ObjectiveTo investigate the mechanism by which Liuwei Dihuang Erzhiwan combination inhibit the lung metastasis of spontaneous breast cancer in mice by regulating the recruitment of myeloid-derived suppressor cells （MDSCs）. MethodThree hundred and eighty SPF-grade 10-month-old female breeders of Kunming mouse were palpated at the mammary gland site once every 3 days. Mice that have not had a lump touched after being raised for 6 months are used as control group. After tumor development， the mice were randomized into model， positive control （paclitaxel， intraperitoneal injection at 0.01 g·kg^-1 every other day for 22 d）， Liuwei Dihuangwan （0.65 g·kg^-1·d^-1 by gavage）， Erzhiwan （5.41 g·kg^-1·d^-1 by gavage）， and Liuwei Dihuang Erzhiwan combination （6.05 g·kg^-1·d^-1 by gavage） groups. The mice were euthanised when the tumor reached a diameter of about 15 mm， and the tumor and lung tissues were collected. The survival time， tumor mass， and lung metastasis rate of tumor-bearing mice were recorded. Hematoxylin-eosin （HE） staining was used to observe the histopathological and morphological changes of mouse tumor and lung tissues. Immunofluorescence （IF） was used to detect the distribution of MDSCs in tissues of mice in each group by double-staining of MDSCs cells with lymphocyte antigen 6 complex site G6D （Ly6G） and CD11 antigen-like family member B （CD11b）. Western blot was employed to determine the protein levels of matrix metalloproteinase-9 （MMP-9）， transforming growth factor-β （TGF-β）， zinc finger transcription factor 1 （Snail1）， and E-cadherin in the tumor tissue and CC motif chemokine 9 （CCL9） and CC motif chemokine receptor 1 （CCR1） in the lung tissue. ResultDuring the modelling period， the paclitaxel group and Chinese medicine intervention groups had longer median number of days of survival and lower tumor weight， lung metastasis rate， and lung nodule than the model group （P<0.05， P<0.01）. HE staining showed an increase in tumor cell necrosis in the paclitaxel group and the Liuwei Dihuang Erzhiwan combination group. The paclitaxel group and Chinese medicine intervention groups had lower fluorescence intensity of MDSCs in the tumor tissue than the model group （P<0.05， P<0.01）. Compared with the normal control group， the model group showed increased fluorescence intensity of MDSCs in the metastatic lung tissue （P<0.01）， which， however， was decreased in the paclitaxel group and Chinese medicine intervention groups （P<0.01）. The model group showed higher protein levels of MMP-9， TGF-β， and Snail1 and lower protein level of E-cadherin in the tumor tissue than in the normal control group （P<0.01）. Compared with model group， paclitaxel and Chinese medicine interventions downregulated the protein levels of MMP-9， TGF-β， and Snail1 （P<0.05， P<0.01） and upregulated the protein level of E-cadherin in the tumor tissue （P<0.01）. Moreover， the Liuwei Dihuang Erzhiwan combination group had lower protein levels of TGF-β and Snail1 than the Liuwei Dihuangwan group and Erzhiwan group （P<0.05）. In the metastatic lung tissue， the expression of CCL9 and CCR1 was higher in the model group than in the normal control group， paclitaxel group， and Chinese medicine intervention groups （P<0.05， P<0.01）. ConclusionLiuwei Dihuang Erzhiwan combination inhibit tumor growth， prolong survival time， and reduce the occurrence of lung metastasis in the mouse model of spontaneous breast cancer by reducing the recruitment of MDSCs in the tumor and lung tissues and modulating the phenotypes of epithelial-mesenchymal transition （EMT）-related molecules and the expression of CCL9/CCR1.

Objective:To investigate the fundus fluorescein angiography (FFA) characteristics of spontaneous regression in retinopathy of prematurity (ROP) and the range of retinal vascularization.Methods:A clinical retrospective study. A total of 82 eyes of 41 infants with ROP, who underwent FFA from January 2019 to December 2021 in Department of Ophthalmology of Xijing Hospital after completion of ROP regression, were included. There were 25 males (50 eyes) and 16 females (32 eyes). ROP was diagnosed in Zone Ⅱ in 44 eyes, with 38 eyes in stage 2 and 6 eyes in stage 3, and in zone Ⅲ in 38 eyes of stage 2. All patients underwent FFA examination under general anesthesia, at postmenstrual age of 70.70±12.25 weeks, after the natural regression of ROP was completed. Focus on the retinal vascular development, as well as choroid circulation and macular abnormalities, and compare and observe the differences between zone Ⅱ and Ⅲ after spontaneous regression. The extent of retinal vascularization was determined by the ratio between the distance of the center of the disc to the border of the vascularized zone (DB) and the center of the disc to the fovea distance (DF). The width of avascular area, recorded as the distance from the ora serrata to the vascular termination, was counted by disc diameters (DD). The measurement data between zone Ⅱ and zone Ⅲ ROP were compared by the independent sample t-test, and the count data were compared by χ2 test or Fisher exact probability test. Results:The linear choroidal pattern was present in 9 eyes (21.95%, 9/41), and the tortuous arteries in the posterior retina were detected in 32 eyes (39.02%, 32/82). It was noted that increased branching of vessels presented in 45 eyes (54.88%, 45/82), straight shape of vessels shown in 27 eyes (32.93%, 27/82), circumferential vessels arisen in 45 eyes (54.88%, 45/82), arteriovenous shunt appeared in 18 eyes (21.95%, 18/82), and capillary bed lost in 46 eyes (56.10%, 46/82) in areas from initial ridge to vascular termini. Punctate or linear dye leakage was observed in 23 eyes (28.05%, 23/82) during the late phase of FFA. Macular abnormalities, such as the absence of foveal avascular zone and hypoperfusion, were observed in 28 eyes (34.15%, 28/82), of which the macular ectopia presented in 1 eye. The mean DB/DF ratio of all the 82 eyes on the temporal side was 4.63±0.29 and 3.77±0.23 in the nasal. The mean avascular area on the temporal retina was 1.74±1.00 DD. Compared with ROP in zone Ⅲ, increased branching of vessels and dye leakage were more common ( χ2=9.303, 10.774; P=0.002, 0.001), the extent of temporal retinal vascularization was smaller ( t=-2.285, P=0.026), and the avascular area of the retina was more significant ( t=5.491, P<0.001) in zone Ⅱ ROP. Conclusions:Even after completion of spontaneous regression in ROP, incomplete retinal vascularization and vascular abnormalities may exist permanently in FFA, including those such as the tortuous arteries in the posterior retina, increased branching and straight shape of vessels, circumferential vessels, capillary bed lost and macular abnormality. Further appropriate follow-up is needed for long-term safety.