OBJECTIVE To explore the intervention mechanism of Shangke Lengtongtie on cold hyperalgesia in KOA mice based on the HMGB1/CXCL12/CXCR4 signaling axis.METHODS Monosodium iodoacetate(MIA)was used for the intra-articular injec-tion into the knee joint to establish mice model of knee osteoarthritis(KOA).Peripheral blood monocytes were extracted from mice,cultured,and then reinfused into the tail vein of the mice.Subsequently,in vivo animal imaging was used to observe the recruitment sites of these monocytes.The cold hyperalgesia threshold was measured at various time points in each group of mice.Hematoxylin and eosin(HE)staining was used to evaluate the level of synovial pathological changes.ELISA was employed to detect the expression of in-flammatory factors IL-1β,TNF-α,and pain mediators CGRP and Substance P in mouse serum.Western blot and qPCR methods were used to detect the protein and gene expression of cold hyperalgesia-related indicators such as TRPA1,TRPM8,HMGB1,CXCL12,CXCR4,Collagen Ⅰ,and Netrin-1 in synovial tissue,as well as DCC in dorsal root ganglia(DRG)tissue.RESULTS In vivo ima-ging showed that after the monocytes were reinfused into KOA mice,they were recruited to the knee joint area,with the HMGB1 group exhibiting a greater recruitment of circulating monocytes at the knee joint.Additionally,compared to the control group,the KOA group and HMGB1 group showed inflammatory pathological changes in the synovium,increased expression of serum inflammatory factors and pain mediators,reduced cold hyperalgesia threshold,and upregulated protein and gene expression of cold hyperalgesia-related indica-tors in synovial and DRG tissues.The changes were more significant in the HMGB1 group compared to the KOA group(P<0.05).Af-ter treatment with Shangke Lengtongtie or GL intervention,synovial inflammation was alleviated,serum inflammatory factors and pain mediators decreased,cold hyperalgesia threshold increased,and the upregulation of cold hyperalgesia-related indicator protein and gene expression levels was significantly reversed(P<0.05).CONCLUSION Shangke Lengtongtie exerts a beneficial effect on the mitigation of synovitis and cold hyperalgesia in KOA mice,a therapeutic mechanism that possibly mediated through the inhibition of the HMGB1/CXCL12/CXCR4 signaling axis.

ObjectiveTo investigate the possible mechanism of action of Xibining Formula （膝痹宁） for cartilage damage in knee osteoarthritis （KOA） through the cyclic guanosine-adenosine monophosphate synthase （cGAS）- stimulator of interferon genes （STING） signaling pathway. MethodsFifty C57BL/6J mice were randomly divided into five groups （10 per group）， sham operation group， KOA model group， low-dose Xibining Formula group， high-dose Xibining Formula group， and high-dose Xibining Formula + agonist group. The KOA models were constructed using the destabilization of the medial meniscus （DMM） method in all groups but the sham surgery group. Two weeks after surgery， the low- and high-dose Xibining Formula groups were administered Xibining Formula at doses of 3.58 g/（kg·d） and 14.32 g/（kg·d） respectively via gavage. The high-dose Xibining Formula + agonist group received 14.32 g/（kg·d） of Xibining Formula via gavage followed by an intraperitoneal injection of Vadimezan （DMXAA） at 25 mg/kg. The sham surgery group and the KOA model group mice were given an equivalent volume of normal saline at 5 ml/（kg·d） via gavage， once daily for four consecutive weeks. Serum levels of tumor necrosis factor-alpha （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） were measured by ELISA； pathological changes in cartilage tissue were observed using hematoxylin-eosin （HE） staining and Safranin O-Fast Green staining. Pathological changes were scored according to the Mankin scoring system； the levels of cartilage tissue matrix regulation-related indicators such as matrix metalloproteinase 3 （MMP3）， matrix metalloproteinase 13 （MMP13）， a disintegrin and metalloproteinase with thrombospondin motifs 5 （ADAMTS）， type-Ⅱ collagen （CⅡ） and aggregated proteoglycan （Aggrecan）， and also cGAS-STING pathway-related protein and mRNA expression levels were detected by Western blot and qPCR methods. ResultsCompared with the sham surgery group， the KOA model group showed severe cartilage edge destruction， significantly increased Mankin scores， significantly decreased protein and mRNA expression levels of COLⅡ and Aggrecan， and significantly increased protein and mRNA expression levels of cGAS， STING， MMP3， MMP13， and ADAMTS5 （P＜0.01）. Compared with the control group， serum level of IL-6， IL-1β， TNF-α in all the intervented groups decreased （P＜0.01）， while compared with high-dose Xibining Formula group， level of IL-6， IL-1β， and TNF-α in low-dose Xibining Formula group and high-dose Xibining Formula + agonist group increased （P＜0.01）. Compared with the KOA model group， all the intervention groups exhibited alleviated cartilage pathological changes， signi-ficantly reduced Mankin scores， significantly increased protein and mRNA expression levels of COLⅡ and Aggrecan， and significantly decreased protein and mRNA expression levels of cGAS， STING， MMP3， MMP13， and ADAMTS5 （P＜0.01）. Compared with high-dose Xibining Formula group， high-dose Xibining Formula + agonist group showed cartilage edge destruction， significantly increased Mankin scores， significantly decreased protein and mRNA expression levels of COLⅡ and Aggrecan， and increased protein and mRNA expression levels of cGAS， STING， MMP3， MMP13， and ADAMTS5 （P＜0.01）. ConclusionXibining Formula may improve KOA cartilage damage by inhibiting the cGAS-STING signaling pathway， decreasing matrix degradation-related proteins， and elevating matrix composition-related proteins.

OBJECTIVE To explore the intervention mechanism of Shangke Lengtongtie on cold hyperalgesia in KOA mice based on the HMGB1/CXCL12/CXCR4 signaling axis.METHODS Monosodium iodoacetate(MIA)was used for the intra-articular injec-tion into the knee joint to establish mice model of knee osteoarthritis(KOA).Peripheral blood monocytes were extracted from mice,cultured,and then reinfused into the tail vein of the mice.Subsequently,in vivo animal imaging was used to observe the recruitment sites of these monocytes.The cold hyperalgesia threshold was measured at various time points in each group of mice.Hematoxylin and eosin(HE)staining was used to evaluate the level of synovial pathological changes.ELISA was employed to detect the expression of in-flammatory factors IL-1β,TNF-α,and pain mediators CGRP and Substance P in mouse serum.Western blot and qPCR methods were used to detect the protein and gene expression of cold hyperalgesia-related indicators such as TRPA1,TRPM8,HMGB1,CXCL12,CXCR4,Collagen Ⅰ,and Netrin-1 in synovial tissue,as well as DCC in dorsal root ganglia(DRG)tissue.RESULTS In vivo ima-ging showed that after the monocytes were reinfused into KOA mice,they were recruited to the knee joint area,with the HMGB1 group exhibiting a greater recruitment of circulating monocytes at the knee joint.Additionally,compared to the control group,the KOA group and HMGB1 group showed inflammatory pathological changes in the synovium,increased expression of serum inflammatory factors and pain mediators,reduced cold hyperalgesia threshold,and upregulated protein and gene expression of cold hyperalgesia-related indica-tors in synovial and DRG tissues.The changes were more significant in the HMGB1 group compared to the KOA group(P<0.05).Af-ter treatment with Shangke Lengtongtie or GL intervention,synovial inflammation was alleviated,serum inflammatory factors and pain mediators decreased,cold hyperalgesia threshold increased,and the upregulation of cold hyperalgesia-related indicator protein and gene expression levels was significantly reversed(P<0.05).CONCLUSION Shangke Lengtongtie exerts a beneficial effect on the mitigation of synovitis and cold hyperalgesia in KOA mice,a therapeutic mechanism that possibly mediated through the inhibition of the HMGB1/CXCL12/CXCR4 signaling axis.

Objective To investigate the effect of Piezo1 activated by mechanical stress on knee osteoarthritis synovial fibrosis via the extracellular signal-regulated kinase(ERK)1/2 signaling pathway.Methods Twenty-five Sprague Dawley rats were divided into blank,exercise,exercise+GsMTx4,exercise+PD98059,and exercise+GsMTx4+PD98059 groups(n=5 per group).After modeling,serum and synovial tissue were extracted and collagen deposition was evaluated by Sirius red and Masson staining.Expression levels of Piezo1,ERK1/2,phospho(p)-ERK1/2,α-smooth muscle actin(SMA),transforming growth factor(TGF)-β,Collagen Ⅰ,and tissue inhibitor of metalloproteinase(TIMP)-1 were detected by Western blot and reverse transcription-quantitative polymerase chain reaction(RT-qPCR),and the interleukin(IL)-1β,IL-6,and tumor necrosis factor(TNF)-α contents were detected by enzyme-linked immunosorbent assay.For cell experiments,synovial cells were divided into blank,pull,pull+GsMTx4,pull+PD98059,and pull+GsMTx4+PD98059 groups and the above indices were detected in the model cells by Western blot,RT-qPCR,and other techniques.Results Mechanical stress increased collagen deposition in synovial tissues in the rats,and increased the protein and mRNA expression levels of the pathway-related and fibrosis-specific indicators Piezo1,p-ERK/ERK,α-SMA,TGF-β,Collagen I,and TIMP-1(P＜0.05).Piezo1 expression was significantly down-regulated by both inhibitors(P＜0.05),but the ERK inhibitor(PD98059)had no significant effect on Piezo1 gene expression.Levels of serum inflammatory factors were significantly higher in the exercise group compared with the blank group(P＜0.05),and levels were significantly reduced by the inhibitors(P＜0.05).The in vitro experiments showed the same trends as the animal experiments.Conclusions The Piezo1 ion channel can sense mechanical stress and activate the ERK 1/2 pathway to mediate knee synovial fibrosis.

ObjectiveTo observe the effects of Xibining （XBN） and adipose stem cell exosome （ADSC-Exos） in the cases of separate or joint application on cartilage degeneration and mitochondrial autophagy and explore its mechanism of action to improve knee osteoarthritis （KOA）. MethodSD rats were divided into a sham operation group （sham group）， a model group， an ADSC-Exos group （Exos group）， an XBN group， and an ADSC-Exos+XBN group （Exos+XBN group）. KOA model was established by using anterior cruciate ligament transection （ACLT）. The pain sensitivity status of rats was evaluated， and the degeneration degree of the knee joint and cartilage tissue was detected by Micro-CT and pathological staining. The expression of p62 and LC3B was observed by immunofluorescence， and the serum levels of TNF-α， IL-1β， IL-6， and IL-15 in rats were detected by ELISA. The Western blot was used to detect the protein expression levels of MMP-3， MMP-13， ADAMTS5， ColⅡ， TIMP， ACAN， PINK1， Parkin， p62， and LC3A/B. ResultCompared with the sham group， rats in the model group showed decreased cold-stimulated foot-shrinkage thresholds and mechanical pain sensitivity thresholds， varying degrees of abrasion and loss of cartilage tissue， degeneration of cartilage tissue， elevated serum IL-1β， IL-6， IL-15， and TNF-α levels （P<0.01）， and increased protein expression of MMP-3， MMP-13， and ADAMTS5 in cartilage tissue. In addition， the protein expression of ColⅡ， TIMP1， and ACAN was decreased （P<0.01）. Compared with the model group， rats in each treatment group showed higher cold-stimulated foot-shrinkage thresholds and mechanical pain sensitivity thresholds， reduced cartilage tissue degeneration， lower serum levels of IL-1β， IL-6， IL-15， and TNF-α （P<0.05，P<0.01）， decreased protein expression of MMP-3， MMP-13， and ADAMTS5， and higher protein expression of Cold， TIMP1， and ACAN in cartilage tissue （P<0.05，P<0.01）. Moreover， the changes were the most obvious in the Exos+XBN group. ConclusionBoth ADSCs-Exos and XBN can increase the level of mitochondrial autophagy in chondrocytes and delay cartilage tissue degeneration by promoting the expression of the PINK1/Parkin signaling pathway， and the combination of the two can enhance the therapeutic effect.

OBJECTIVE To explore the effect and mechanism of warming channels and activating blood circulation external treat-ment to alleviate peripheral hyperalgesia in knee osteoarthritis(KOA)based on NGF/TrKA signaling pathway.METHODS 30 SD rats were randomly divided into normal group,KOA group and Yiceng group.KOA model was established by anterior cruciate ligament transection(ACLT).14 days after model establishment,rats in Yiceng group were treated with Yiceng patch.The peripheral pain threshold of rats was measured at different time points.The cartilage sections were stained with HE,Aggrecan and type II collagen.The synovial sections were stained with HE,Sirius red,silver and performed with immunostaining.The protein expression of key molecules NGF and TrKA of NGF/TrKA signaling pathway,inflammatory index IL-1β,pain mediator TRPV1,pan-neural mark-ers PGP9.5 and S100 in synovium and complexes transported to dorsal root ganglia(DRG)tissues via nerve endings was determined by Western Blot.The corresponding gene expression was determined by qPCR.The levels of NGF and SP in peripheral blood of rats were determined by ELISA.RESULTS Compared with the KOA group,the cold allodynia and mechanical allodynia thresholds of the rats in the Yiceng group increased(P<0.05,P<0.01);the protein and gene expression of NGF,TrKA,TRPV1,IL-1β,PGP9.5 in the synovial tissue decreased(P<0.05,P<0.01);the protein and gene expression levels of TRPV1,PGP9.5,S100 in the DRG tissue were downregulated(P<0.05,P<0.01).CONCLUSION The warming channels and activating blood circulation external treatment can inhibit the NGF/TrKA signaling pathway,downregulate the gene and protein expressions of NGF,TrKA,TRPV1,IL-1β,PGP9.5,and may inhibit the sprouting of sensory nerve fibers and improve the peripheral hyperalgesia state of rats with KOA.

Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.

Objective:To observe the differences in clinical efficacy of Jieyu Anshen Decoction combined with auricular points and oral tibolone in the treatment of patients with perimenopausal sleep disorders, and provide effective treatment for patients with contraindications to hormone supplement therapy in clinicalMethods:Using a randomized trial design, from July 2018 to August 2020,102 perimenopausal insomnia patients in International Peace Maternity and Child Health Hospital of China Welfare Institutewith kidney deficiency and liver depression who met the inclusion criteria were randomly divided into a treatment group and a control group with 51 cases each. The treatment group took Jieyu Anshen Recipe. At the same time, unilateral auricular point pressing treatment was given, and the opposite ear was changed in 5 d. The control group was treated with tiburon for a period of 3 months. The changes in the scores of each scale were observed in the two groups after 1 month and 3 months treatment. The scale included Pittsburgh sleep quality index (PSQI), modified Kupperman score (KMI), Generalized Anxiety Disorder Scale (GAD-7) and Patient Health Questionnaire Depression Screening Scale (PHQ-9). Its effectiveness and differences were evaluated and analyzed comprehensively through the above scale.Results:PSQI, KMI, GAD-7, PHQ-9 scores decreased significantly in the control and treatment groups after 1 month and 3 months of treatment, and the difference were statistically significant: PSQI: (8.58 ± 1.94) and (5.81 ± 1.93) scores vs. (13.10 ± 2.53), (9.15 ± 2.59) and (6.33 ± 1.98) scores vs.(13.52 ± 2.27) scores; KMI: (19.92 ± 2.16) and (14.67 ± 4.11) scores vs. (28.54 ± 7.65) scores, (19.02 ± 5.92) and(14.10 ± 4.37) scores vs. (27.42 ± 7.34) scores; GAD-7: (4.54 ± 2.03) and (3.81 ± 1.63) scores vs. (5.69 ± 2.95) scores, (3.71 ± 2.48) and (3.32 ± 1.73) scores vs. (4.90 ± 3.17) scores; PHQ-9:(6.90 ± 2.52) and (4.98 ± 1.96) scores vs. (9.83 ± 3.71) scores, (6.15 ± 2.62) and (4.44 ± 1.81) scores vs. (9.02 ± 3.73) scores ( P<0.01). PSQI, KMI, PHQ-9, and GAD-7 scores declined between the two groups, but there were no significant differences between the two groups ( P>0.05). After 1 month and 3 months of treatment, using PSQI scale and KMI score, the total efficiency of patients in the control group was slightly higher than that of the treatment group, but the difference was not statistically significant ( P>0.05); after 1 month and 3 months of treatment, using PHQ-9 score and GAD-7 score, the total efficiency of patients in the treatment group was slightly higher than that of the control group, but the difference was not statistically significant ( P>0.05). Conclusions:Traditional Chinese medicine combined with ear acupoint pressing has similar effects to tibolone in treating perimenopausal insomnia with kidney deficiency and liver depression. It can significantly improve the quality of sleep and quality of life of patients, and has good safety. For patients who are not suitable for hormone, Chinese medicine can be used as an alternative. The therapies are worthy of clinical application.

A double burden of overweight/obesity and malnutrition during childhood is a major concern in China. Dietary intakes in this critical period affect children's physical and cognitive development, and also have health consequences in later life. Therefore, establishing healthy eating habits that will endure is crucial for children. Nutrition education is an effective way in improving nutrition knowledge and attitudes, and healthy eating behaviors. Diverse forms of nutrition improvement programs that targeting children, family, teachers, and school settings have been conducted in many developed countries. However, due to the differences of genetic background, household environment as well as dietary patterns between Chinese children and children from other countries, the existing nutrition education programs for children abroad might not be appropriate for children in China. Thus, nutrition education programs that consider Chinese nutrition-related policies and food supply as well as the local educational resources are required for Chinese children. This review summarized nutrition-related policies and legislations in China and developed countries. A series of evidence-based nutrition education programs that combined educational strategies and environmental supports conducted in the Southwest China Childhood Nutrition and Growth Study were presented. These programs can serve as example models for adopting nutrition interventions to improve nutrition and health status of children in different regions of China.

Objective: To effectively reduce the concentration of poisons in cleanroom, protect the health of workers, realize the optimization and automatic control of the new return air device. And the influence of initial concentration, air volume, temperature and relative humidity of formaldehyde on the purification effect of the new return air device was explored. Methods: The purification effect of the new return air device installed with the activated carbon and the photocatalyst purification net or ordinary activated carbon purification network was tested in a 60 m³ simulated cleanroom. The concentration of formaldehyde was determined by solution absorption-phenol reagent spectrophotometry. Based on the single factor experiment to determine the combination of two purification nets. The effects of air volume, initial formaldehyde concentration, temperature and relative humidity on the purification effect of the new return air device were investigated by orthogonal test. Then, the performance parameters of the return air device to purify formaldehyde were determined. Results: The formaldehyde purification efficiency of the two types of purification nets in the new return air device was higher than that of the ordinary activated carbon purification network (P<0.05) . The combination of activated carbon and photocatalyst purification net has no effect on the formaldehyde purification efficiency of the return air device (P>0.05) . According to the direct analysis and variance analysis, air volume was the most sensitive factor (F value is 18.894, P<0.05) , followed by initial concentration (F value is 16.128, P<0.05) , while temperature and relative humidity have little effect (F value is 0.041 and 0.599, respectively, P>0.05) . LSD analysis showed that there was no significant difference in the purification efficiency of formaldehyde between 475 m³/h and 626 m³/h (P>0.05) . From the perspective of formaldehyde purification efficiency and energy saving, when the air volume is set to 475 m³/h, the new return air device has higher purification efficiency for high concentration of formaldehyde. Conclusion The new return air device consisting of activated carbon and photocatalyst purification net can play a good purification role in cleanroom with different temperatures and different humidity. Its formaldehyde purification efficiency is affected by air volume and initial concentration.