Abstract Respiratory diseases represent prevalent conditions that pose a significant threat to public health. The pathogenesis of these diseases is complex involving a variety of cells and cytokines. Cell culture technology serves as a fundamental tool for investigating pathological mechanisms and drug efficacy. However, traditional monolayer cell culture fail to mimic the intricate cell-cell interactions, limiting its applicability. In contrast, cell co-culture offers a more physiologically relevant approach by closely mimicking the multicellular microenvironment. This advancement provides a robust model for elucidating pathological pathways and evaluating pharmacological interventions in respiratory diseases. In this review, the applications of cell co-culture technologies in common respiratory diseases, including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, lung cancer, asthma, and pneumonia are summarized, in order to propose strategies for the prevention and treatment of these diseases.

Objective:To prepare aldolylated hyaluronic acid-modified antimicrobial carbon dots (AHA-CDs) eye drops and evaluate its antibacterial activity against Staphylococcus aureus in vitro and in vivo. Methods:AHA-CDs eye drops were synthesized by modifying small particle size positively charged carbon dots and introducing aldehyde-based hyaluronic acid.The AHA-CDs were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy.The antibacterial activity of AHA-CDs against Staphylococcus aureus was evaluated using the microdilution method, plate counting, and live-dead bacteria fluorescence staining in vitro.The mouse bacterial keratitis model was established by removing the corneal epithelium after ring drilling and inoculating Staphylococcus aureus.Eighteen conventional female C57BL/6 mice aged 6-8 weeks were selected and divided into a blank control group, an AHA-CDs-treated group and a tobramycin-treated group of 6 mice each using random number table method, and the mice were treated with phosphate buffer saline, 80 μg/ml AHA-CDs, and 80 μg/ml tobramycin eye drops three times daily for 5 consecutive days accordingly to assess antibacterial activity against Staphylococcus aureus in vivo.Another 12 mice were divided into an AHA-CDs group and a normal control group using the random number table method, with 6 mice in each group, which were treated with 80 μg/ml AHA-CDs and phosphate buffer saline for 7 days.The mice were then sacrificed.Their eyeballs were removed and stained with hematoxylin-eosin to observe and compare the morphology and integrity of the eyeballs between the two groups to evaluate the treatment's biosafety.The use and care of the animals complied with the principles of the ARRIVE guidelines.The study protocol was approved by the Ethics Committee of the Experimental Animal Care of Henan Eye Hospital (No.HNEECA-2022-17). Results:The successful preparation of AHA-CDs was demonstrated by transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. In vitro studies showed that the minimum inhibitory concentration of AHA-CDs against Staphylococcus aureus was approximately 8 μg/ml, which effectively inhibited bacterial growth at a lower concentration.Plate counting results showed that only 20% of the bacteria survived after 10 minutes of treatment with AHA-CDs.Fluorescence staining of live/dead bacteria showed obvious red fluorescence signals after 4 hours of treatment with AHA-CDs and SP-CDs. In vivo studies showed that, after 5 days of treatment with AHA-CDs eye drops, the corneas of mice with bacterial keratitis were obviously transparent, and the corneal epithelium was basically repaired.In contrast, the tobramycin-treated group exhibited incomplete epithelial repair and mild corneal edema. In vivo safety evaluation revealed that the eye tissue morphology remained intact and no structural abnormalities were observed after AHA-CDs treatment. Conclusions:AHA-CDs eye drops have superior antibacterial effects in vivo and in vitro, and inactivate bacteria rapidly and effectively.

Objective To compare the early clinical efficacy of arthroscopic bridge suture combined with strengthening thread fixation for posterior cruciate ligament(PCL)tibial insertion avulsion frac-tures and traditional open double-row anchor suture fixation,and to explore a safe,effective,and min-imally invasive reduction and fixation method for PCL tibial insertion fractures in knee joint under ar-throscopy.Methods This study was a retrospective study.Sixty patients with PCL tibial insertion frac-tures(Meyers&McKeever types II-IV)and admitted to the Department of Orthopedics of Kunming Hospital of Traditional Chinese Medicine between June 2021 and June 2023 were included,and ran-domly divided into a traditional group(age:39.56±3.24 years,male/female:16/14)and a minimally in-vasive group(age:40.32±4.38,male/female:18/12),each of 30,according to their admission or-der.All patients were of Grade III positive in the drawer test before surgery.The traditional group un-derwent posterior medial inlay incision,reduction under direct vision and double-row anchor suture fix-ation,while the minimally invasive group underwent reduction under total arthroscopy,suture-bridge suture at the tendon-bone junction of PCL,and one anchor suture placed at the femoral insertion of PCL,with the anchor suture line serving as the reinforcing suture.Then,the anchor and suture-bridge suture lines were respectively pulled into the two tunnels behind the tibia from the front and then pulled out from the front of the tibia,and the reinforcing suture was knotted in front of the tib-ia.The bridge suture line was fixed in front of the tibia with an external row anchor.Six months after surgery,both groups were evaluated the efficacy using the posterior drawer test and Lysholm score.Re-sults At the follow-up six months after the surgery,both groups had good fracture healing,with one wound infection and delayed healing in the traditional group and total one-stage heal in the other group.According to the physical examination 6 months after surgery,all were negative in the posterior drawer test,except one of degree II positive in the traditional group.Moreover,the average Lysholm score of the traditional group increased from 65.23±3.48 before surgery to 87.64±4.58 points after surgery(P<0.05),while that of the minimally invasive group increased from 64.35±2.52 to 86.82±2.58(P<0.05),showing no significant differences between the two groups(P>0.05).Conclusion Both techniques in this study can achieve excellent surgical outcomes in the treatment of posterior cruciate ligament avulsion fractures at the tibial insertion site.

Objective: To explore the association between vitamin D levels and sleep in children and adolescents，so as to provide a reference for promoting the sleep health of children and adolescents. Methods: From October to December, 2021, 4 827 primary and middle school students aged 6-17 in Shenzhen were selected by multistage cluster random sampling method, and their demographic information, family background, lifestyle and sleep status were obtained by facetoface questionnaire survey, and their fasting venous blood in the morning was collected to detect the serum 25(OH)D level. The relationship between serum vitamin D level and sleep characteristics was analyzed by binary Logistic regression, and stratified analysis was carried out according to gender. Results: The proportion of vitamin D deficiency was 41.1%, and the proportion of sleep deficiency was 19.4%. With the increase of vitamin D level, daily sleep duration of children and adolescents tended to increase (r=0.10,P<0.01). After adjusting for covariates such as gender and age, it was found that children and adolescents with insufficient vitamin D levels were more likely to experience sleep insufficiency, social jetlag, and late sleep on weekdays, with ORs being 1.32(95%CI=1.12-1.56), 1.35(95%CI=1.19-1.54), and 1.26(95%CI=1.05-1.52)(P<0.05). Sexstratified analysis showed that, among boys, vitamin D deficiency was associated with sleep deficiency, social jetlag, and late bedtime on weekdays and weekends[OR(95%CI)=1.42(1.14-1.77),1.25(1.04-1.49),1.39(1.06-1.82),1.86(1.19-2.92),P<0.05]. In girls, however, serum vitamin D levels were only associated with social jetlag with OR being 1.47 (95%CI=1.21-1.79, P<0.05). Conclusion Vitamin D levels are associated with various sleep characteristics in children and adolescents, with this association being more pronounced among boys.

ObjectiveBased on modern analytical techniques and a depressed mouse model established by chronic unpredictable mild stress（CUMS）， to evaluate the quality marker（Q-Marker） and pharmacodynamic difference of Baihe Dihuangtang prepared by different processes. MethodsHigh performance liquid chromatography（HPLC） was used to establish the characteristic profiles of Baihe Dihuangtang， and determine the content of Q-Marker in the samples prepared by ancient and modern processes. Seventy C57BL/6J mice were randomly divided into the normal group， model group， fluoxetine group（3 mg·kg^-1）， low and high dose groups of ancient process（6.5， 26 g·kg^-1）， and low and high dose groups of modern process（6.5， 26 g·kg^-1）， with 10 mice in each group. Except for the normal group， CUMS was used to induce depression in mice from the other groups for 28 d. After successful modeling， administration groups were given the corresponding drugs by gavage every day， and the normal and model groups were given an equal volume of pure water by gavage for 21 consecutive days. Change in body mass of mice was recorded， tail suspension test and open field test were used to evaluate the depressive behavior of mice， and enzyme-linked immunosorbent assay（ELISA） was employed to determine the contents of tumor necrosis factor-α（TNF-α）， interleukin（IL）-1β and IL-6 in serum. ResultsCharacteristic profiles of Baihe Dihuangtang prepared by the two processes were established， the similarity between the two was 0.951， and 8 characteristic peaks were recognized with the reference peak of regaloside A. The results of quantitative analysis showed that the Q-Marker content was similar in Baihe Dihuangtang prepared by ancient and modern processes. The results of pharmacodynamics showed that， compared with the normal group， the model group showed increased immobility time in the tail suspension test， reduced total movement distance in the open field test， and elevated IL-1β， IL-6 and TNF-α levels in the serum（P<0.01）. Compared with the model group， the behavioral indicators of mice in the Baihe Dihuangtang treatment group were significantly improved in terms of tail suspension time and open field exercise， and the levels of IL-1β， IL-6 and TNF-α in serum were significantly reduced（P<0.05， P<0.01）. Baihe Dihuangtang prepared by the two processes both had antidepressant effects， and the difference between the two was not statistically significant in improving depressive symptoms. ConclusionQ-Marker of Baihe Dihuangtang prepared by modern and ancient methods are equivalent in content， and the pharmacological effects are consistent， indicating that dried Lilii Bulbus can replace fresh products in the preparation of Baihe Dihuangtang. This study provides a scientific basis for the development of new drugs of Baihe Dihuangtang and a reference for its rational application and clinical use.

ObjectiveBased on modern analytical techniques and a depressed mouse model established by chronic unpredictable mild stress（CUMS）， to evaluate the quality marker（Q-Marker） and pharmacodynamic difference of Baihe Dihuangtang prepared by different processes. MethodsHigh performance liquid chromatography（HPLC） was used to establish the characteristic profiles of Baihe Dihuangtang， and determine the content of Q-Marker in the samples prepared by ancient and modern processes. Seventy C57BL/6J mice were randomly divided into the normal group， model group， fluoxetine group（3 mg·kg^-1）， low and high dose groups of ancient process（6.5， 26 g·kg^-1）， and low and high dose groups of modern process（6.5， 26 g·kg^-1）， with 10 mice in each group. Except for the normal group， CUMS was used to induce depression in mice from the other groups for 28 d. After successful modeling， administration groups were given the corresponding drugs by gavage every day， and the normal and model groups were given an equal volume of pure water by gavage for 21 consecutive days. Change in body mass of mice was recorded， tail suspension test and open field test were used to evaluate the depressive behavior of mice， and enzyme-linked immunosorbent assay（ELISA） was employed to determine the contents of tumor necrosis factor-α（TNF-α）， interleukin（IL）-1β and IL-6 in serum. ResultsCharacteristic profiles of Baihe Dihuangtang prepared by the two processes were established， the similarity between the two was 0.951， and 8 characteristic peaks were recognized with the reference peak of regaloside A. The results of quantitative analysis showed that the Q-Marker content was similar in Baihe Dihuangtang prepared by ancient and modern processes. The results of pharmacodynamics showed that， compared with the normal group， the model group showed increased immobility time in the tail suspension test， reduced total movement distance in the open field test， and elevated IL-1β， IL-6 and TNF-α levels in the serum（P<0.01）. Compared with the model group， the behavioral indicators of mice in the Baihe Dihuangtang treatment group were significantly improved in terms of tail suspension time and open field exercise， and the levels of IL-1β， IL-6 and TNF-α in serum were significantly reduced（P<0.05， P<0.01）. Baihe Dihuangtang prepared by the two processes both had antidepressant effects， and the difference between the two was not statistically significant in improving depressive symptoms. ConclusionQ-Marker of Baihe Dihuangtang prepared by modern and ancient methods are equivalent in content， and the pharmacological effects are consistent， indicating that dried Lilii Bulbus can replace fresh products in the preparation of Baihe Dihuangtang. This study provides a scientific basis for the development of new drugs of Baihe Dihuangtang and a reference for its rational application and clinical use.

Objective:To prepare aldolylated hyaluronic acid-modified antimicrobial carbon dots (AHA-CDs) eye drops and evaluate its antibacterial activity against Staphylococcus aureus in vitro and in vivo. Methods:AHA-CDs eye drops were synthesized by modifying small particle size positively charged carbon dots and introducing aldehyde-based hyaluronic acid.The AHA-CDs were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy.The antibacterial activity of AHA-CDs against Staphylococcus aureus was evaluated using the microdilution method, plate counting, and live-dead bacteria fluorescence staining in vitro.The mouse bacterial keratitis model was established by removing the corneal epithelium after ring drilling and inoculating Staphylococcus aureus.Eighteen conventional female C57BL/6 mice aged 6-8 weeks were selected and divided into a blank control group, an AHA-CDs-treated group and a tobramycin-treated group of 6 mice each using random number table method, and the mice were treated with phosphate buffer saline, 80 μg/ml AHA-CDs, and 80 μg/ml tobramycin eye drops three times daily for 5 consecutive days accordingly to assess antibacterial activity against Staphylococcus aureus in vivo.Another 12 mice were divided into an AHA-CDs group and a normal control group using the random number table method, with 6 mice in each group, which were treated with 80 μg/ml AHA-CDs and phosphate buffer saline for 7 days.The mice were then sacrificed.Their eyeballs were removed and stained with hematoxylin-eosin to observe and compare the morphology and integrity of the eyeballs between the two groups to evaluate the treatment's biosafety.The use and care of the animals complied with the principles of the ARRIVE guidelines.The study protocol was approved by the Ethics Committee of the Experimental Animal Care of Henan Eye Hospital (No.HNEECA-2022-17). Results:The successful preparation of AHA-CDs was demonstrated by transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. In vitro studies showed that the minimum inhibitory concentration of AHA-CDs against Staphylococcus aureus was approximately 8 μg/ml, which effectively inhibited bacterial growth at a lower concentration.Plate counting results showed that only 20% of the bacteria survived after 10 minutes of treatment with AHA-CDs.Fluorescence staining of live/dead bacteria showed obvious red fluorescence signals after 4 hours of treatment with AHA-CDs and SP-CDs. In vivo studies showed that, after 5 days of treatment with AHA-CDs eye drops, the corneas of mice with bacterial keratitis were obviously transparent, and the corneal epithelium was basically repaired.In contrast, the tobramycin-treated group exhibited incomplete epithelial repair and mild corneal edema. In vivo safety evaluation revealed that the eye tissue morphology remained intact and no structural abnormalities were observed after AHA-CDs treatment. Conclusions:AHA-CDs eye drops have superior antibacterial effects in vivo and in vitro, and inactivate bacteria rapidly and effectively.

Objective To compare the early clinical efficacy of arthroscopic bridge suture combined with strengthening thread fixation for posterior cruciate ligament(PCL)tibial insertion avulsion frac-tures and traditional open double-row anchor suture fixation,and to explore a safe,effective,and min-imally invasive reduction and fixation method for PCL tibial insertion fractures in knee joint under ar-throscopy.Methods This study was a retrospective study.Sixty patients with PCL tibial insertion frac-tures(Meyers&McKeever types II-IV)and admitted to the Department of Orthopedics of Kunming Hospital of Traditional Chinese Medicine between June 2021 and June 2023 were included,and ran-domly divided into a traditional group(age:39.56±3.24 years,male/female:16/14)and a minimally in-vasive group(age:40.32±4.38,male/female:18/12),each of 30,according to their admission or-der.All patients were of Grade III positive in the drawer test before surgery.The traditional group un-derwent posterior medial inlay incision,reduction under direct vision and double-row anchor suture fix-ation,while the minimally invasive group underwent reduction under total arthroscopy,suture-bridge suture at the tendon-bone junction of PCL,and one anchor suture placed at the femoral insertion of PCL,with the anchor suture line serving as the reinforcing suture.Then,the anchor and suture-bridge suture lines were respectively pulled into the two tunnels behind the tibia from the front and then pulled out from the front of the tibia,and the reinforcing suture was knotted in front of the tib-ia.The bridge suture line was fixed in front of the tibia with an external row anchor.Six months after surgery,both groups were evaluated the efficacy using the posterior drawer test and Lysholm score.Re-sults At the follow-up six months after the surgery,both groups had good fracture healing,with one wound infection and delayed healing in the traditional group and total one-stage heal in the other group.According to the physical examination 6 months after surgery,all were negative in the posterior drawer test,except one of degree II positive in the traditional group.Moreover,the average Lysholm score of the traditional group increased from 65.23±3.48 before surgery to 87.64±4.58 points after surgery(P<0.05),while that of the minimally invasive group increased from 64.35±2.52 to 86.82±2.58(P<0.05),showing no significant differences between the two groups(P>0.05).Conclusion Both techniques in this study can achieve excellent surgical outcomes in the treatment of posterior cruciate ligament avulsion fractures at the tibial insertion site.

ObjectiveTo construct traditional Chinese medicine （TCM） diagnostic scale of turbid toxin syndrome in order to provide corresponding reference for the standardization of TCM syndromes and studies. MethodsWe systematically searched the Chinese Medical Dictionary （CMD）， China Knowledge Network （CNKI）， Wanfang Data Knowledge Service Platform （WF） and VIP database for TCM classics and modern literature on turbid toxin syndrome， and initially screened the four diagnosis information of turbid toxin syndrome， established a pool of information entries， and conducted a cross-sectional clinical survey. Discrete trend method， correlation coefficient method， Cronbach's coefficient method， and factor analysis method were applied to objectively screen the entries. The diagnostic scale of turbid toxin syndrome were constructed through three rounds of Delphi method expert survey to determine the scale entries， using hierarchical analysis to get the judgement matrix scores and relative weight of each entry， after passing consistency test and then isometric expansion of the relative weight of the entries to get the weight of each entry and assign the value. ResultsA total of 35 articles were included， 45 entries were obtained after the initial screening. After the clinical investigation， 12 entries were not suitable by the discrete trend method， 23 entries not suitable by correlation coefficient method， 13 entries by the internal consistency screening were removed with the Cronbach's alpha coefficient rising， and 10 entries not suitable by the factor analysis method. Twenty-two entries were retained after objective screening by the combined use of the four statistical methods. The positive coefficients of experts in the three rounds of Delphi method of expert consultation were 96.67%， the coefficients of expert authority were 0.834， 0.856， and 0.867， and the coefficients of co-ordination were 0.126， 0.326， and 0.312， respectively. After consulting with clinical experts， and three rounds of Delphi method survey and hierarchical analysis method weight assignment， the diagnostic scale entries of turbid toxin syndrome were finally established. Primary symptoms： dark red or purple and dusky tongue， yellowish greasy or dry coating （10 points）； sticky and unpleasant stools （8 points）； disharmony of tastes including halitosis， sticky and greasy taste in the mouth， dry mouth and bitter taste in the mouth （6 points）； unfavourable or yellowish or red urination （5 points）； and dark complexion （4 points）. Secondary symptoms： heavy body （3 points）； dizziness （3 points）； profuse， sticky， foul-smelling secretions （2 points）； wiry and slippery， or slippery， or slippery and rapid pulse （2 points）； feeling of hardness in the abdomen （1 point）. ConclusionUsing Delphi method combined with the hierarchical analysis method， combining qualitative and quantitative study， a diagnostic scale of turbid toxin syndrome was initially developed.

Objective:To observe the effect of lipopolysaccharide (LPS) induced conditioned medium of alveolar epithelial cells on the inflammatory response and cell damage of vascular endothelial cells, and explore its mechanism.Methods:The LPS induced type Ⅱ alveolar epithelial cells (A549) conditioned medium was used as a stimulus to induce human umbilical vein endothelial cells (HUVEC) damage. The cell counting kit-8 (CCK-8) was used to detect the effect of 0% (blank group), 12.5%, 25%, 50%, 75% and 100% A549 cell conditioned medium cultured for 6, 12, 24 and 48 hours on the cell viability of HUVEC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] and vasoactive substances [vascular endothelial growth factor (VEGF), endothelin-1 (ET-1)] in the supernatant. Phalloidin staining was used to observe the effects of A549 cells conditioned medium on cell morphology. The expressions of protein kinase B/nuclear factor-κB (AKT/NF-κB) pathway in HUVEC induced by conditioned medium was detected by Western blotting.Results:Compared with the blank group, A549 cells conditioned medium with concentrations of 12.5%, 25%, and 50% had no significant effects on cell viability of HUVEC after 6, 12, and 24 hours, but the activity of HUVEC decreased significantly after 48 hours. Therefore, 12.5%, 25%, 50% A549 cell conditioned medium stimulated for 24 hours was selected as the induction condition for follow-up experiments. Compared with the blank group, the level of IL-6 was significantly increased in 12.5% and 50% conditioned medium groups (ng/L: 2?438.95±64.89, 3?036.41±96.69 vs. 1?736.75±20.99, both P < 0.05), the level of TNF-α was significantly increased in 12.5% and 25% conditioned medium groups (ng/L: 174.08±11.09, 81.37±8.17 vs. 50.03±0.26, both P < 0.01), the levels of VEGF and ET-1 were significantly increased in 12.5%, 25% and 50% conditioned medium groups [VEGF (ng/L): 173.60±41.44, 192.49±12.38, 318.89±27.90 vs. 66.68±19.65; ET-1 (ng/L): 54.88±1.37, 36.69±0.29, 24.07±0.73 vs. 10.67±0.25, all P < 0.01]. Phalloidin staining showed that HUVEC induced by 25% A549 cells conditioned medium were irregular in shape, uneven in size, disordered in arrangement, widened in gap, dense and unclear in microfilament structure and serrated in cell membrane. Furthermore, the average fluorescence intensity of 25% conditioned medium group significantly increased compared to the blank group (67?205.60±3?430.40 vs. 56?272.67±7?650.95, P < 0.05). Western blotting showed that compared with the blank group, the expression of HUVEC cells phosphonated inhibitor α of NF-κB (p-IκBα) was significantly decreased in the 12.5%, 25%, and 50% conditioned medium groups (p-IκBα/IκBα: 0.38±0.08, 0.67±0.12, 0.31±0.07 vs. 1.00±0.00, all P < 0.01), the expressions of phosphonated-AKT (p-AKT) and VEGF were significantly increased (p-AKT/AKT: 1.50±0.18, 1.42±0.27, 1.61±0.14 vs. 1.00±0.00, VEGF/GAPDH: 1.37±0.10, 1.53±0.22, 1.40±0.12 vs. 1.00±0.00, all P < 0.05), the expression of phosphonated NF-κB p65 (p-P65) was significantly increased in the 25% conditioned medium group (p-P65/P65: 1.45±0.14 vs. 1.00±0.00, P < 0.05). Conclusion:LPS induced conditional culture medium of alveolar epithelial cells induced endothelial cell damage via activating AKT/NF-κB pathway.