Increasing studies have found that PM2.5 has large adverse effects on stroke mortality. We want to investigate the long-term trends in the mortality of stroke attributable to ambient particulate matter pollution and household air pollution to provide evidence facilitating the design of policy.The deaths data of stroke and its subtypes attributable to PM2.5 were obtained from the Global Burden of Disease (GBD) 2019, analyzed by Joinpoint regression software and the age-period-cohort (APC) method to assess the magnitude of the trends in mortality and the temporal trends in the mortality rate by age, period, and cohort.From 1990 to 2019, the age-standardized mortality rate (ASMR) attributable to PM2.5 exposure trended downwards, but the trends of ambient particulate matter pollution and household air pollution were opposite. The trends varied among subtypes, the AAPC of intracerebral hemorrhage, ischemic stroke, and subarachnoid hemorrhage attributable to PM2.5 were 0.7, 2.5, and−3.3%, respectively. The longitudinal age curve of the APC model showed that the mortality rates due to PM2.5 exposure increased with age. The period RRs of ischemic stroke due to ambient particulate matter pollution increased significantly. The cohort RRs of ambient particulate matter pollution increased among those born from 1905 to 1990. The net drifts of all subtypes attributable to PM2.5 were below 0, but owing to the increase of ambient particulate matter pollution, the range of the decline was small. Males had higher net drift values, compared with females.Ambient particulate matter pollution has become the main type of PM2.5 leading to stroke in China. PM2.5 exposure is more harmful to ischemic stroke, males, and elderly. Chinese government should pay attention to the long-term impact of ambient air pollution on stroke and take effective public health policies and interventions. Stroke is a leading cause of mortality in middle-income countries, especially in China . AccordiPM2.5, the particulate matter with an aerodynamic diameter ≤ 2.5 μm, has become one of the three risk factors resulting in more than 1% of DALYs globally , causing3 increase in environmental PM2.5 per year, the risk of stroke increased by 19% , 20% , and 12% , respectively  of stroke and three subcategories of mortality attributable to ambient particulate matter pollution and household air pollution, and analyzed the independent effects by an age-period-cohort (APC) mode. The finding of our study may help public health managers to make evidence-based policies and assess specific interventions.We extracted the mortality rate of stroke, intracerebral hemorrhage, ischemic stroke, and subarachnoid hemorrhage attributable to PM2.5 in China from 1990 to 2019 from the Global Burden of Disease (GBD) 2019 at GBD Data tool.The original stroke mortality data were obtained from the Cause of Deaths Reporting System of the Chinese Centers for Disease Control and Prevention (CDC) and Disease Surveillance Points (DSPs) . IschemiThe mortality rates were age-standardized as follows:Based on published trials or cohort studies, GBD2019 provides a standardized and comprehensive assessment of the magnitude of risk factor exposure, relative risk, and attributable burden of disease by age, sex, and geographies for specific points in a series of time by using spatiotemporal Gaussian process regression, DisMod-MR 2.1, Bayesian meta-regression method and other alternative methods , 22.In the GBD study, PM2.5 included ambient particulate matter pollution and household air pollution. Ambient particulate matter pollution was defined as the population-weighted annual average mass concentration of outdoor PM2.5 exposure, which was obtained from satellite observations of aerosols in the atmosphere, ground measurements, chemical transport model simulations, population estimates, and land-use data. Exposure to household air pollution (HAP) from solid fuels is estimated from both the proportion of individuals using solid cooking fuels and the level of PM2.5 air pollution exposure. Solid fuels in GBD2019 include coal, wood, charcoal, dung, and agricultural residues .3 (* The number of deaths due to stroke or the subtypes.Population attributable fraction (PAF) represents the proportion of related risk that would be reduced if the exposure to a risk factor were reduced to the theoretical minimum exposure level, which is known as the theoretical minimum risk exposure level (TMREL). In GBD 2019, the TMREL of ambient and household air pollution was between 2.4 and 5.9 μg /m3 , 17, 23.3 : AD = PAThe age-standardized rates of PM2.5-ADs, ambient particulate matter pollution -ADs, and household air pollution-ADs were calculated by the world standard population . A comprIn our studies, JoinPoint software (Version 4.9.1.0) was used to calculate the average annual percentage change (AAPC), as well as 95% CIs, which can analyze the magnitude and direction of trends of the mortality rate of stroke and different subtypes over 30 years. The JoinPoint software estimated the mortality data by applying the grid search method with 5 Joinpoints and the Monte Carlo permutation test to optimize the model.1, y1),..., , of which, x1 ≤ ... ≤ xn, the JoinPoint regression model can be written in log-liner form asSuppose there is a sequence of observations  is over 1, where y-Υk)+= 0 .It is known that there is collinearity between age, period, and cohort . Like prThe age effect is the age-related physiological and pathological changes, causing epidemiological differences. To assess the age effect, the longitudinal age curve fitted longitudinal age-specific rates, which is relative to the reference cohort adjusted for period deviations. The period effect refers to changes in mortality rate due to human factors, such as the development of diagnosis technology, screening, and early detection, changes in disease definition and registration, treatment improvement, etc. These human factors may affect the disease rate in different periods, resulting in a period effect. The period effect, shown in the period ate ratios (period RRs), refers to changes in disease mortality caused by different human factors, such as the development of disease diagnosis technology, screening, and early detection, changes in disease definition and registration, treatment improvement and the conservation and emission reduction policies introduced by the Chinese government. When RR is over 1, the relative risk of death is higher in this period compared with the reference period, and when RR is below 1,the relative risk of death in this period is lower than that in the reference period. The cohort effects, shown in the cohort rate ratios (cohort RRs), refer to differences in disease mortality rates caused by various lifestyle changes or different exposure to risk factors among generations. Net drift is an overall log-linear trend by calendar period and cohort effects, indicating overall the annual percentage change. Local drift is the log-linear by period and birth cohort for each age group, representing annual percentage changes for each age group . Like prAPC model can be written in linear regression form as follow –36:i age group during j period; αi denotes age effect of the i age group; βj represents period effect of the j period; γk denotes cohort effect of the k (k = I + j - 1) birth cohort; μ is intercept or adjusted mean death rate, and ε is the residual or a random error.M represents the death rate for age https://analysistools.cancer.gov/apc/). p < 0.05 was considered significant  and the age-period-cohort web tool .We further analyzed the ASMRs of stroke attributable to ambient particulate matter pollution and household air pollution. For ambient particulate matter pollution, the number of deaths increased 3.3 times over, from 161,939 in 1990 to 541,796 in 2019. The ASMR of stroke attributable to ambient particulate matter pollution increased by 1% annually, with similar trends observed among both sexes . Among different stroke subtypes, the ASMR of subarachnoid hemorrhage decreased , but the ASMR of ischemic stroke  and intracerebral hemorrhage  similarly increased from 1990 to 2019.For household air pollution, the number of deaths decreased from 382, 505 in 1990 to 136,950 in 2019. It also decreased significantly among both sexes . The ASMRs of different stroke subtypes attributable to household air pollution were all decreased.The ASMR due to ambient particulate matter pollution was higher than that due to household air pollution after 2002. For males, the ASMR due to ambient particulate matter pollution was higher than that due to household air pollution after 1999, and for females, the ASMR due to ambient particulate matter pollution was higher than that due to household air pollution after 2005 .For the same birth cohort, the mortality rate of stroke attributable to PM2.5 increased with age. The mortality rate of stroke among males was higher than that among females ,E.Among different subtypes, for subarachnoid hemorrhage, the mortality rate was lower than others, and it increased with age before 55 and slightly decreased after that. Other subtypes of stroke had increasing trends with age attributable to PM2.5, they trended upwards rapidly after the 55–60 age groups –D.In terms of ambient particulate matter pollution and household air pollution, the mortality rates due to ambient particulate matter pollution and household air pollution were low in younger group, but the mortality rate due to ambient particulate matter pollution significantly increased with age among those older groups. Similar changes were observed in males and females ,F.The period RRs of all stroke subtypes attributable to PM2.5 exposure trended downwards. The downward trend among females was steeper than that among males ,E,F.In terms of ambient particulate matter pollution, the period RRs of stroke increased from 1990 to 2019. For household air pollution, it trended downwards significantly. Similar trends were observed among those of intracerebral hemorrhage and ischemic stroke. The period RRs of subarachnoid hemorrhage attributable to ambient particulate matter pollution and household air pollution decreased from 1990 to 2019 –D.Cohort RRs of all stroke subtypes attributable to PM2.5 exposure decreased among those born from 1905 to 1990. The cohort effects were opposites for ambient particulate matter pollution and household air pollution: for ambient particulate matter pollution, the cohort effects increased, for household air pollution, they declined. Similar trends were observed among both males and females. The downward trend among females was steeper than that among males ,E,F.In terms of different subtypes, the cohort RRs for household air pollution trended downwards obviously, while for ambient particulate matter pollution, the cohort RRs of the mortality rate of intracerebral hemorrhage and ischemic stroke trended upwards –D.P < 0.001). However, the net drift values of intracerebral hemorrhage and ischemic stroke attributable to ambient particulate matter pollution were above 0 . In terms of sexes, the net drift value of stroke for males was−1.97%, and for females, it was−3.98% (P < 0.001). Among different stroke subtypes, males had higher net drift values, compared with females.The overall net drift values of all stroke subtypes attributable to PM2.5 were below 0 , Natural Science Foundation of Guangdong Province (Grant Number: 2019A1515011496), Social Science Foundation of Guangdong Province (Grant Number: GD19CSH04), and the Key Projects of Philosophy and Social Sciences Research of Education Department of Hubei in China (Grant Number: 17ZD024).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Idiopathic pulmonary fibrosis is a fatal interstitial disease characterized by fibroblast proliferation and differentiation and abnormal accumulation of extracellular matrix, with high mortality and an increasing annual incidence. Since few drugs are available for the treatment of pulmonary fibrosis, there is an urgent need for high-efficiency therapeutic drugs and treatment methods to reduce the mortality associated with pulmonary fibrosis. The interstitium, a highly efficient transportation system that pervades the body, plays an important role in the occurrence and development of disease, and can be used as a new route for disease diagnosis and treatment. In this study, we evaluated the administration of hydroxysafflor yellow A phytosomes via intervaginal space injection (ISI) as an anti-pulmonary fibrosis treatment. Our results show that this therapeutic strategy blocked the activation of p38 protein in the MAPK-p38 signaling pathway and inhibited the expression of Smad3 protein in the TGF-β/Smad signaling pathway, thereby reducing secretion of related inflammatory factors, deposition of collagen in the lungs of mice, and destruction of the alveolar structure. Use of ISI in the treatment of pulmonary fibrosis provides a potential novel therapeutic modality for the disease. Idiopathic pulmonary fibrosis is an irreversible and fatal interstitial lung disease with a high mortality rate, increasing annual incidence, and extremely poor prognosis for patients, with a survival time of 3–5 years after diagnosis . The curThe interstitium is composed of a fibrous network and matrix, which is located between cells, substantive functional tissues, and organs, forming a multi-level network structure from a loose fibrous network to a dense fibrous stroma structure, making the body a flat and connected organic whole ,11. The Hydroxysafflor yellow A (HSYA), a compound with a monochalcone glycoside structure, is the most effective water-soluble ingredient of safflower that has been widely used in the treatment of cardiovascular diseases because of its effective anticoagulant, anti-myocardial ischemic, and vasorelaxant effects ,19,20,21Herein, hydroxysafflor yellow A phytosome (HYAP), as an anti-pulmonary fibrosis therapeutic agent, was prepared to improve the lipid solubility and bioavailability of HSYA, to treat bleomycin-induced pulmonary fibrosis in mice by ISI. This therapeutic strategy alleviates the severity of pulmonary fibrosis, elaborates the mechanism of interstitial treatment, and provides a novel therapeutic approach for the treatment of pulmonary fibrosis.HSYA has poor lipid solubility, which results in low oral bioavailability. Hence, we prepared the HYAP to increase its lipid solubility and prolong its release time to improve its efficacy. Phytosomes are similar to liposomes in many ways: both can alter the physicochemical properties of the parent drug and improve the bioavailability of the drug, but they differ greatly in their principles of formation. A liposome is a drug encapsulated in a closed vesicle formed by a phospholipid, and the drug is either free in the solution within the vesicle or dispersed between multiple membranes of the phospholipid. In contrast, phytosomes are more stable compounds or complexes formed by charge migration between the drug and the phospholipid. The oxygen atom in the hydroxyl group on the phospholipid atom in the phospholipid structure has a strong tendency to gain electrons, while the nitrogen atom has a strong tendency to lose electrons, so under certain conditions, it can generate complexes with drug molecules of a certain structure. In the process of HYAP formation, mainly HSYA acts by forming hydrogen bonds or van der Waals forces with phospholipid molecules A. As forThe prepared HYAP was characterized by transmission electron microscopy (TEM). HYAP was uniformly spherical, and the sizes were mainly concentrated around 101.67 nm B. The hyThe compounding rate of HYAP was 91.4% and the encapsulation rate was 57.86%, as measured by the ultraviolet spectrophotometer (UV). The cumulative release rate of HSYA reached 73.48% at 2 h, 75.85% at 4 h, and leveled off around 6 h, with a cumulative release rate of 83.84% at 48 h. The cumulative release rate of HYAP was 45.88% at 2 h, followed by a rapid release period for the first 4 h, with a cumulative release rate of 57.65% at 4 h, followed by a slow release, which leveled off at 24 h. The cumulative release rate was 83.88% after 48 h F. This sWe then analyzed the physicochemical properties of the HYAP by UV and Fourier transform infrared spectrometer (FT-IR). As shown in -1 (-OH), 1515 cm-1 (C=O), and 1079 cm-1 (C-O), and the characteristic absorption peaks of phospholipids were 3526 cm-1 (-OH), 2926 and 2855 cm-1 (C-H), 1740 cm-1 (C=O), 1239 cm-1 (P=O), and 1067 cm-1 (P-O-C)  H. The chThe pulmonary fibrosis model was established by intratracheal injection of BLM, and HYAP was administered by ISI for 14 days after the successful establishment of pulmonary fibrosis on day 7 A. To comHE staining showed that the alveolar structure in the saline group was clear and intact, the alveolar septum was not thickened, and no inflammatory cell infiltration was observed, whereas the alveolar structure in the model group was seriously damaged, fibrous tissue proliferated, and the septum between alveoli was significantly thickened. Compared with the BLM group, the BLM + pirfenidone and BLM + HYAP groups showed greater improvement in the degree of lung tissue destruction, thinner alveolar septum, and complete recovery of alveolar structure, and this phenomenon became more obvious with the increase in days of administration B. The AsMasson trichrome staining showed that only a small amount of collagen was deposited in the lung tissue of the mice in the saline group, whereas a large amount of blue collagen was diffusely deposited in the interstitium of the lung tissue of mice in the BLM group. Compared with the BLM group, the extent of lung tissue lesions was reduced in both the BLM + pirfenidone and BLM + HYAP groups, and the blue collagen deposition was significantly reduced B. The frTo assess the disease development in mice in each group after model establishment and administration, we performed computed tomography (CT) imaging analysis A. The grTo detect the expression of related inflammatory factors in mice after model establishment and administration, we determined the levels of inflammatory factors in mouse plasma using ELISA. TGF-β1 plays an important role in lung fibrosis by promoting the proliferation and differentiation of fibroblasts into myofibroblasts, which further leads to the deposition of collagen and ultimately to fibrosis. Compared with the saline group (4961.1 pg/mL), TGF-β1 was significantly elevated in the plasma in animals of the BLM group, with plasma levels reaching 9147.2 pg/mL. After treatment, the BLM + HYAP group recovered to 4891.5 pg/mL, which was significantly lower than that of the BLM group B. At theTo further understand the inflammatory infiltration and fibroblast activation, we performed immunofluorescence staining A. LeukocCD45 was only slightly expressed in the saline group, whereas the BLM group showed a large amount of leukocyte interstitial infiltration. Compared with the BLM group, inflammatory infiltration in the BLM + pirfenidone and BLM + HYAP groups was significantly lower B. α-SMA To further investigate the mechanism through which HYAP ameliorated pulmonary fibrosis, we investigated the signaling pathways associated with pulmonary fibrosis using Western blotting. Deposition of extracellular matrix is the most direct manifestation of pulmonary fibrosis; therefore, we first examined collagen deposition in the BLM and BLM + HYAP groups A. ConsisThe MAPK signaling pathway mainly includes ERK, JNK, and p38MAPK, which promote fibrosis by regulating inflammatory responses, mediating apoptosis, and participating in angiogenesis. This signaling pathway is triggered by a variety of extracellular stimuli. We analyzed the activation of p38 in mouse lung tissue using Western blotting A. There We also determined the effects of the TGF-β/Smad signaling pathway on pulmonary fibrosis. TGF-β1 is an important factor in promoting pulmonary fibrosis, and the TGF-β/Smad signaling pathway plays an important regulatory role in the disease by promoting excessive deposition of extracellular matrix. Smad3 is an important protein in TGF-β1 intracellular signal transduction, which is the hub of the signaling pathway between the cell membrane and the nuclei and is the most critical and major signaling protein in the TGF-β1 signaling pathway. We found that the expression of Smad3 in the lung tissue of mice with BLM-induced pulmonary fibrosis was higher than that in the saline group D, and thISI is a novel drug delivery method for injecting drugs or nanoparticles into inter-stitial pathways using the ankle or carpal tunnel as ISI sites, which contain tendons, arteries, veins, and nerves. This transport pathway allows gold nanoparticles (AuNPs) to reach the central nervous system without crossing the blood–brain barrier, providing a new avenue for more effective treatment of central nervous system diseases . SubsequHSYA has antioxidant, anti-inflammatory, and anti-cancer activities and a wide range of pharmacological effects on the cardiovascular, hematological, and nervous systems. However, it exhibits poor lipid solubility and low bioavailability when orally administered. We selected HSYA as the target drug for interstitial treatment of antifibrosis and prepared phytosomes to improve the lipid solubility of HSYA, thereby improving its bioavailability. The prepared HYAP had a uniform particle size, good stability, and a good, sustained effect. Subsequently, we compared the different administration concentrations. When the administration concentration of HYAP was 60 mg/kg, the degree of pulmonary fibrosis did not increase after 14 days of administration, and the alveolar structure tended to recover; however, the pharmacological effect was not obvious. The recovery of the alveolar structure increased significantly, and collagen accumulation decreased significantly, with increasing administration concentration. Combined with the clinical dosage, we selected 120 mg/kg for the treatment of the mouse model of pulmonary fibrosis.Repair of lung tissue after injury is a complex phenomenon involving intricate mechanisms. In general, wound healing undergoes three dynamic and interrelated stages that overlap over time: inflammatory, proliferative, and remodeling phases of maturation and scar formation . After rThe role of the interstitium in pulmonary fibrosis has rarely been described. It has been shown that the subcutaneous interstitium is a major reservoir of fibroblasts, endothelial cells, and macrophages. In deep injury, fibroblasts in the subcutaneous interstitium direct their local complex matrix into the wound, coordinating with the coagulation cascade response and forming a prefabricated matrix that subsequently develops into mature scar tissue . The intHSYA , lecithin , bleomycin , pirfenidone , ELISA kits , 4% paraformaldehyde , hematoxylin and eosin staining kit , Weigert hematoxylin staining solution , Ponceau S and Magenta staining solution , phosphomolybdic acid solution , Aniline blue solution , DAPI , RIPA buffer , antibodies , anti-mouse IgG , anti-rabbit IgG ), BCA Protein Assay Reagent Kit , and primary antibodies ), FITC-conjugated goat anti-rabbit IgG , CY3-conjugated goat anti-rat IgG , and CY5-conjugated goat anti-mouse IgG , were purchased.C57 BL/6J mice  were purchased from Beijing Vital River Laboratory Animal Technology . C57 BL/6J mice were randomly divided into four groups: saline, BLM, BLM + pirfenidone, and BLM + HYAP groups. The experimental animals were anesthetized by inhalation of isoflurane, and the gas flow rate of the anesthesia machine was adjusted to 300 mL/min. After the anesthetic agent filled the anesthesia box, the animals were put into the anesthesia box with an anesthetic concentration of 3% and anesthesia time of 3 min. After the mice were completely anesthetized, the anesthesia mask was switched to continue the anesthesia on the experimental animals, followed by the next operation. The skin of the neck of the mice was dissected to expose the trachea, and 0.05 mL of BLM was injected at a dose of 5 mg/kg. An equal volume of saline was administered to the saline-treated group. This study was approved by the Institutional Animal Care and Use Committee .The thin-film hydration method was used to prepare HYAP. HSYA powder and soybean lecithin were placed in a round-bottomed flask at a mass ratio of 1:3, ethanol was used as the solvent, and the mass concentration of HSYA was adjusted to 2 mg/kg. The samples were placed on a constant temperature magnetic stirrer and incubated at 40 °C for 2 h. After the reaction was complete, the unreacted precipitate was removed by suction filtration, and the organic solvent was removed by rotary evaporation under reduced pressure at 45 °C. An appropriate amount of chloroform and cholesterol (the mass ratio of phospholipid and cholesterol was 5:1) was added, shaken slightly to dissolve it, and any insoluble precipitates were removed via filtration. Continued rotary evaporation under reduced pressure at 45 °C formed a phytosome film on the bottom of the bottle. An appropriate amount of the saline was added to hydrate the phospholipid film for 30 min at 60 °C, followed by sonication for 15 min in a bath sonicator at 42 kHz and 100 W. The resulting phytosomes were subsequently extruded 10 times through a 100 nm polycarbonate porous membrane using an extruder .The size, polydispersity index, and zeta-potential of HYAP were measured by DLS  in triplicate at 25 °C. The morphology was characterized by TEM . A drop of diluted HYAP was placed on a carbon-coated grid and air-dried, and a drop of 1% uranyl acetate was deposited onto the grid for 3 min. Excess uranyl acetate stain was removed from the edge with filter paper, dried overnight at room temperature, and used for TEM. Phospholipids and HYAP are soluble in chloroform, so the compounding rate of HSYA with phospholipids can be measured by dissolving HYAP in chloroform. The finished compounded solution was dissolved by rotary evaporation out of anhydrous ethanol, and the appropriate amount of chloroform was added. The free HSYA precipitated because it was insoluble in chloroform, the precipitate was removed, and the filtrate was broken by rotary evaporation with the addition of quantitative methanol ultrasonication. The content of the compound HSYA was measured at 403 nm using UV .A certain amount of HYAP squeezed by the extruder was placed in an ultrafiltration tube with a MWCO of 10,000 Da, centrifuged at 15,000 r/min for 30 min, and the nanoparticles were trapped in the inner tube, then the nanoparticles were collected and added to the quantitative methanol ultrasonication to break the emulsion.The compounding rate and encapsulation rate were calculated using the following equations:The sustained release rate of the drug was determined by dialysis sampling and analyzed by UV. Using PBS as the release medium, stirring at 37 °C, 3 mL of HYAP was precisely measured in a pretreated dialysis bag. Samples (3 mL) were taken from the release medium at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h and supplemented with equal volumes of PBS. The samples were filtered through a 0.22 μm microporous membrane and used for UV analysis. Absorbance values were measured at 403 nm and cumulative release rates were calculated.The HSYA, phospholipids, physical mixture of HSYA and phospholipids, and HYAP were dissolved in methanol as the solvent and methanol was used as the blank, then the absorbance was measured in the wavelength range of 200–600 nm and the curves were plotted.−1.The mixture of HSYA and phospholipids, phospholipids, and HYAP with KBr was ground at 1:100, pressed into tablets, and then measured by FT-IR at wavelengths of 4000–500 cmSeven days after BLM injection, pulmonary fibrosis model mice were randomly divided into three groups of six mice each. The BLM + pirfenidone group was intragastrically treated with pirfenidone solution (200 mg/kg), daily for 14 days. The BLM + HYAP group was treated with HYAP in 0.1 mL of saline solution (120 mg/kg) by ISI in the right upper limb for 14 consecutive days, once a day. The chosen ISI injection point was the carpal tunnel of the wrist bone. The carpal tunnel is composed of the flexor retinaculum and carpal groove, inside which are the superficial and deep flexor tendons, flexor tendon sheath, flexor hallucis longus tendon and its sheath, and the median nerve and vessels running through it. The injection point was a convergent point for tendons, vessels, and nerve fibers connected to the fascia surrounding them. The saline and BLM groups were injected with an equal amount of saline via ISI in the right upper limb for 14 consecutive days, once a day.On day 14 of administration, mice were randomly selected from the saline, BLM, BLM + pirfenidone, and BLM + HYAP groups, and changes in lung structure during the establishment of fibrosis and drug treatment were recorded using CT in vivo imaging. CT was preheated for 15 min. The setting parameters were as follows: voltage, 90 kV; imaging current, 180 µA; real-time observation current, 180 µA; FOV, 24 mm; scanning program, 4.5 min; scanning angle, 360. The anesthesia machine was connected to an oxygen bottle, isoflurane was added, the gas source valve was adjusted at the front end of the oxygen flowmeter so that the output gas reached the required flow rate (300 mL/min), and the mice were placed in the anesthesia box. The anesthetized mice were fixed on the instrument cavity operating table, and the chamber was closed for scanning and analysis of the structure.2 asphyxiation method was adopted to execute the animal, whereby the animal was placed into the euthanasia box and CO2 was infused into the box at a rate of 20% of the volume of the box per minute replacement. It was confirmed that the animal was not moving and not breathing, the CO2 was turned off after the pupils were dilated, and then the animal was observed for 2 min to ensure that it was dead. Then, the left lower lung tissues were collected from each group on days 7 and 14 after administration and then fixed in 4% paraformaldehyde for 72 h. The samples were subsequently dehydrated using a series of gradients of ethanol, made transparent with xylene, and then embedded in paraffin. The samples were then cut into thin slices using an automated microtome, mounted on slides, and stored at room temperature for hematoxylin and eosin (HE) and Masson trichrome staining. After dewaxing, hydration, staining, dehydration, and sealing, pathological analysis was performed. Semi-quantitative analysis of pulmonary fibrosis was performed with reference to the Ashcroft scoring criteria, and the collagen volume fraction of Masson trichrome staining was calculated for different groups using Image J software.The COThe largest lobe of the left lung was fixed with 4% paraformaldehyde, sliced using a frozen microtome, and stained with immunofluorescence. The sections were incubated overnight at 4 °C after adding the primary antibodies against CD45, α-SMA, and collagen I (1:50), and the slices were washed three times with TBST and incubated with FITC-labeled secondary antibody (1:200), CY3-labeled secondary antibody (1:300), and CY5-labeled secondary antibody (1:400) at room temperature for 3 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 3 min, washed three times with TBST, dehydrated with gradient alcohol, made transparent with xylene, and sealed. Images were detected and collected by a slice scanner: DAPI glows blue by UV excitation wavelength 330–380 nm and emission wavelength 420 nm, FITC glows green by excitation wavelength 465–495 nm and emission wavelength 515–555 nm, CY3 glows red by excitation wavelength 510–560 nm and emission wavelength 590 nm, and CY5 glows pink by excitation wavelength 608–648 nm and emission wavelength 672–712 nm . The staining results were analyzed using the Image J software.On day 14 of treatment, blood was collected from the ocular venous sinus and placed in blood collection tubes with EDTA. The upper plasma was collected after centrifugation at 4 °C and 3000 rpm for 5 min. ELISA kits were used to quantify the levels of TGF-β1 and IL-6 in the plasma according to the manufacturer’s instructions.g at 4 °C for 10 min, and the supernatant was kept at −80 °C. Protein concentration was determined using the BCA method. Equal amounts of total protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane using the semi-dry transfer method. After being sealed with 5% skim milk powder for 1 h at room temperature, the membrane was incubated with primary antibody diluted in phosphate-buffered saline overnight at 4 °C. After washing thrice with TBST, the proteins were incubated with horseradish peroxide-coupled IgG diluted in PBS at room temperature for 1 h. The proteins collagen I, p38, p-p38, and Smad3 were detected using enhanced chemiluminescence reagents, and the optical density values of the target bands were determined.A section (100 mg) of the lower lobe of the right lung was cut into small pieces on ice. After adding 1 mL of RIPA lysis solution and 10 μL of PMSF (100 mM), the sample was ground until there was no visible tissue and was left standing for 30 min or until complete lysis. The cells were centrifuged at 10,000× p < 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.Group comparisons were performed using the GraphPad Prism (version 9.0) software . Ordinary one-way ANOVA was used for comparisons between more than two groups. Statistical significance was set at In conclusion, our study showed that the delivery of HYAP via ISI ameliorated bleomycin-induced pulmonary fibrosis compared to oral positive drugs, which was associated with blocking the activation of p38 in the MAPK-p38 signaling pathway and reducing the content of Smad3 in the TGF-β/Smad signaling pathway, which in turn decreased the levels of relevant inflammatory factors and ameliorated the effects of pulmonary fibrosis. Whether interstitial administration can work better than traditional injection methods needs to be further explored. In the absence of an effective treatment, ISI does offer a reference strategy for the treatment of pulmonary fibrosis as a new potential method of drug delivery.


Rhizoma drynariae, whereas the model and sham groups received 0.9% saline solution twice daily for 12 weeks. Subsequently, the rats were sacrificed, and the left femur of each group was removed for computerized tomography testing, while right femurs were used for hematoxylin and eosin staining. High-throughput RNA sequencing and functional and pathway enrichment analyses were performed. Comparing the gene expression between the experimental and model groups, 149 differential genes were identified, of which 44 were downregulated and 105 were upregulated. The criteria for statistical significance were |log2 Fold Change| > 1 and P < 0.05. Gene ontology analysis showed that the differentially expressed genes were enriched in cell component terms such as cell part and outer cell membrane part, and the genes were associated with cell process, biological regulation, metabolic processes, DNA transcription, and catalytic activity. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways showed significantly enriched pathways associated with systemic lupus erythematosus, herpes simplex infection, circadian rhythm, vascular smooth muscle contraction, the AGE-RAGE signaling pathway in diabetic complications, and the TNF, Apelin, and Ras signaling pathways. Our results revealed that the Npas2, Dbp, Rt1, Arntl, Grem2, H2bc9, LOC501233, Pla2g2c, Hpgd, Pde6c, and Dner genes, and the circadian rhythm, lipid metabolism, inflammatory signaling pathway, and immune pathways may be the key targets and pathways for traditional Chinese medicine therapy of Rhizoma Drynariae in osteoporosis.Osteoporosis is increasingly becoming a serious problem affecting the quality of life of the older population. Several experimental studies have shown that Chinese medicine has a definite effect on improving osteoporosis. Based on transcriptome sequencing, we analyzed the differential gene expression and mechanism of the related signaling pathways. Fifteen rats were randomly divided into an experimental group, a model group, and a sham surgery group. The rat model for menopausal osteoporosis was established using an ovariectomy method. One week after modeling, the experimental group was administered(intragastric administration)8.1 g/kg of  Osteoporosis, a disease with increased bone fragility and risk of fracture due to decreased bone formation and increased destruction of bone resorption , 2,is prIn the study of the pathophysiology of osteoporosis, we have found that factors associated with bone strength and damage include a weakened microstructure and tissue of the trabecular bone and decreased bone formation with excessive bone resorption. These factors are closely related to age and changes in estrogen levels .In termsCurrent drug treatments for osteoporosis include basic supplements , bone absorption inhibitors ;and bonPostmenopausal osteoporosis, also known as primary osteoporosis, is common in older, postmenopausal women, presenting bone pain, adverse limb movement, and an increased risk of bone fracture. Estrogen deficiency is the major cause of postmenopausal bone loss, while older age, smoking, chronic disease, and other related illnesses and drugs are additional causes , 8.Chinese herbal medicine has an apparent effect on osteoporosis, as it can ameliorate bone health through multiple targets and pathways .Rhizoma Female Sprague-Dawley rats  were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The feeding environment was clean and well ventilated, and water was freely available. All animals were housed at the SPF Grade Animal Experimental Center of Shandong Provincial Hospital of Traditional Chinese Medicine at 25°C with a constant humidity of 50% for 12 h per day and given a standard diet. The experimental animals were bred for 7 d, and 15 rats were divided into the experimental group (OVXDF),the model group (OVX), and the sham operation group (SHAM). In the OVXDF and OVX groups, a preoperative diet was not provided for 12 h, and intraperitoneal pentobarbital sodium (10g/L) was used for anesthesia. The OVXDF group was subjected to surgical resection of their bilateral ovaries, and only small peripheral adipose tissue around the ovary was removed in the SHAM group . After tThe preparation of drug decoction was followed the preliminary preparation protocol of our research group , 18, GraFollowing the animal dose conversion formula , RhizomaRats were sacrificed following a sodium pentobarbital overdose(60mg/kg) , and bilThe right femur of each group was taken, cleaned and fixed in 10% formaldehyde solution for 48 h, washed in running water and distilled water for 24 h. The specimen was decalcified, and the decalcification was stopped after 3~4 h. The alkaline solution was washed for 12h, then dehydrated, transparent and wax, respectively, embedded and sliced in paraffin, and the drying time was about 10~15 min.The right femoral paraffin sections of each group were dewaxed for 5 to 10 min for 5min with absolute ethanol, 90% ethanol, and 70% ethanol for 2min each. After washing and drying naturally for 2min, the fixative was fixed for 30s~3min at room temperature, stained with appropriate staining solution, covering the tissue, placed in a 37°C temperature box, and washed for 45~60min. Compound staining was performed for 5 to 8 min or methyl green staining for 2 to 3 min. Finally, it was washed, dried and micro examine.Total RNA was extracted from the bone tissue samples selected from each group after using TRIzol , Samples are sequenced on the platform to get image files, which are transformed by the software of the sequencing platform, and the original data is generated. we use Cutadapt (v1.15) software to filter the sequencing data to get high quality sequence for further analysis.Finally obtained the expression of differential genes between the OVX group and OVXDF group.P < 0.05 as criteria for statistical significance. DEGs were represented using graphs to calculate the number of upregulated and downregulated DEGs in each comparison group. Volcano maps showing gene distribution of the DEGs were obtained using ggplot2 (R package). Two-way clustering analysis of the union and samples of DEGs across all comparison groups was performed using Pheatmap (R package).Differential gene expression was analyzed using DESeq (R package). Differentially expressed genes (DEGs) were selected with |log2 Fold Change| > 1 and P < 0.05) was performed to determine the significantly enriched GO terms for DEGs and identify their main biological functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the clusterProfiler v3.4.4 software, focusing on significantly enriched pathways with P < 0.05.Gene ontology (GO) enrichment analysis using topGO (https://string-db.org/) to reveal the functional relationship between the target genes. When PPI information for this species was included in the STRING database, PPI action pairs containing DEGs and scores > 0.95 were screened based on the results of the DEG analysis. Differential gene correlation was also assessed by visual analysis using the Cytoscape v3.7.2 software.Protein interaction analysis was performed employing the Search Tool for the Retrieval of Interacting Genes (STRING) database  was added, PCR thermocycler amplification, amplification conditions: 95°C pre-denaturation for 3 min; 95°C was denatured for 20s, 60°C annealed for 15s, 72°C extended for 30s, and amplified in 45 cycles. The last 72°C was extended for 1 min. For SDS polyacrylamide gel electrophoresis, the image analysis software analyzed the band density, and the relative expression of the genes was calculated using the 2-△△Ct method. Five DEGs with different expression patterns were randomly chosen to validate the accuracy of the transcriptome sequencing results, qRT-PCR was verified using actin as the internal reference. The primers used are listed below Table 1.P < 0.05 was considered significant. GraphPad Prism v7.0  was utilized. All assays were conducted three times or more.All the data were expressed as the mean ± standard deviation. An independent sample t-test evaluated the significant differences between two groups using SPSS v24.0 , and P < 0.05). In contrast, the bone mineral density of the OVXDF group was significantly higher than that of the OVX group (P < 0.05). Moreover, the bone volume score (BV/TV), surface area volume fraction (BS/TV), and number of trabecular bones (Tb.N) for the OVXDF and SHAM groups were significantly higher than those of the OVX group  structural constituent and protein heterodimerization activity. DEGs enriched in cellular component terms, facilitated by the major enrichment in the nucleosome, included DNA and protein-DNA packaging complex, extracellular space, and collagen-containing ECM. Furthermore, the DEGs were involved in biological processes such as negative regulation of smooth muscle cell-matrix adhesion, negative regulation of megakaryocyte differentiation, nucleosome organization, DNA replication-dependent and -independent nucleosome assembly, stimulatory C-type lectin receptor signaling pathway, and chromatin assembly or disassembly. Regulation of smooth muscle and cell-matrix adhesion contribute a large proportion of the biological process classification and changes in both states are closely related to the aging process. Whether the appearance of vascular calcification or cell-matrix adhesion affects the Wnt signaling pathway, they all cause bone loss to some extent. The outer part of the cell membrane accounted for the most proportion of DEGs in the cell group classification. In the molecular function classification, ECM structural constituent, mainly by protein heterodimerization activity, can promote stem cell proliferation and maturation, the formation of matrix scaffold, and bone remodeling by bone-forming cells. It has been shown that changes in the functional properties of the ECM may be involved in diseases such as osteoporosis and oral bone loss.Enrichment analysis of DEGs Figure 5P < 0.05), with 110 downregulated and 118upregulated pathways. Top-scoring pathways included systemic lupus erythematosus, herpes simplex infection, alcoholism, the AGE-RAGE signaling pathway in diabetic complications, and graft-versus-host disease. The diseases associated with the DEGs included rheumatoid arthritis, amoebiasis, and organismal systems. Moreover, the DEGs were strongly correlated with protein digestion and absorption, circadian rhythm, vascular smooth muscle contraction, aldosterone synthesis and secretion, antigen processing and presentation, salivary secretion, complement and coagulation cascades, and cellular processes. The most enrichment was in focal adhesion and environmental information processing. Furthermore, the DEGs were primarily enriched in inflammatory immune signaling pathways such as the TNF, Apelin, and Ras signaling pathways and ECM-receptor interaction.DEGs from the OVXDF and OVX groups were mapped to the KEGG pathway database for enrichment analysis Figure 5https://string-db.org/) and imported into Cytoscape v3.7.2. The results showed 130 nodes and 111 edges, with an average node degree of 1.71. Genes with a large correlation between them were Col1a1 , Edn1 , Mmp3 , Serpine1 , Acta2 , Eln , Ciart , Arntl , Npas2 , and Sox10   Figure 6Following our transcriptomic sequencing results, five DEGs with apparent upregulation and downregulation were selected as qRT-PCR validation targets, namely Npas2, Dbp, Arntl, Grem2, and Dner. Dbp was significantly upregulated in the OVXDF group compared with that in the OVX group. However, the expression of Npas2, Arntl, Grem2, and Dner was significantly downregulated. These results were consistent with our RNA-seq results, thus reaffirming them Figure 7In this study, we aimed to identify DEGs and their expression patterns for promoting bone repair in osteoporosis and further reveal the specific mechanism of genes in osteoporosis treatment through transcriptomic analysis of osteoporosis model rats (OVX) and experimentally treated osteoporosis rats (OVXDF). A total of 149 DEGs were identified between the two groups. The 10 most altered DEGs were Npas2, Dbp, RT1, Arntl, Grem2,H2bc9, LOC501233, Pla2g2c, Hpgd, Pde6c, and AABR07065750. these may be a potential target for treatment in ovariectomized rats with osteoporosis. We confirmed the positive role of Rhizoma Drynariae in improving osteoporosis by an experimental analysis of rat femoral tissue.Combined with the results of the DEG analysis, we found that DEGs involved in Rhizoma Drynariae regulation are mainly related to the circadian rhythm, lipid metabolism-related diseases, osteoporosis disease, inflammation, and tumors. Osteoporosis treatment with Rhizoma Drynariae can play a significant role through multiple pathways, and we confirmed that the Ras signaling pathway is important to regulate bone homeostasis and bone formation . Ras sigThe mechanisms of the inflammatory signaling and immune pathways in osteoporosis have been widely studied. Our analysis confirmed that Rhizoma Drynariae could regulate the tumor inflammatory pathway by changing the levels of inflammatory factors and reducing the inflammatory response to regulate the immune status. The prostaglandin dehydrogenase gene Hpgd also plays a role in tumor development and inflammation . In our Notably, multiple key genes associated with the circadian rhythm were significantly expressed in our results, including Arntl, Npas2, Dbp, Per1, and Per2. Our analysis confirmed the important role of the circadian rhythm in osteoporosis and the positive regulatory effect of Rhizoma Drynariae. Circadian rhythms are controlled by a molecular clock system located in the suprathalamic optic chiasmus  and the peripheral tissue . Some studies have revealed that the disruption of circadian rhythms can lead to bone remodeling disorders and eventually osteoporosis because various bone regulatory growth factors are associated with clock genes . Fu 36)36) showein vivo. NPAS2 usually cooperates with Arnt-1 (BMAL1) to regulate the transcription of circadian genes.BMAL1 also acts as a core component gene of circadian rhythms, and increasing evidence indicates that a key role of BMAL1 in bone development can promote fracture healing and improve bone. Animal experiments confirm that when BMAL1 receives inhibition, bone and cartilage development is hindered and thus caused by a decrease in bone mass , confirming its involvement in regulating the expression of genes related to lipid metabolism .As the mIn this analysis, it was found that many genes about fat metabolism were also significantly expressed in differential genes and enriched pathways. It shows a positive regulatory role of Rhizoma Drynariae in regulating osteogenic differentiation and fat differentiation. Gremlin-2, a key bone morphogenetic protein (GREM2) (BMP) antagonist, can competitively bind BMP receptors to regulate the activity of BMP ligands, thus it plays a crucial role in regulating bone homeostasis . In the Several genes related to lipid metabolism, such as HPGD and DNER, were also confirmed to be significantly regulated in this analysis. It shows that Rhizoma Drynariae can regulate proteins and pathways related to lipid metabolism, improve the anti-oxidative stress resistance, thus promoting osteogenic expression and reversing osteoporosis.Lisa et al.  showed tIt also shows that osteoporosis is closely linked to T2DM. The AGE-RAGE signaling pathway is important in diabetes complications, including reduced bone mineral density and impaired bone quality. Our study confirmed that AGE-RAGE signaling has a role in promoting increased osteogenic function in bones , MoreoveAlthough previous bioinformatics studies have been carried out on the effect of Rhizoma Drynariae in postmenopausal osteoporosis (15), no further verification has been conducted. In our DEG analysis, we have verified that Rhizoma Drynariaer in osteoporosis model rats occurs through multiple pathways and targets. However, this study is limited by its small sample size, the difficulty of RNA extraction from bone tissue, and some errors that may affect the experimental results to a certain extent. Although the mechanisms of Chinese herbal medicine in the treatment of osteoporosis have been widely studied, the lack of a more comprehensive analysis necessitates further research. In this regard, transcriptome analysis can broaden the scope of research and provide new directions to these studies.https://www.ncbi.nlm.nih.gov/bioproject/PRJNA862708, Accession: PRJNA862708; ID: 862708.The original contributions presented in the study are publicly available. This data can be found here: The animal study was reviewed and approved by Affiliate Hospital of Shandong University. Written informed consent was obtained from the owners for the participation of their animals in this study.ZX, GT, and HS designed and developed the experiments. HS and HX prepared the draft of the manuscript. HS, HX, GT, ZX, SG, BY and RW participated in all the experiments. HS, BY, RW and HX analyzed the data and drafted the manuscript.HX, GT, LZ, and ZX supervised all research and revised the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by National Natural Science Foundation of China (82174410); Natural Science Foundation of Shandong Province, ZR2020KH011 Natural Science Foundation of Shandong Province, ZR2020MH362.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Haloferax are interesting models, as a reliable genetic system and versatile microscopy methods are available. However, only one virus infecting the Haloferax species is currently available. In this study, we tested ~100 haloarchaeal virus isolates for their infectivity on 14 Haloferax strains. From this, we identified 10 virus isolates in total capable of infecting Haloferax strains, which represented myovirus or siphovirus morphotypes. Surprisingly, the only susceptible strain of all 14 tested was Haloferax gibbonsii LR2-5, which serves as an auspicious host for all of these 10 viruses. By applying comparative genomics, we shed light on factors determining the host range of haloarchaeal viruses on Haloferax. We anticipate our study to be a starting point in the study of haloarchaeal virus–host interactions.Viruses can infect members of all three domains of life. However, little is known about viruses infecting archaea and the mechanisms that determine their host interactions are poorly understood. Investigations of molecular mechanisms of viral infection rely on genetically accessible virus–host model systems. Euryarchaea belonging to the genus  Caudoviricetes [Sphaerolipoviridae) [Pleolipoviridae) [Halspiviridae) [Hafunaviridae is the largest family of archaeal tailed viruses and its myoviruses have a broad host range [Microbial viruses are widespread and able to infect members of all three domains of life, including archaea. Archaea are ubiquitous microorganisms that can be found in extreme environments, such as salt lakes as well as in mesophilic surroundings such as the oceans and the human body ,2. The siricetes ,10,11,12viridae) , pleomorviridae) , or spinviridae) . Archaeaviridae) . All isoviridae) . Howeverviridae) . The strviridae) . While tviridae) . Previoust range . Other hst range .Sulfolobales are currently the most common archaeal models with genetic systems available [Halorubrum and Haloarcula strains that are infected by a substantial number of known viruses [Haloferax has become increasingly popular in the archaeal scientific community, and it is the euryarchaeal model for which the most advanced tools for genetic engineering, imaging, and molecular biology are available [Haloferax tools entail a versatile genetic system for overexpression and genomic knock-out, the availability of several plasmids and different markers, and a CRISPR-based repression system to downregulate gene expression [Haloferax as a central euryarchaeal model, has led to a substantial increase in the understanding of its cell biology [Haloferax as a model, is of great added value in studies of viral infection mechanisms.The study of the virus–host relationships and infection mechanisms of archaeal viruses would greatly benefit from the availability of genetically accessible virus–host models, for which molecular biology tools are available. For crenarchaeal virus hosts, several members of the genus vailable ,22,23,24vailable ,26,27,28 viruses ,19,29. T viruses ,32,33,34vailable . The Halpression ,37,38,39pression . It is apression . The dev biology ,54,55,56Haloferax strains. Almost 30 years ago, the HF1 virus was reported to infect Haloferax lucentense and Hfx. volcanii, and a defective provirus of Haloferax mediterranei has been identified, both of which are no longer available  [Haloferax host, and it was recently isolated together with its host Haloferax gibbonsii LR2-5 from the hypersaline Lake Retba in Senegal [In contrast to some other haloarchaea, there are far less viruses known that infect ication) ,58. At t Senegal ,60.Haloferax for molecular studies, we aim to identify viruses that infect Haloferax strains. In this study, we used the largest available collection of isolated and characterized haloarchaeal virus isolates, and we tested the infectivity of 95 viruses on 14 Haloferax strains. The virus collection contains viruses from all current haloarchaeal virus families, and these viruses represent the majority of haloarchaeal virus isolates isolated to date. This endeavor resulted in an extended virus–host matrix for Haloferax from which we could identify a promising model that is the host to a substantial number of viruses, Haloferax gibbonsii LR2-5. In addition, we used comparative genomics to identify viral factors that allow for the infection of this Haloferax host. With this work, we pave the way to using Haloferax as a model in virus–host interactions.Due to the attractiveness of Haloferax strains  was diluted to obtain 18, 20, or 23% SW in the top-agar media, plates, and liquid media, respectively. MGM also contained 5 g of peptone (Oxoid), 1 g of Bacto yeast extract , and Bacto agar  per liter. Viruses were grown on their own host strain listed in All  strains , virus h strains , and vir strains  were groHaloferax strains , and for more complex motifs a manual search was carried out in Geneious v 8.1.9..The genomic sequences of the viruses were retrieved from NCBI . PhylogeThe phylogenetic analyses of the adhesin and tail fiber gene sequences were carried out as follows. For both datasets, sequences were aligned using MAFFT v. 7.450 , and a mHaloferax, a collection of 95 haloarchaeal viruses  or Retbasiphovirus   . It is idetected .Haloferax host, which is an indication of the efficiency of infection. As a summary, we did not find any true virus–host pairs on any of the Haloferax strains, except on strain Haloferax gibbonsii LR2-5, which was isolated a few years ago from Lake Retba in Senegal at the same time as HFTV1 [Halorubrum or Haloarcula as their own host and their titers varied between 103 and 1010 PFU/mL on LR2-5  system . These aOne strategy of viruses to escape host recognition is the avoidance of certain motifs in their genomes, which are the targets for the RM systems . In lineMethylated motifs in the genome of LR2-5 were previously predicted . We searHafunaviridae family members showed the same genome organization within their own group   . There wviruses) . Many ofCaudoviricetes [Another highly variable region in the viral genomes is located around genes encoding the viral tail fiber and adhesin . Adhesiniricetes . Long tairicetes ,80. Moreiricetes . All haliricetes . HFTV1 iHaloarcula, Halobacterium, Halobellus, Halorubrum, and Haloterrigena strains [A tree of the hafunaviruses  tail fiber adhesin proteins showed that they are divided in four clades , as also strains . We aimeWe investigated the hypothetical relationship between the susceptibility of the LR2-5 strain to infection and diversification of the hafunavirus tail fiber and adhesin genes by comparing the number of Markov jumps  through the adhesin and the tail fiber gene evolution . A “nullHfx. gibbonsii LR2-5. However, it should be emphasized that our dataset was limited to Haloferax hosts. Such a correlation between host range and adhesin type might only become apparent when a more diverse set of hosts is used, as the Haloferax strains are generally not very susceptible for viral infection. However, we did find a few case examples, which seems to pinpoint the adhesin as a host determining factor. First, HRTV-26, one of the few haloferacalesviruses that infects LR2-5, encodes a Clade 1 adhesin, similar to most mincapviruses. Since the adhesins of haloferacalesviruses that are closely related to HRTV-26 all belong to Clade 3, it is likely that HRTV-26 picked up an adhesin gene via horizontal gene transfer from one of the Clade 1 viruses. This might be the explanation for why HRTV-26, even though it is an haloferacalesvirus, is still capable of infecting LR2-5.Based on this analysis, we did not observe any clear correlation between the type of adhesin or tail fiber gene and the viral ability to infect Second, viruses encoding a Clade 3 adhesin do not infect LR2-5, with the exception of HRTV-10. To gain more insight into the HRTV-10 tail fiber adhesin, a multiple sequence alignment was performed. We identified one mutated codon resulting in an amino acid change (T380A) in a conserved motif at the 3′end of the adhesin gene . This suHaloferax strains, as the most advanced molecular and genetic tools are available for these organisms. Due to the low number of known viruses that infect Haloferax, we set up a large-scale assay to identify viruses capable of infecting Haloferax strains. Our extensive screening of approximately 100 isolated and mostly characterized haloarchaeal viruses showed that only a small subset was capable of infecting Haloferax. Specifically, Hfx. gibbonsii LR2-5 showed itself as an auspicious host, as we identified 10 virus isolates that can infect this host and all other hosts could not be infected. Previously, we studied potential factors that make Hfx. gibbonsii LR2-5 (DSM No. 112399) so much more receptive to virus infection in contrast to related strains. For its virus susceptibility, several possible factors were identified as potential explanations. The most prominent was an absence of CRISPR-cas virus defense systems in LR2-5 [Hfx. gibbonsii LR2-5 has a different RM system from some other Haloferax strains, and the cell surface might show a different composition than that of other Haloferax strains because LR2-5 encodes divergent surface proteins that might serve as viral receptors, such as pili and S-layer proteins [We aimed to develop an euryarchaeal virus–host model system with attractive molecular, genetic, and imaging tools to dive deeper into virus–host relationships in archaea. We focused on in LR2-5 . In addiproteins . These lproteins ,82.Hfx. gibbonsii LR2-5 and transferred to a lysogenic cycle. Consequently, these types of interactions would not show clear plaques, and they could have been missed by our approach. However, further experimental research on the host range implications of these proteins is needed.Because we used plaque assays for the host range determination, we cannot distinguish at which step of the infection cycle viruses are not successful in infecting a host: entry, replication, or release. However, the results of the present study might indicate that adhesins, which are required for entry, are an important factor determining host range specificity, whereas other factors such as the viral egress proteins and restriction–modification systems might also play a role. Moreover, it should be noted that one limitation of the approach utilized is the assumption that the successful infection of the host results in plaque formation. It could be that some of the viruses studied here might have integrated their genome when infecting Hfx. gibbonsii LR2-5 as the only virus susceptible Haloferax strain. This host can be infected by 10 different viruses with sequenced genomes [Hfx. gibbonsii LR2-5. We conclude that this species is an extremely promising model host to study virus–host interactions in haloarchaea, and we anticipate that this work serves as a stepping stone for future in-depth molecular characterization of archaeal viral infection mechanisms.Our analysis has identified  genomes , and it  genomes . We are


Trichoderma harzianum (TH) and an organic fertilizer (OF) was investigated in two consecutive tomato crops for its effect on the root-knot nematode Meloidogyne incognita and plant growth and yield. The application of 1,3-D was only performed on the first crop, while TH and OF were provided to both crops. Almost all treatments significantly reduced nematode infestation in both crops, though the greatest nematicidal effect was caused by a combination of the three products. The treatment with 1,3-D limited its nematicidal efficacy to the first crop only. Fumigant integration with TH and OF also resulted in the greatest increases of plant growth and yield. Therefore, the integrated management of root-knot nematodes with a soil fumigant, a bionematicide as T. harzianum and a source of organic matter demonstrated effective nematode suppression though limiting the number of chemical applications.Environmental concerns raised by synthetic nematicides are encouraging integrated management strategies based on their combination with non-chemical control tools, such as biocontrol agents and/or organic amendments. In this study, the combination of the fumigant 1,3-dichloropropene  with a commercial formulation of the biocontrol agent  Meloidogyne, are the cause of huge yield losses on a large spectrum of horticultural and ornamental crops in all tropical and subtropical regions of the world [Phytoparasitic nematodes, and particularly the root-knot species of genus he world . Crop lohe world ,3. Moreohe world ,5,6.Control of phytoparasitic nematodes has been traditionally based on soil treatments with synthetic nematicides, mainly fumigants, but concerns for their environmental and health effects have led to a complete ban or restrictions on the use of most nematicides while also adhering to the European Community Regulations ,8. AmongTrichoderma are among the most promising biocontrol agents, in addition to mycorrhizal and endophytic fungi [Trichoderma spp. has been recently proved by both semi-field studies [Trichoderma fungi can enhance plant resistance against nematode attacks through the activation of hormone-mediated defence mechanisms [Trichoderma species were also acknowledged as plant biostimulants, due to the larger availability of water and nutrients related to modified root morphology and rhizosphere interactions [Trichoderma varies among the fungal strains and is strongly affected by soil physicochemical and biological properties. In particular, a key role is played by the soil content of organic matter (OM) that ensures a feeding substrate for Trichoderma development also in the absence of host plants [Trichoderma [The need of nematode control tools as an alternative to chemicals has strongly encouraged research on the use of biocontrol agents, generally approved by public opinion and encouraged by EU policy. Fungi of genus ic fungi . The nem studies ,14 and f studies ,16,17. Ichanisms , the synchanisms ,21,22,23ractions . The nemt plants , demonstchoderma ,27.Trichoderma spp. to soil treatments with fumigants and their rapid recolonization of soil after chemical treatment, mainly due to the biological vacuum caused by fumigation and the consequent absence of Trichoderma predators [Trichoderma after the treatment with a fumigant nematicide could ensure a rapid and permanent establishment of the fungus in the soil, thus, enhancing its nematicidal effectiveness. According to this hypothesis, we investigated the nematicidal performance of combined treatments with the fumigant 1,3-D and a formulate of Trichoderma harzianum (TH) in two consecutive tomato crops, either with or without the addition of an organic fertilizer (OF) as a source of OM, against the root-knot nematode Meloidogyne incognita.Interestingly, literature data indicated the high resistance of redators ,29. TherM. incognita ranged from 2.1 to 2.6 J2 mL−1 soil, without statistical difference among the experimental plots (data not shown). At the end of the first tomato crop, all the treatments significantly reduced soil nematode population and root gall infestation index, compared to the non-treated control (NT)  . SuppresYield of the first tomato crop was significantly increased by all the tested treatments, though the yield increase provided by OF and TH was statistically greater only when used in combination or with 1,3-D .In the second tomato crop, tomato yield was always almost double that in the first cycle. As in the first cycle, all the treatments provided a significantly higher yield performance, compared to the non-treated control, though the highest yield values were recorded for the combined application of 1,3-D and TH and, to a lesser extent, of OF and TH.Compared to NT, all the treatments applied in the first crop resulted in a significantly heavier plant biomass and chlorophyll content, though not affecting the root system .Conversely, the growth of plants from the second crop was only significantly increased in the plots previously treated with 1,3-D in combination with OF and/or TH. Significantly larger root systems and higher chlorophyll contents were also recorded for the combined application of TH and OF.M. incognita was able to reproduce on tomato Shiren F1 in both crop cycles, in spite of the generally stated resistance of this hybrid to RKN. This can be explained by the virulence of the nematode population present in the greenhouse and its ability to break the tomato resistance, as confirmed by the susceptibility of Shiren F1 to RKN observed in the previous tomato crops of the surrounding areas. A higher nematode reproduction and gall formation on tomato roots was observed in the second crop cycle, as a consequence of the higher soil temperatures occurring in spring–summer conditions.The RKN T. harzianum, and an organic fertilizer can be an effective strategy for root-knot nematode management by enhancing nematicidal effect and yield performance of each of its components and extending the nematicidal effect of the fumigant to a second crop cycle. Our findings agree with recent studies [T. harzianum, B. subtilis) and OFs following 1,3-D fumigation, such as significant reductions of soilborne phytopathogens , positive changes of soil physicochemical properties, a larger abundance of beneficial microorganisms and an increased crop yield and quality.The increasing attention to environmental safety and human health has led to a dramatic reduction in synthetic pesticides, in favour of more environmentally friendly alternatives, such as the integrated control strategy positively tested in this study. Data presented here indicate that the combined use of soil fumigation with a biocontrol agent, such as  studies ,31 documSoil fumigation with 1,3-D has demonstrated to be highly effective for controlling root-knot nematode infestation and increasing yield in the first tomato crop, as also previously documented by many other studies either on tomato or other horticultural crops ,10,11. IM. javanica, root galling was reduced and top fresh weight increased following soil pre-treatment with peat-bran preparations of T. harzianum [M. javanica infestation on green gram (Vigna radiata L.), maximum reductions in the number of nematode galls and eggs were observed for T. viride and T. harzianum while the lowest suppression was recorded for T. pseudokoningii and T. atroviride [M. incognita on potted tomato following soil application of indigenous isolates of T. viride. Moreover, field assessment of the antagonistic potential of seventeen isolates of Trichoderma spp. against M. incognita on tomato indicated a strong inhibition of nematode reproduction and root galling suppression by T. asperellum T-16 and T. brevicompactum T-3 [T. longibrachiatum and also on the cereal cyst nematode Heterodera avenae.The genus Trichoderma includes several species of free-living soil-borne fungi commonly present in roots, often reported as effective against phytoparasitic nematodes and phytopathogenic fungi . Literatarzianum . In a reroviride . Analogoroviride  documentctum T-3 . In addictum T-3  proved pMeloidogyne spp. eggs and J2 by T. harzianum was attributed to the secretion of several hydrolytic enzymes  able to degrade the nematode cell wall [H. avenae by T. longibrachiatum [T. harzianum to an increased production of enzymes, such as phenylalanine ammonia-lyase and peroxidase, known to be involved in systemic resistance. In addition, the data of Martinez Medina et al. [M. incognita in tomato roots. Competition for space and nutrients can be also an important mechanism of the phytonematode suppression by Trichoderma species, due to their high and rapid growth capacity [Trichoderma spp. were also reported for producing volatile organic metabolites tested as toxic to different nematode species such as Panagrellus redivivus, Caenorhabditis elegans and Bursaphelenchus xylophilus [Several mechanisms were documented for the nematicidal activity of Trichoderma species, such as direct parasitism, antibiosis, food competition, induction of plant resistance, and enzymatic hydrolysis ,24,37,38ell wall ,40. Analachiatum . Singh eachiatum  related a et al.  indicatecapacity . In addilophilus .M. incognita infestation and its pressure on tomato plants. Plant growth stimulation by Trichoderma spp. can be attributed to various mechanisms, such as an enhanced nutrient uptake, a larger solubilization of phosphates and sequestration of inorganic nutrients [T. atroviride or T. longibrachiatum favoured fungal adhesion to tomato roots and nematicidal efficacy of treatments [Sclerotinia sclerotiorum and Chalara thielavioides were strongly suppressed by soil treatments with T. viride carried on granulated waste fruit pomace [Most of treatments with TH included in this study resulted also in a significant enhancement of growth and yield parameters of both tomato crops compared to the non-treated control. However, both tomato yield performance and plant growth were reasonably influenced by seasonal factors, as the higher soil temperatures can have contributed to the larger tomato yield occurred in the second crop yield. Adversely, the higher temperatures in the spring summer crop could have negatively affected the plant growth, favouring the utrients ,46. Howeeatments . Moreovet pomace .M. incognita in both tomato crops, while the effects on crop yield were limited only to the first tomato cycle. A previous study of Amir-Ahmadi et al. [M. javanica and stimulation of kidney bean plant growth by T. harzianum in soils amended with higher amounts of OM. More generally, organic materials, such as composts, were indicated as suitable carrier medium for T. harzianum, as stimulating nitrogen mineralization, soil enzyme activity, and fungal growth [This study also indicated that the combination of TH and OF improved the suppressiveness to i et al.  reportedl growth ,50.This study confirmed that the addition of a OM source plays a key role for a successful application of biocontrol agents as Trichoderma. According to their composition, OM sources can directly release nematicidal compounds, increase plant tolerance and resistance and increase soil populations of antagonistic microorganisms ,27,51. MBoth autumn and spring tomato cycles were carried out in an unheated greenhouse with a soil of medium texture and a slightly basic reaction (pH 7.2), located at Vitulazio . The timetable of all cultural practices, as well as of experimental treatments, is reported in M. incognita, with a 7.5 average root gall infestation index [2 (6 × 1.6 m) plots, aligned along four 1 m spaced parallel rows, according to a randomized complete block design with four replicates per each of the eight treatments.Tomato crop of the previous spring–summer cycle was heavily and uniformly infested by the root-knot nematode on index  at the h®) of the fumigant nematicide 1,3-D, alone or combined with a commercial pellet OF formulation, Stalfert N5® , as a source of organic matter (OM) and/or a commercial formulate of TH, strain ITEM 908 (Auget®). The remaining plots were treated only with OF and TH, either alone or in combination, while four nontreated plots were used as a control (NT). The 1,3-D was applied by drip irrigation at the rate of 180 L ha−1, while TH was manually distributed at a 1 kg ha−1 rate two days before tomato transplant and again 30 and 60 days after tomato transplant at 0.5 kg ha−1. OF  was distributed on the plot surface at a 2.5 T ha−1 dose and then incorporated into the soil one week before transplanting. All treatments barring soil fumigation with 1,3-D were also repeated in the spring tomato crop, at the same rates and in the same plots of the first cycle.In the first tomato crop, half of experimental plots were treated with a commercial formulation . This hybrid had repeatedly proven to be susceptible to ® CHL PLUS chlorophyll meter combined with atLEAFSoft 1.0 software .Tomato fruits were harvested four and five times in the first and second crop, respectively. At each harvest, fruit weight was recorded on the ten central plants from each plot. At the final harvest, chlorophyll content (SPAD index), plant top and root weight and Zeck’s root gall infestation index  were evaM. incognita was determined on a 10-subsample composite soil sample collected from each plot before the treatments and at harvest, respectively, by extracting nematode juveniles, either already present in soil or emerged from nematode eggs, with the nematode cotton wool filter method [In both crops, initial and final soil population density of r method . During p ≤ 0.05). All the statistical analyses were performed using the software PlotIT 3.2 .All data were statistically analysed by the analysis of variance (ANOVA) and means compared by the least significant difference test (T. harzianum can be an effective model of integrated management of root-knot nematodes. Beyond the well-known biocidal effects of fumigants, the strength of this IPM model also relies on the direct nematicidal activity of the two additional non-chemical components, as well as on their side effects on plant resistance, root system development, nutrient adsorption and interaction among rhizosphere components. As it is not systemic in plants, 1,3-D has low adverse impacts on pollinator insects and has risks to earthworms that are limited to the first growing season after the fumigation treatments [The combination of soil fumigation with a source of organic matter and a bionematicide as eatments ,57. ThouAs resistance to fumigation treatments can largely vary among different Trichoderma species and strains ,29, the


To determine the incidence of fundus hemorrhage (FH) not associated with retinopathy of prematurity (ROP) during ocular screening and report their clinical features, risk factors, therapies, and prognosis in a large population of premature newborns.The medical records of all preterm newborns admitted to the Department of Ophthalmology, Peking University People's Hospital, Beijing, from January 1, 2016 through August 31, 2021 were retrospectively reviewed. Fundus examinations were carried out by experienced retinal experts. Examination under anesthesia was carried out in newborns with abnormal fundus including vitreous hemorrhage (VH) or retinal hemorrhage (RH) >2 disks' diameter by a Retcam 2 system. A lens-preserving vitrectomy was performed in infants requiring a vitrectomy. A comprehensive medical history was also recorded and analyzed.During the 5-year period, a total of 7,260 preterm babies were screened. There were 82 (1.13%) newborns and 104 (0.72) eyes with FH, including VH or RH.P = 0.22).Twelve (14.63%) newborns  had VH; 56 (68.29%)  had flame-shaped, superficial hemorrhages; six (7.31%)  had small, round, deep hemorrhages (<2 disk diameters in size); and eight (9.76%)  had large, round hemorrhages (>2 disk diameters). In all, there were 10 (12.20%) cases of intracranial hemorrhage. The mode of delivery was not found to be a significant factor in the occurrence of birth-related retinal hemorrhage (Six newborns (eyes) with large, round retinal hemorrhage at the posterior pole while the macular was not impacted and 11 cases (15 eyes) with vitreous hemorrhage were required to receive close follow-up with average follow-up time of 105 days. A lens-sparing vitreous surgery was conducted in three patients without any complications.Preterm newborns with FH that are not caused by ROP are more likely to have superficial, peripheral hemorrhages. Vaginal delivery compression and forceps may be associated with hemorrhage. A lens-preserving vitrectomy is required and considered safe for dense FH involving the refractive media. Fundus hemorrhage (FH) is an ocular abnormality and one of the important neonatal ocular complications in infants, with an incidence rate ranging from 2.6 to 50.0% , 2. TherIn premature newborns, FH in ROP patients have been extensively investigated –8. HowevConsidering the possible visual impairment secondary to FH in newborns, early detection and intervention in newborns with FH are critical; however, there are significant challenges involved. Screening all newborns for FH in China is not only difficult to perform but also less efficient, given the current scenario that China is still a developing country , 10. ForIn this study, we analyzed non-ROP-related FH in all premature newborns during ocular screening at our hospital from 2016 to 2021, and described their clinical features, risk factors, therapies, and prognosis.In this retrospective study, the medical records of all preterm newborns admitted to the Department of Ophthalmology, Peking University People's Hospital, Beijing, from January 1, 2016 through August 31, 2021 were reviewed. Consent was obtained from the parents or legal guardians of all newborn patients for inclusion in the study. The study was approved by the ethics committee of Peking University People's Hospital. All newborn patients were referrals from pediatricians or obstetricians in Beijing in accordance to the screening guidelines for ROP in China .Every newborn patient underwent a systemic ocular examination. Outpatient screening was carried out first. The ophthalmologic examinations, which included assessment of the anterior and posterior segments and a red light reflex test were carried out by experienced retinal experts. Fundus examination was made after pupil dilation with an indirect fundoscopy. All the ophthalmologic examinations were recorded carefully. A comprehensive medical history including birth weight, gestational weeks at birth, mode of delivery, history of oxygen inspiration, and the length until follow-up was reviewed. We also reviewed the newborn's health records to see whether there were any complications at birth such as anemia or intracranial hemorrhage. The mother's perinatal complications were also recorded and analyzed.A specially designed questionnaire recorded all the above mentioned information for all newborns as well as perinatal information for the mothers, and we reviewed the questionnaires for all newborns and collected the results. We performed a detailed retrospective chart review. Based on this, all preterm newborns found to have FH were reviewed. The eligibility criteria were infants whose gestational weeks at birth was <37 weeks and FH not associated with ROP. Newborns with ROP-related fundus changes were excluded. Fundus hemorrhage secondary to additional diseases such as retinal vasculitis and retinoblastoma were also excluded.For newborns with superficial and peripheral RH, further fundus examination was not required; the parents were instead asked to monitor their child's vision development. For other newborns, close ophthalmologic follow-up was required until the RH was totally absorbed. Examination under anesthesia (EUA) was carried out in newborns with abnormal fundus including VH and RH >2 disks' diameter by a Retcam 2 system . In three patients, we performed a lens-preserving vitrectomy to avoid further complications due to the presence of dense posterior pole FH as soon as the systemic condition was stabilized, as previously described . For patP < 0.05.Data was analyzed by SPSS software (version 12.0). Statistical comparisons between the newborns with or without fundus hemorrhage were carried out with a chi-square test. The difference between different mode of delivery were carried out with a ANOVA analysis. The statistical significance level was set at During the 5-year follow-up period, a total of 7,260 preterm babies were screened. Forty-eight of the tested newborns had VH or RH secondary to active ROP and were thus excluded. Among 82 (1.13%) newborns, 104 (0.72) eyes showed FH (VH or RH), 22 (26.83%) of which were bilateral. According to the morphological characteristics of FH, 12 (14.63%) newborns  had VH. Vitreous hemorrhage was not dense but dispersed in the vitreous cavity in all newborns but one (1 eye). Among the 70 (85.37%) newborns  with RH, 56 (68.29%)  had flame-shaped, superficial hemorrhages; six (7.31%)  had small, round, deep hemorrhages (<2 disks' diameters in size) at the posterior pole; and eight (9.76%)  had large, round hemorrhages (>2 disks' diameters) located in the posterior pole .In all, 47 (57.32%) of neonates with FH had a history of oxygen supplementation, with an average length of 6.03 ± 1.42 days, and 35 (42.68%) neonates had no history of oxygen supplementation. The perinatal history showed that 10 (12.20%) patients had intracranial hemorrhage, 2 (2.44%) had congenital heart disease, 3 (3.65%) had anemia, 3 (3.65%) had hypoxic-ischemic encephalopathy, 3 (3.65%) had hyperbilirubinemia, and 3 (3.65%) experienced intrauterine fetal anoxia. The mode of delivery for all newborns studied is shown in For different newborns, different management options were selected depending on the morphology and location of FH fundus as stated in the methods. We believed that frequent fundus inspections were not necessary for 56 newborns (74 eyes) with superficial RH, and 6 (6 eyes) with small, round RH; instead, the parents were asked to monitor the child's visual growth. Close follow-up of newborns with less dense FH, including 6 newborns (6 eyes) with large, round RH at the posterior pole (non-impacted macula) and 11 cases (15 eyes) with VH was necessary until the hemorrhage was entirely absorbed. The average time between follow-ups was 105 ± 45.7 days, with intervals ranging from 9 days to 1.5 years. A lens-sparing vitreous surgery was conducted in two cases where the macula was impacted by dense hemorrhage and in one case where the macula was affected by dense vitreous hemorrhage . PatientP = 0.74)  .In this paper, we reported FH not associated with ROP in a large population. Most cases of FH were superficial RH that did not need further intervention. Although there was no significant association between the mode of delivery and FH, the rates of vaginal delivery and forceps use were greater in the among newborns with FH.As previously indicated, birth-related RH occur at a rate of 2.6–50%, depending on factors such as inspection time and examination methods. In our investigation, the rate was 1.13%. The intensity of the hemorrhages varied greatly with diverse morphologies of hemorrhage. They are mostly located in the posterior pole and are usually intraretinal, but can also be subretinal or preretinal. Retinal hemorrhages are “typically a mixture of splinter-shaped, flame-shaped, dot and blot hemorrhage, with uncommon sub retinal and preretinal hemorrhage,” per Kaur and Taylor . SimilarP > 0.05), vaginal delivery and forceps use was associated with a higher rate of FH than cesarean delivery. Furthermore, we found a higher incidence of intracranial hemorrhage than other complications, which suggested that compression of the eye and brain during delivery is simultaneous. No neonates in this study were delivered with a vacuum based on the following facts: (i) Most obstetricians are aware of the side effects of using vacuum pumps, such as hemorrhage; and (ii) The forceps technique is widely used, technically mature, and with few complications in most hospitals in Beijing.When we analyzed these risk factors for FH in newborns, we discovered that none of the neonates in our study exhibited systemic coagulation dysfunction, vitamin K insufficiency, or shaken baby syndrome, as previously reported in the literature as possible causes. Based on our findings, a higher percentage of children with FH were born vaginally, particularly those with forceps-assisted delivery, leading us to believe that the etiology of FH in these children was linked to the mode of delivery. Williams et al., for example, discovered a link between the severity of hemorrhage and the mode of delivery, with consecutive vacuum and forceps-assisted deliveries posing the greatest risk . Whitby Close monitoring and observation should be considered for most neonates with FH, because the hemorrhage is usually localized in the peripheral retina, is mostly superficial, and can be quickly absorbed with little effect on the refractive medium. Regardless of the cause of fundus hemorrhage in newborns, there are usually no sequelae. In general, published literature in consistent that birth-related RH clear up quickly . EmersonThe timing of intervention is mainly determined by the density and duration of the media opacification. For dense FH, vitrectomy is both essential and safe. Antonio Capone et al.  documentOur study has some limitations. Screening in this study was only performed in preterm newborns, not full-term infants, owing to the constraints of the screening circumstances. Therefore, it may not be fully representative of the full-term infant population. However, our conclusions can still be extrapolated to all newborns. Furthermore, because this was a retrospective study, the follow-up duration was insufficient. Hence, to track changes in children who have undergone vitrectomy, a longer follow-up period is required in the future.In conclusion, preterm newborns with FH that are not caused by ROP are more likely to have superficial, peripheral hemorrhages, with birth-related factors being the leading cause. Vaginal delivery compression and forceps use rate were higher in newborns with RH A lens-preserving vitrectomy is required and considered safe for dense FH involving the refractive media. In future, fundus screening may also be required for full-term newborns with similar risk factors.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the Program of Development and Cultivation of Medical Innovative Varieties and Industrial Support, Beijing Municipal Science and Technology Commission (Grant Number Z191100007619041), and Beijing Residency Training Quality Improvement Project (No. Zhupei2021043).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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