BACKGROUND:Carnosic acid,a bioactive compound found in rosemary,has been shown to reduce inflammation and reactive oxygen species(ROS).However,its mechanism of action in osteoclast differentiation remains unclear. OBJECTIVE:To investigate the effects of carnosic acid on osteoclast activation,ROS production,and mitochondrial function. METHODS:Primary bone marrow-derived macrophages from mice were extracted and cultured in vitro.Different concentrations of carnosic acid(0,10,15,20,25 and 30 μmol/L)were tested for their effects on bone marrow-derived macrophage proliferation and toxicity using the cell counting kit-8 cell viability assay to determine a safe concentration.Bone marrow-derived macrophages were cultured in graded concentrations and induced by receptor activator of nuclear factor-κB ligand for osteoclast differentiation for 5-7 days.The effects of carnosic acid on osteoclast differentiation and function were then observed through tartrate-resistant acid phosphatase staining,F-actin staining,H2DCFDA probe and mitochondrial ROS,and Mito-Tracker fluorescence detection.Western blot and RT-PCR assays were subsequently conducted to examine the effects of carnosic acid on the upstream and downstream proteins of the receptor activator of nuclear factor-κB ligand-induced MAPK signaling pathway. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining and F-actin staining showed that carnosic acid dose-dependently inhibited in vitro osteoclast differentiation and actin ring formation in the cell cytoskeleton,with the highest inhibitory effect observed in the high concentration group(30 μmol/L).Carnosic acid exhibited the most significant inhibitory effect during the early stages(days 1-3)of osteoclast differentiation compared to other intervention periods.Fluorescence imaging using the H2DCFDA probe,mitochondrial ROS,and Mito-Tracker demonstrated that carnosic acid inhibited cellular and mitochondrial ROS production while reducing mitochondrial membrane potential,thereby influencing mitochondrial function.The results of western blot and RT-PCR revealed that carnosic acid could suppress the expression of NFATc1,CTSK,MMP9,and C-fos proteins associated with osteoclast differentiation,and downregulate the expression of NFATc1,Atp6vod2,ACP5,CTSK,and C-fos genes related to osteoclast differentiation.Furthermore,carnosic acid enhanced the expression of antioxidant enzyme proteins and reduced the generation of ROS during the process of osteoclast differentiation.Overall,carnosic acid exerts its inhibitory effects on osteoclast differentiation by inhibiting the phosphorylation modification of the P38/ERK/JNK protein and activating the MAPK signaling pathway in bone marrow-derived macrophages.

ObjectiveTo investigate the effect of Danggui Shaoyaosan on ferroptosis in the rat model of non-alcoholic fatty liver disease （NAFLD） and explore the underlying mechanism based on the nuclear factor E₂-related factor 2 （Nrf2）/solute carrier family 7 member 11 （SLC7A11）/glutathione peroxidase 4 （GPX4） signaling pathway. MethodsThe sixty SD rats were randomly grouped as follows： control， model， Yishanfu （0.144 g·kg^-1）， and low-， medium-， and high-dose （2.44， 4.88， and 9.76 g·kg^-1， respectively） Danggui Shaoyaosan. A high-fat diet was used to establish the rat model of NAFLD. After 12 weeks of modeling， rats were treated with corresponding agents for 4 weeks. Then， the body weight and liver weight were measured， and the liver index was calculated. At the same time， serum and liver samples were collected. The levels or activities of total cholesterol （TC）， triglycerides （TG）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， and Fe²⁺ in the serum and TC， TG， free fatty acids （FFA）， malondialdehyde （MDA）， superoxide dismutase （SOD）， glutathione peroxidase （GPX）， and Fe²⁺ in the liver were measured. Hematoxylin-eosin staining and oil red O staining were employed to observe the pathological changes in the liver. Immunofluorescence was used to assess the reactive oxygen species （ROS） content in the liver. Mitochondrial morphology was observed by transmission electron microscopy. The protein levels of Nrf2， SLC7A11， GPX4， transferrin receptor 1 （TFR1）， and divalent metal transporter 1 （DMT1） in the liver were determined by Western blot. ResultsCompared with the control group， the model group showed increases in the body weight， liver weight， liver index， levels or activities of TC， TG， ALT， AST， and Fe²⁺ in the serum， levels of TC， TG， FFA， MDA， Fe²⁺， and ROS in the liver， and protein levels of TFR1 and DMT1 in the liver （P<0.01）， and decreases in the activities of SOD， GPX and the protein levels of Nrf2， SLC7A11， and GPX4 in the liver （P<0.05， P<0.01）. Meanwhile， the liver tissue in the model group presented steatosis， iron deposition， mitochondrial shrinkage， and blurred or swollen mitochondrial cristae. Compared with the model group， all doses of Danggui Shaoyaosan reduced the body weight， liver weight， liver index， levels or activities of TC， TG， ALT， AST， and Fe²⁺ in the serum， levels of TC， TG， FFA， MDA， Fe²⁺， and ROS in the liver， and protein levels of TFR1 and DMT1 in the liver （P<0.01）， while increasing the activities of SOD and GPX and the protein levels of Nrf2， SLC7A11， and GPX4 in the liver （P<0.01）. Furthermore， Danggui Shaoyaosan alleviated steatosis， iron deposition， and mitochondrial damage in the liver. ConclusionDanggui Shaoyaosan may inhibit lipid peroxidation and ferroptosis by activating the Nrf2/SLC7A11/GPX4 signaling pathway to treat NAFLD.

ObjectiveTo explore the effects of Renshentang on atherosclerosis （AS） in mice based on the role of transient receptor potential vanilloid1 （TRPV1） in regulating cholesterol metabolism in foam cells. MethodsNine SPF-grade 8-week-old C57BL/6J mice were set as a normal group， and 60 ApoE^-/- mice were randomized into model， positive drug （simvastatin， 0.02 g·kg^-1·d^-1）， and low-， medium-， and high-dose （1.77， 3.54， 7.08 g·kg^-1·d^-1， respectively） Renshentang groups （n=12） according to body weight. The normal group was fed with a normal diet， and the other groups were fed with a high-fat diet and given corresponding drugs by oral gavage for the modeling of AS. The mice were administrated with corresponding drugs once a day for 12 weeks. After the last administration and fasting for 12 h， the aorta was collected. Plaque conditions， pathological changes， levels of total cholesterol （TC）， triglcerides （TG）， low-density lipoprotein-cholesterol （LDL-C）， and high-density lipoprotein-cholesterol （HDL-C）， and the expression of TRPV1， liver X receptor （LXR）， inducible degrader of the low-density lipoprotein receptor （IDOL）， and low-density lipoprotein receptor （LDLR） in the aortic tissue were observed and detected by gross oil red O staining， HE staining， Western blot， immunohistochemistry， and real-time PCR. ResultsCompared with the normal group， the model group presented obvious plaque deposition in the aorta， raised levels of TC， TG， and LDL-C in the serum （P<0.01）， up-regulated expression level of LDLR in the aorta （P<0.01）， lowered level of HDL-C in the serum， and down-regulated expression levels of TRPV1， LXR， and IDOL in the aorta （P<0.05， P<0.01）. Compared with the model group， the positive drug and Renshentang at different doses alleviated AS， elevated the levels of HDL-C， TRPV1， LXR， and IDOL （P<0.05， P<0.01）， while lowering the levels of TC， TG， LDL-C， and LDLR （P<0.05， P<0.01）. ConclusionRenshentang has a lipid-lowering effect on AS mice. It can effectively reduce lipid deposition， lipid levels， and plaque area of AS mice by activating TRPV1 expression and regulating the LXR/IDOL/LDLR pathway.

ObjectiveTo explore the effects of Renshentang on atherosclerosis （AS） in mice based on the role of transient receptor potential vanilloid1 （TRPV1） in regulating cholesterol metabolism in foam cells. MethodsNine SPF-grade 8-week-old C57BL/6J mice were set as a normal group， and 60 ApoE^-/- mice were randomized into model， positive drug （simvastatin， 0.02 g·kg^-1·d^-1）， and low-， medium-， and high-dose （1.77， 3.54， 7.08 g·kg^-1·d^-1， respectively） Renshentang groups （n=12） according to body weight. The normal group was fed with a normal diet， and the other groups were fed with a high-fat diet and given corresponding drugs by oral gavage for the modeling of AS. The mice were administrated with corresponding drugs once a day for 12 weeks. After the last administration and fasting for 12 h， the aorta was collected. Plaque conditions， pathological changes， levels of total cholesterol （TC）， triglcerides （TG）， low-density lipoprotein-cholesterol （LDL-C）， and high-density lipoprotein-cholesterol （HDL-C）， and the expression of TRPV1， liver X receptor （LXR）， inducible degrader of the low-density lipoprotein receptor （IDOL）， and low-density lipoprotein receptor （LDLR） in the aortic tissue were observed and detected by gross oil red O staining， HE staining， Western blot， immunohistochemistry， and real-time PCR. ResultsCompared with the normal group， the model group presented obvious plaque deposition in the aorta， raised levels of TC， TG， and LDL-C in the serum （P<0.01）， up-regulated expression level of LDLR in the aorta （P<0.01）， lowered level of HDL-C in the serum， and down-regulated expression levels of TRPV1， LXR， and IDOL in the aorta （P<0.05， P<0.01）. Compared with the model group， the positive drug and Renshentang at different doses alleviated AS， elevated the levels of HDL-C， TRPV1， LXR， and IDOL （P<0.05， P<0.01）， while lowering the levels of TC， TG， LDL-C， and LDLR （P<0.05， P<0.01）. ConclusionRenshentang has a lipid-lowering effect on AS mice. It can effectively reduce lipid deposition， lipid levels， and plaque area of AS mice by activating TRPV1 expression and regulating the LXR/IDOL/LDLR pathway.

Humans ; Vascular Stiffness ; Coal Mining ; Occupational Diseases/epidemiology* ; Risk Factors ; Coal ; Miners

ObjectiveTo explore the effect and mechanism of Zhishi Xiebai Guizhitang on the progression of atherosclerosis （AS） mice based on the regulation of cholesterol metabolism in foam cells by transient receptor potential channel ankyrin 1 （TRPA1）. MethodThe AS model was established on apolipoprotein E knockout （ApoE^-/-） mice with a high-fat diet. The mice were randomly divided into low-dose， middle-dose， and high-dose groups of Zhishi Xiebai Guizhitang （2.97， 5.94， 11.88 g·kg^-1） and simvastatin group （0.002 g·kg^-1）， and the drug was administered along with a high-fat diet. C57BL/6J mice were fed an ordinary diet as a normal group. After the above process， the aorta and serum of mice were taken. The pathological changes of the aortic root were observed by hematoxylin-eosin （HE） staining. The lipid plaques in the aorta were observed by gross oil redness. Serum levels of total cholesterol （TC）， triglyceride （TG）， low density lipoprotein cholesterol （LDL-C）， and high density lipoprotein cholesterol （HDL-C） were detected， and the levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot and immunohistochemical method were used to analyze the expression of TRPA1， ATP-binding cassette transporter A1 （ABCA1）， ATP-binding cassette transporter G1 （ABCG1）， and mannose receptor （CD206）. ResultFrom the perspective of drug efficacy， compared with the normal group， pathological changes such as plaque， a large number of foam cells， and cholesterol crystals appeared in the aorta of the model group， and the serum levels of TC， LDL-C， IL-1β， and IL-18 were significantly increased （P<0.01）. The HDL-C level was significantly decreased （P<0.01）， and the CD206 level in aortic tissue was significantly decreased （P<0.01）. Compared with the model group， the lipid deposition in the aorta was alleviated in all drug administration groups. In addition， except for the high-dose group of Zhishi Xiebai Guizhitang， all drug administration groups could significantly decrease the levels of TC and LDL-C （P<0.01）. In terms of inflammation， except for the middle-dose group of Zhishi Xiebai Guizhitang， the levels of IL-1β and IL-18 were significantly decreased in all drug administration groups （P<0.05）. Moreover， Zhishi Xiebai Guizhitang could also up-regulate the levels of CD206， and the difference was significant in the middle-dose and high-dose groups （P<0.05）. From the perspective of mechanism， the expression levels of TRPA1， ABCA1， and ABCG1 in the aorta in the model group were lower than those in the normal group （P<0.05）. Compared with the model group， all drug administration groups significantly increased the expression of TRPA1 in the aorta （P<0.05）， and the expressions of ABCA1 and ABCG1 were increased. The differences in the middle-dose and high-dose groups and the simvastatin group were significant （P<0.05）， which was basically consistent with the trend of immunohistochemical results. ConclusionZhishi Xiebai Guizhitang can effectively reduce blood lipid and inflammation levels and inhibit the formation of aortic plaque. The mechanism may be explained as follows： the expressions of ABCA1 and ABCG1 downstream are increased through TRPA1， which promotes cholesterol outflow in foam cells， thereby regulating cholesterol metabolism， intervening in inflammation level to a certain extent， and finally treating AS.

Umbilical cord mesenchymal stem cells (UC-MSCs) have been widely used in regenerative medicine, but there is limited research on the stability of UC-MSCs formulation during production. This study aims to assess the stability of the cell stock solution and intermediate product throughout the production process, as well as the final product following reconstitution, in order to offer guidance for the manufacturing process and serve as a reference for formulation reconstitution methods. Three batches of cell formulation were produced and stored under low temperature (2-8 ℃) and room temperature (20-26 ℃) during cell stock solution and intermediate product stages. The storage time intervals for cell stock solution were 0, 2, 4, and 6 h, while for intermediate products, the intervals were 0, 1, 2, and 3 h. The evaluation items included visual inspection, viable cell concentration, cell viability, cell surface markers, lymphocyte proliferation inhibition rate, and sterility. Additionally, dilution and culture stability studies were performed after reconstitution of the cell product. The reconstitution diluents included 0.9% sodium chloride injection, 0.9% sodium chloride injection + 1% human serum albumin, and 0.9% sodium chloride injection + 2% human serum albumin, with dilution ratios of 10-fold and 40-fold. The storage time intervals after dilution were 0, 1, 2, 3, and 4 h. The reconstitution culture media included DMEM medium, DMEM + 2% platelet lysate, 0.9% sodium chloride injection, and 0.9% sodium chloride injection + 1% human serum albumin, and the culture duration was 24 h. The evaluation items were viable cell concentration and cell viability. The results showed that the cell stock solution remained stable for up to 6 h under both low temperature (2-8 ℃) and room temperature (20-26 ℃) conditions, while the intermediate product remained stable for up to 3 h under the same conditions. After formulation reconstitution, using sodium chloride injection diluted with 1% or 2% human serum albumin maintained a viability of over 80% within 4 h. It was observed that different dilution factors had an impact on cell viability. After formulation reconstitution, cultivation in medium with 2% platelet lysate resulted in a cell viability of over 80% after 24 h. In conclusion, the stability of cell stock solution within 6 h and intermediate product within 3 h meets the requirements. The addition of 1% or 2% human serum albumin in the reconstitution diluent can better protect the post-reconstitution cell viability.

Objective To explore the feasibility and operability in identifying the therapeutic dominant stages of traditional Chinese medicine(TCM)based on subdivision model of disease course.Methods The hierarchical Bayesian model was used to differentiate the disease course of 125 cases of premature ovarian failure(POF),and the disease course of POF were divided into the occult stage,diminished ovarian reserve(DOR)stage,premature ovarian insufficiency(POI)stage,and POF stage.An then the paired sample t-test,Pearson correlation analysis and expert in-depth interview were used for the analysis of the therapeutic effects of TCM for POF at various stages.Results(1)Compared with POF stage,DOR and POI stages were frequently intervened by Chinese patent medicine.(2)In DOR(complicated with POI)stage and POF stage,there was significant difference between the degree of TCM intervention and the therapeutic effect(t =-3.70,P＜0.001).(3)The degree of TCM intervention was positively correlated with treatment outcomes in the DOR stage(r = 0.679,P＜0.001),so did in the POF stage(r = 0.432,P＜0.001),but the correlation in the POF stage was slightly lower than that in the DOR stage.(4)The results of in-depth interviews with experts of TCM gynecology showed that in the concealed phase of POF,the prognosis would be most favorable if TCM regulation and intervention were performed.In the DOR stage and POI stage,treatment with Chinese medicine prescriptions usually brought about better curative effect and prognosis.For the patients at POF stage,the therapeutic effect of TCM depended on the patients'compliance and the treatment course,and the effect was relatively not as good as that of the previous stages.Conclusion In the DOR stage and POF stage,the higher the degree of TCM intervention,the better the prognosis will be achieved for the patients treated with western medicine.In the POF stage,the efficacy of TCM intervention is reduced to a certain extent compared with the DOR stage.The results indicated that it is feasible and operable to identify the TCM therapeutic dominant stages based on the subdivision model of disease course.

Osteoporosis （OP） is a common bone disease affecting the quality of life and causing huge medical burden to the patients and society. The occurrence of OP is mainly caused by excessive bone resorption and insufficient bone formation， which are directly influenced by external calcium ion balance. Calcium imbalance can impair bone integrity， reduce the calcium supply to the bone， and lower the calcium content in the bone， thus triggering OP. Drugs are the main anti-OP therapy in modern medicine， which， however， may cause adverse reactions and drug dependence. Chinese medicines have good clinical effects and high safety in treating OP， being suitable for long-term use. Recent studies have shown that Chinese medicines can alleviate estrogen deficiency， regulate bone cell and calcium metabolism， which is crucial for the formation and development of OP. The transient receptor potential cation channel superfamily V members 5 and 6 （TRPV5 and TRPV6， respectively） affect bone homeostasis by mediating the transmembrane calcium ion transport in the intestine （TRPV6） and kidney （TRPV5）. Therefore， TRPV5/6 is one of the key targets to understand the anti-OP mechanisms of the effective parts of Chinese medicines， which is worthy of further study. This paper summarizes the research results about the anti-OP effects of Chinese medicines in the last two decades， especially the mechanism of regulating calcium metabolism， aiming to provide new ideas for the basic research， clinical application， and drug development of OP treatment.

Objective To observe the effects of amyloid-β(Aβ)receptor PirB on mouse astrocyte proliferation and reactive astrogliosis in vitro.Methods Mouse primary astrocytes were cultured,and divided into control group,Aβ group,Aβ+0.2 μmol/L PEP group,Aβ+0.4 μmol/L PEP group,Aβ+Fluspirilene group,Aβ+GFP-LV group,and Aβ+mPirB-LV group.The mouse astrocytes were treated with soluble PirB extracellular peptide PEP or PirB inhibitor Fluspirilene,respectively,to inhibit endogenous PirB receptor,or overexpressed PirB gene via lentivirus transfection and then treated with Aβ1-42 oligomers.The proliferation of astrocytes was observed by RTCA and EdU methods,and the mRNA expression levels of S-100 calcium-binding protein B(S-100β),Vimentin,Nestin and amyloid precursor protein(APP)associated with reactive astrogliosis of astrocytes were observed by real-time PCR,and the expression level of glial fibrillary acid protein(GFAP)was detected by Western-blotting.Results The results of RTCA monitoring showed that normalized cell index(NCI)values of each group decreased sharply after treatment,and then increased gradually and tended to be stable.The results of EdU staining showed that the proliferative activity of astrocytes was significantly enhanced in the Aβ group(P<0.05)compared with control group;Compared with Aβ group,cell proliferation activity in Aβ + 0.2 μmol/L PEP group,Aβ+0.4 μmol/L PEP group and Aβ+Fluspirilene group were significantly decreased(P<0.01 or P<0.001).The results of real-time PCR showed that compared with control group,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβ group were significantly increased(P<0.05);Compared with Aβ group,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβ+0.4 μmol/L PEP group were significantly decreased(P<0.01);Compared with Aβ+GFP-LV group,mRNA expressions of GFAP,S-100β,Vimentin,Nestin,APP and PirB in Aβ +mPirB-LV group were significantly increased(P<0.05).The results of Western blotting showed that compared with control group,the expression of GFAP in Aβ group was significantly increased(P<0.05);Compared with Aβ group,the expression of GFAP in Aβ+0.4 μmol/L PEP group was significantly decreased(P<0.05).Conclusions PirB is an upstream molecule which could regulate astrocyte proliferation and reactive astrogliosis,and inhibiting PirB receptor in astrocytes may be a potential treatment for Alzheimer's disease.