ObjectiveTo investigate the mechanism of Xiaozhi Qinggan decoction （XQD） in preventing and treating metabolic dysfunction-associated steatotic liver disease （MASLD） by regulating ferroptosis， network pharmacology， in vitro and in vivo experiments. MethodsIn the in vivo experiment， mouse MASLD models were established by high-fat diet （HFD） induction. The model mice were randomly assigned to a positive control group （silybin， 50 mg·kg^-1）， low-， medium- and high-dose XQD groups （4.725， 9.45， 18.9 g·kg^-1）， with a normal control group. After 4 weeks of modeling， mice except the normal group were administered intragastrically for 8 consecutive weeks. Liver function， serum lipid levels， hepatic histopathology， as well as the levels of malondialdehyde （MDA）， superoxide dismutase （SOD）， reduced glutathione （GSH） and oxidized glutathione （GSSG） and Fe²⁺ were detected. The mRNA and protein expression of p53， SLC7A11 and GPX4 were determined by quantitative Real-time quantitative polymerase chain reaction（Real-time PCR） and Western blot. In the network pharmacology analysis， active components and potential targets of XQD for MASLD were screened， followed by functional and pathway enrichment analyses， and molecular docking was performed to verify the target binding activity. In the in vitro experiment， the optimal concentration of XQD-containing serum was screened by cytotoxicity assay. HepG2 cells were transfected with ov-NC or ov-p53 plasmid， and a lipid accumulation model was induced by free fatty acid （FFA， 1.0 mmol·L^-1）. Cells were divided into a normal group， FFA model group， ov-NC+XQD （15%） group and ov-p53+XQD （15%） group. Intracellular Fe²⁺ level and lipid accumulation were evaluated， and the protein expression of p53， SLC7A11 and GPX4 was measured by Western blot. ResultsCompared with the normal group， the model group exhibited markedly elevated body weight， liver weight， liver index， fasting blood glucose， AUC of glucose tolerance test， serum liver function and blood lipid levels at week 12 （P<0.01）. Hepatic steatosis and inflammatory infiltration were observed by pathological staining. Additionally， hepatic levels of MDA， SOD and Fe²⁺ were increased （P<0.01）， while GSH， GSSG and the GSH/GSSG ratio were decreased （P<0.01）. The mRNA and protein expression of hepatic p53 was upregulated （P<0.01）， whereas the expression of SLC7A11 and GPX4 was downregulated （P<0.01）. Compared with the model group， the low- and medium-dose XQD groups showed significantly decreased body weight at week 12 （P<0.05）. The silybin group， together with the medium- and high-dose XQD groups， presented reduced liver weight and liver index （P<0.05）. Fasting blood glucose and the AUC of glucose tolerance test were lowered in all four treatment groups （P<0.05， P<0.01）. Pathological staining revealed alleviated hepatic steatosis and inflammation， accompanied by decreased serum liver function and blood lipid levels （P<0.05， P<0.01）. Moreover， hepatic MDA and SOD levels were markedly reduced， while GSH， GSSG and the GSH/GSSG ratio were significantly elevated （P<0.05， P<0.01）. Hepatic Fe²⁺ level was decreased （P<0.01）. The mRNA and protein expression of hepatic p53 was downregulated， and the expression of SLC7A11 and GPX4 was upregulated （P<0.05， P<0.01）. Network pharmacology analysis identified quercetin， kaempferol， luteolin， tanshinone IIA and isorhamnetin as the core active components of XQD， with p53 serving as the key target. Stable binding was verified between these active components and the p53 protein. The optimal concentration of XQD-containing serum in vitro was determined to be 15%. Compared with the normal group， the model group showed increased intracellular Fe²⁺ and lipid accumulation， significantly upregulated p53 protein expression （P<0.01）， and markedly downregulated SLC7A11 and GPX4 protein expression （P<0.01）. Compared with the model group， the ov-NC group exhibited reduced Fe²⁺ and lipid accumulation， downregulated p53 expression， and upregulated SLC7A11 and GPX4 expression. In the ov-p53 group， p53 expression was upregulated （P<0.01）， while SLC7A11 and GPX4 expression was downregulated （P<0.01）. ConclusionXQD inhibits ferroptosis by downregulating p53 and upregulating SLC7A11 and GPX4， thereby alleviating oxidative stress and lipid peroxidation in hepatocytes and improving MASLD.

ObjectiveTo investigate the effect of Toxoplasma gondii infection on copper metabolism in the kidneys of mice. MethodsA total of 80 7-8-week-old C57BL/6 female mice were randomly divided into four groups of 20 mice in each group after one week of adaptation， including Control group， Cu group， TgCtwh6 group and Cu+TgCtwh6 group. Mice that were not infected and fed with normal diet and water were used as the Control group； Mice fed with 1 g/kg of copper chloride processing diet and 0.1% copper chloride water for 60 consecutive days were used as Cu group； Mice infected with 25-30 TgCtwh6 cysts （one of the predominant genotype Chinese 1 in China） fed with normal diet and water were used as the TgCtwh6 group； mice infected with 25-30 TgCtwh6 cysts and fed with a processed diet containing 1 g/kg of copper chloride and water with 0.1% copper chloride for 60 consecutive days were used as the Cu+TgCtwh6 group. ICP-MS was used to determine the changes in copper content in kidney tissues. Hematoxylin-eosin （HE） staining was used to observe the pathological changes of mouse kidney tissue. The number of apoptotic cells was observed by PI staining. Western blot was used to detect the protein expression levels of glutathione peroxidase 4 （GPX4） and superoxide dismutase （SOD1， SOD2）. RT-qPCR was used to detect the mRNA expression of cuproptosis-related genes. ResultsPathological manifestations such as inflammatory cell infiltration in the Cu group and TgCtwh6 group were seen under the microscope， and the inflammatory infiltrating cells of the renal interstitial were reduced in the Cu+TgCtwh6 group， and the pathological manifestations

ObjectiveTo investigate and manage an imported dengue fever (DF) outbreak in Shanghai in 2024, to summarize the experience and lessons learned from the on-site management, and to provide a reference basis for future prevention and control of DF. MethodsEpidemiological investigation and case search were carried out for an imported DF outbreak in Shanghai, 2024. Real-time fluorescence polymerase chain reaction (RT-PCR) was used to detect dengue virus nucleic acid in the serum samples from cases. Meanwhile, emergency vector surveillance and mosquito control measures were carried out in the affected areas, and the effectiveness of the management was evaluated. ResultsAccording to the epidemiological investigation, it was confirmed that this epidemic was a family cluster of imported DF, with both cases infected in Thailand and developed symptoms successively after returning to Shanghai. Laboratory testing identified the pathogens as dengue virus serotype-3 (DENV-3). In the core and precautionary area, ultra-low-volume space spraying and residual spraying were combined to kill adult mosquitoes, and at the same time, comprehensive cleaning and elimination of mosquito breeding sites was carried out. After 2 weeks, the Breteau Index (BI) in the core area decreased from 20 to 5, and the mosquito net trap index decreased from 2 mosquitoes (net·hour)^-1 to 0.67 mosquitoes (net·hour)^-1. Continuous implementation of mosquito control measures kept the BI and net trap index below the safety thresholds [BI<5 and mosquito net trap index <2 mosquitoes (net·hour)^-1] both in the core and precautionary area. ConclusionEarly diagnosis and isolation of patients, combined with rapid suppression of the density of vector Aedes mosquitoes, are the key measures to prevent the transmission of imported DF cases.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

This paper explores the differentiation and treatment strategies for pediatric IgA vasculitis based on the correlation between blood trubidity theory and oxidative stress. It is proposed that pediatric IgA vasculitis follows an evolution of pathogenesis characterized by "deficiency of healthy qi leading to turbidity generation， accumulation of turbid toxin， and toxin damage to the collateral vessels"， which corresponds to the pathological process of oxidative stress， namely decreased antioxidant capacity with accumulation of reactive oxygen species， metabolite deposition， and endothelial cell injury. A staged treatment strategy is proposed. In the acute stage， wind-toxin invading the colla-terals and reckless movement of heat in the blood are the main manifestations， for which the treatment should focus on dispelling wind and venting pathogens， resolving toxins and unblocking the collaterals， with a modified Yinqiao Powder （银翘散） and Xijiao Dihuang Decoction （犀角地黄汤） to regulate oxidative stress burst. In the prolonged stage， intermingling of turbidity and stasis with collateral obstruction is emphasized； treatment should focus on resolving turbidity and dispelling stasis， harmonizing the blood and stabilizing the collaterals， and a modified Simiao Powder （四妙散） and Taohong Siwu Decoction （桃红四物汤） can be used to improve microcirculatory dysfunction. In the remission stage， when healthy qi remains insufficient and residual pathogens persist， treatment should focus on strengthening the root and clearing the source， reinforcing healthy qi and nourishing the collaterals， for which modified Zhibai Dihuang Pill （知柏地黄丸） and Yupingfeng Powder （玉屏风散） is suggested to rebuild the antioxidant defense system.

ObjectiveTo investigate the possible mechanism of Pibai Yucuo Formula （枇柏愈痤方， PYF） in treating acne from the perspective of skin barrier damage. MethodsThirty-two mice were randomly divided into blank group， model group， minocycline group， and PYF group， with 8 mice in each group. Except for the blank group， mice were induced by intradermal injection of Cutibacterium acnes （C.acnes） combined with topical application of artificial sebum to establish acne model. The blank group and model group received intragastric administration of 0.2 ml of distilled water， while the PYF group received intragastric administration of 22.75 g/（kg·d）of PYF， and the minocycline group received 0.013 g/（kg·d）of minocycline suspension， all once daily for 5 consecutive days. On day 0 and day 6 of the experiment， the body weight of mice in each group was recorded， and the absolute value of the body weight difference during the experiment was calculated. Skin conditions were assessed with multifunctional skin imaging system on the 2nd， 4th and 6th day of the experiment. Skin barrier function indicators including transepidermal water loss （TEWL）， and the water content of the stratum corneum and epidermis on day 0， 2， 4 and 6 of the experiment. Optical coherence tomography （OCT） was used to observe stratum corneum and skin thickness on the 1st， 3rd and 5th day of the experiment. Hematoxylin-eosin （HE） staining was performed to observe histopathological changes， while ELISA was used to detect interleukin-17A （IL-17A） levels， and immunofluorescence staining was used to assess skin barrier-related proteins filaggrin （FLG） and loricrin （LOR） levels of skin lesions on day 6 of the experiment. ResultsCompared to the blank group， the model group showed a decrease in body weight on day 6， and an increase in the absolute value of the difference in body weight before and after the experiment （P＜0.05）. On day 4 and 6， TEWL values increased， while water content in the skin stratum corneum and epidermis decreased （P＜0.05）， accompanied by elevated IL-17A level and reduced immunofluorescence intensity of FLG and LOR proteins （P＜0.05）. The model group mice showed papules or pustules at the skin modeling site with progressively worsening desquamation under multifunctional skin imaging system. OCT revealed focal epidermal protrusions， blurred epidermal-dermal boundaries， and disorganized structural layers. HE staining showed significant epidermal hyperkeratosis and incomplete keratinization in the skin， with keratin plug formation in hair follicles and glandular lumens， thickened stratum corneum， hyperplasia of the stratum spinosum， as well as dense dermal inflammatory cell infiltration， and capillary dilation. Compared to the model group， both the minocycline group and the PYF group showed a reduced difference in body weight before and after experiment （P＜0.05）. On day 4 and 6， the TEWL value decreased， and water content of the skin stratum corneum increased （P＜0.05）； on day 6， the IL-17A level in the skin lesions decreased and immunofluorescence intensity of FLG and LOR proteins increased （P＜0.05）. On day 4 and 6， the severity of the skin lesions and range of redness and swelling were lighter than those in the model group， with reverted epidermal thickness， smoother surface and clearer epidermis-dermis boundary. HE staining showed that the degree of skin keratinization was reduced， and the inflammatory infiltration and vascular dilation in the dermis were improved compared to the model group. The PYF group showed better results than the minocycline group in reducing TEWL value on day 4 （P＜0.05）. ConclusionPYF may improve inflammation and skin barrier damage by downregulating IL-17A levels in lesion tissue and increasing skin barrier-related proteins， which could be one of the potential mechanism of action on acne.

ObjectiveTo analyze the epidemiological characteristics of 21 respiratory pathogens in influenza-like illness (ILI) cases in Jing’an District, Shanghai in 2024, and to provide a scientific basis for the prevention and control of respiratory infectious diseases. MethodsData of1 907 ILI cases at four sentinel hospitals in Jing’an District were collected from January to December 2024. Nasopharyngeal swab samples were collected and tested for 21 respiratory pathogens using polymerase chain reaction (PCR) methods. Chi-square test and Cochran-Armitage trend test were used for data analyses. ResultsAmong the 1 907 ILI cases, 1 340 were tested positive (70.27%), including 1 160 (60.83%) virus-positive cases, 424 (22.23%) bacteria-positive cases , and 86 (4.51%) positive cases of other pathogens (fungi, mycoplasma, and chlamydia). The top five viruses by detection rate were: influenza virus (14.84%), SARS-CoV-2 (14.47%), rhinovirus (12.69%), adenovirus (7.08%), and parainfluenza virus (6.71%). The top two bacteria by detection rate were Streptococcus pneumoniae (14.47%) and Haemophilus influenzae (10.33%). Among other pathogens (fungi, mycoplasma, and chlamydia), Mycoplasma pneumoniae showed the highest detection rate (4.30%). In terms of age distribution, statistically significant differences were observed in the detection rates of SARS-CoV-2, Legionella, and Klebsiella pneumoniae (P<0.05), with the highest rates found in individuals aged 65 years and above. Statistically significant differences were also found in the detection rates of rhinovirus, adenovirus, enterovirus, common coronavirus, respiratory syncytial virus, bocavirus, parainfluenza virus, human metapenu-movirus, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae among different age groups (P<0.05), all showing the highest detection rates in the 0‒<15 years age group. In terms of seasonal distribution, SARS-CoV-2, adenovirus, parainfluenza virus, enterovirus, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae showed epidemic peaks in summer; rhinovirus, common coronavirus, bocavirus, and Klebsiella pneumoniae had higher detection rates in autumn. Influenza virus exhibited a peak incidence during winter, while human metapenu-movirus peaked in winter and spring. Significant differences in co-infection detection rates were observed among age groups, with the rate in children aged 0‒<15 years (34.81%) being the highest. The co-infection detection rate was higher in males than in females (P=0.019). Both the single-pathogen detection rate and the co-infection detection rate (P<0.001) varied significantly across seasons: the single-pathogen detection rate was highest in winter (62.06%), while the co-infection detection rate peaked in summer (31.20%) and was lowest in winter (14.52%). ConclusionBased on detection rates, the main pathogens in the ILI population of Jing’an District, Shanghai, 2024 were influenza virus, SARS-CoV-2, rhinovirus, adenovirus, parainfluenza virus, common coronavirus, enterovirus, Human metapenu-movirus, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae. Pathogen detection rates varied by age and season. Coinfection rates were much higher in children than in adults, higher in males than in females, and peaked in summer while being lowest in winter.

Background Regulatory interactions between microRNAs (miRNAs) and messenger RNAs (mRNAs) are involved in the progression of pulmonary fibrosis, which can either promote or inhibit the development of this disease. Objective To explore the miRNA-mRNA regulatory network during the progression of silica (SiO₂)-induced pulmonary fibrosis in mice using integrated mRNA-seq and miRNA-seq analysis. Methods A mouse model of pulmonary fibrosis was established by dynamic SiO₂ dust exposure. The experimental design included a blank control group and four SiO₂-exposed groups (7, 14, 28, and 56 d, n=10 per group). Successful model induction was confirmed by histopathological analysis (HE and Masson staining), hydroxyproline (HYP) quantification, and expression of key fibrosis-related cytokines [fibroblast growth factor (FGF), interleukin-6 (IL-6), transforming growth factor-β (TGF-β), and tumor necrosis factor-α (TNF-α)]. Lung tissues from mice in each group were subjected to sequencing, and Mfuzz was used for time-series gene clustering to identify dynamic progression patterns. DESeq2 was utilized to identify differentially expressed genes (DEGs) and differentially expressed miRNAs. Enrichment analysis of DEGs was performed to identify critical signaling pathways and biological processes underlying pulmonary fibrosis progression. Expression of four selected miRNAs was subsequently validated by real-time quantitative polymerase chain reaction (RT-qPCR). The target mRNAs of key miRNAs were comprehensively predicted by integrating miRBase, starBase, and miRTarBase to construct the regulatory networks and investigate potential functions. Results SiO₂ exposure led to time-dependent aggravation of pulmonary fibrosis in mice, evidenced by increased fibrous deposition, elevated HYP levels (P < 0.01), and up-regulation of four kinds of pro-fibrotic cytokines (P < 0.01) compared with the NT group. Mfuzz clustering revealed the stage-specific characteristics. Compared to controls, 231, 662, 448, and 1020 DEGs were identified after SiO₂ exposure at 7, 14, 28, and 56 d, respectively, primarily enriched in immune responses and chemokine signaling. During critical fibrotic phases—7 d (acute inflammation and initiation) and 28 d (chronic inflammation and establishment)—18 differentially expressed miRNAs were identified; notably mmu-miR-135b-5p was significantly dysregulated at both time points. The expression trends of the four key miRNAs (mmu-miR-135b-5p, mmu-miR-708-5p, mmu-miR-21a-3p, and mmu-miR-205-5p) were consistent with the sequencing results. Furthermore, bioinformatics databases were used to predict the target mRNAs of key miRNAs. The constructed network highlighted critical miRNA-mRNA pairs—including mmu-miR-135b-5p and Meis1, mmu-miR-708-5p and Mmp25, mmu-miR-21a-3p and Cacna1d, mmu-miR-205-5p and Ereg which were closely associated with inflammatory response, extracellular matrix deposition, and fibroblast activation. Conclusion The progression of pulmonary fibrosis is accompanied by dynamic changes in miRNA-mRNA regulatory networks. The identified miRNA-target axes (e.g., miR-135b-5p and Meis1, mmu-miR-708-5p and Mmp25, mmu-miR-21a-3p and Cacna1d, and mmu-miR-205-5p and Ereg—) may play important roles in fibrogenesis and provide potential therapeutic targets for pulmonary fibrosis.

Background Regulatory interactions between microRNAs (miRNAs) and messenger RNAs (mRNAs) are involved in the progression of pulmonary fibrosis, which can either promote or inhibit the development of this disease. Objective To explore the miRNA-mRNA regulatory network during the progression of silica (SiO₂)-induced pulmonary fibrosis in mice using integrated mRNA-seq and miRNA-seq analysis. Methods A mouse model of pulmonary fibrosis was established by dynamic SiO₂ dust exposure. The experimental design included a blank control group and four SiO₂-exposed groups (7, 14, 28, and 56 d, n=10 per group). Successful model induction was confirmed by histopathological analysis (HE and Masson staining), hydroxyproline (HYP) quantification, and expression of key fibrosis-related cytokines [fibroblast growth factor (FGF), interleukin-6 (IL-6), transforming growth factor-β (TGF-β), and tumor necrosis factor-α (TNF-α)]. Lung tissues from mice in each group were subjected to sequencing, and Mfuzz was used for time-series gene clustering to identify dynamic progression patterns. DESeq2 was utilized to identify differentially expressed genes (DEGs) and differentially expressed miRNAs. Enrichment analysis of DEGs was performed to identify critical signaling pathways and biological processes underlying pulmonary fibrosis progression. Expression of four selected miRNAs was subsequently validated by real-time quantitative polymerase chain reaction (RT-qPCR). The target mRNAs of key miRNAs were comprehensively predicted by integrating miRBase, starBase, and miRTarBase to construct the regulatory networks and investigate potential functions. Results SiO₂ exposure led to time-dependent aggravation of pulmonary fibrosis in mice, evidenced by increased fibrous deposition, elevated HYP levels (P < 0.01), and up-regulation of four kinds of pro-fibrotic cytokines (P < 0.01) compared with the NT group. Mfuzz clustering revealed the stage-specific characteristics. Compared to controls, 231, 662, 448, and 1020 DEGs were identified after SiO₂ exposure at 7, 14, 28, and 56 d, respectively, primarily enriched in immune responses and chemokine signaling. During critical fibrotic phases—7 d (acute inflammation and initiation) and 28 d (chronic inflammation and establishment)—18 differentially expressed miRNAs were identified; notably mmu-miR-135b-5p was significantly dysregulated at both time points. The expression trends of the four key miRNAs (mmu-miR-135b-5p, mmu-miR-708-5p, mmu-miR-21a-3p, and mmu-miR-205-5p) were consistent with the sequencing results. Furthermore, bioinformatics databases were used to predict the target mRNAs of key miRNAs. The constructed network highlighted critical miRNA-mRNA pairs—including mmu-miR-135b-5p and Meis1, mmu-miR-708-5p and Mmp25, mmu-miR-21a-3p and Cacna1d, mmu-miR-205-5p and Ereg which were closely associated with inflammatory response, extracellular matrix deposition, and fibroblast activation. Conclusion The progression of pulmonary fibrosis is accompanied by dynamic changes in miRNA-mRNA regulatory networks. The identified miRNA-target axes (e.g., miR-135b-5p and Meis1, mmu-miR-708-5p and Mmp25, mmu-miR-21a-3p and Cacna1d, and mmu-miR-205-5p and Ereg—) may play important roles in fibrogenesis and provide potential therapeutic targets for pulmonary fibrosis.