognosis of LNM in the PAFP.Methods:We retrospectively analyzed the clinicopathological data on 1 003 cases of PCa treated by RARP in the Department of Urolo-gy of PLA General Hospital from January 2017 to December 2022.All the patients underwent routine removal of the PAFP during RARP and pathological examination,with the results of all the specimens examined and reported by pathologists.Based on the pres-ence and locations of LNM,we grouped the patients for statistical analysis,compared the clinicopathological features between different groups using the Student's t,Mann-Whitney U and Chi-square tests,and conducted survival analyses using the Kaplan-Meier and Log-rank methods and survival curves generated by Rstudio.Results:Lymph nodes were detected in 77(7.7％)of the 1 003 PAFP samples,and LNM in 11(14.3％)of the 77 cases,with a positive rate of 1.1％(11/1 003).Of the 11 positive cases,9 were found in the upgraded pathological N stage,and the other 2 complicated by pelvic LNM.The patients with postoperative pathological stage≥T3 constituted a significantly higher proportion in the PAFP LNM than in the non-PAFP LNM group(81.8％[9/11]vs 36.2％[359/992],P=0.005),and so did the cases with Gleason score ≥8(87.5％[7/8]vs 35.5％[279/786],P=0.009).No statisti-cally significant differences were observed in the clinicopathological features and biochemical recurrence-free survival between the pa-tients with PAFP LNM only and those with pelvic LNM only.Conclusion:The PAFP is a potential route to LNM,and patients with LNM in the PAFP are characterized by poor pathological features.There is no statistically significant difference in biochemical recur-rence-free survival between the patients with PAFP LNM only and those with pelvic LNM only.Routine removal of the PAFP and inde-pendent pathological examination of the specimen during RARP is of great clinical significance.

OBJECTIVE To establish a closed-loop management system for the whole-process traceability of outpatient drugs based on internet of things(IoT)and blockchain technology,and evaluate its implementation effects.METHODS A closed-loop management system for the whole-process traceability of outpatient drugs covering the entire drug lifecycle was designed using drug traceability codes integrated with IoT and blockchain technology.System effectiveness was evaluated from three dimensions:work efficiency,medication management quality and data safety by comparing indicators such as the acceptance time of incoming drugs and the number of collected drug traceability codes before the system implementation(October to December 2024)and after the system implementation(January to March 2025).RESULTS A closed-loop management system for the whole-process traceability of outpatient drugs,centered around the drug traceability code management system,was successfully established.The acceptance time for incoming drugs was shortened from(4.65±0.26)h before implementation to(0.34±0.08)h after implementation(P＜0.05).The number of collected drug traceability codes increased from 419 018 to 1 236 522,and the coverage rate of traceability codes rose from 28.36%to 89.88%(P＜0.05).The time pharmacists spent on drug expiry management per week decreased from(128.40±19.20)min to(0.56±0.13)min(P＜0.05),and the dispensing time for a single prescription(excluding a part of injections and repackaged drugs)was reduced from(143.25±17.67)s to(15.24±10.08)s(P＜0.05).The time for drug return was reduced from 129.90(122.32,137.00)s to 104.36(89.91,117.33)s(P＜0.05);the number of drug dispensing errors decreased from 2 cases to 0 cases.After the system was launched,there were no data security incidents in our outpatient pharmacy.CONCLUSIONS The constructed closed-loop management system for the whole-process traceability of outpatient drugs can significantly enhance drug traceability accuracy and drug management quality,improve pharmacist work efficiency,and reduce drug management risks,thus providing a feasible solution for the digital transformation of hospital pharmaceutical services.

Objective: To explore the feasibility of synthetic magnetic resonance imaging (syMRI) combined with clinicopathological characteristics for the pre-treatment prediction of chemoradiotherapy (CRT) response in advanced nasopharyngeal carcinoma (ANPC). Materials and Methods: Patients with ANPC treated with CRT between September 2020 and June 2022 were retrospectively enrolled and categorized into response group (RG, n = 95) and non RGs (NRG, n = 32) based on the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. The quantitative parameters from pre-treatment syMRI (longitudinal [T1] and transverse [T2] relaxation times and proton density [PD]), diffusion-weighted imaging (apparent diffusion coefficient [ADC]), and clinicopathological characteristics were compared between RG and NRG. Logistic regression analysis was applied to identify parameters independently associated with CRT response and to construct a multivariable model. The areas under the receiveroperating characteristic curve (AUC) for various diagnostic approaches were compared using the DeLong test. Results: The T1, T2, and PD values in the NRG were significantly lower than those in the RG (all P < 0.05), whereas no significant difference was observed in the ADC values between these two groups. Clinicopathological characteristics (Epstein–Barr virus [EBV]-DNA level, lymph node extranodal extension, clinical stage, and Ki-67 expression) exhibited significant differences between the two groups. Logistic regression analysis showed that T1, PD, EBV-DNA level, clinical stage, and Ki-67 expression had significant independent relationships with CRT response (all P < 0.05). The multivariable model incorporating these five variables yielded AUC, sensitivity, and specificity values of 0.974, 93.8% (30/32), and 91.6% (87/95), respectively. Conclusion SyMRI may be used for the pretreatment prediction of CRT response in ANPC. The multivariable model incorporating syMRI quantitative parameters and clinicopathological characteristics, which were independently associated with CRT response, may be a new tool for the pretreatment prediction of CRT response.