The chemical constituents from the n-butanol fraction of ethanol extract of safflower (Carthamus tinctorius L.) were isolated and purified using chromatographic process including MCI Gel CHP-20, ODS, Sephadex LH-20 column chromatography, and semi-preparative HPLC method, and one alkyne and two phenylpropanoid derivants were obtained. Their structures were identified as (5R,E)-tetradecane-12-ene-8,10-diyne-1,5,14-triol (1), (E)-8-O-β-D-glucopyranosyl n-butyl cinnamate (2), and (7S,8S)-7-(4-hydroxy-3-methoxybenzene)-7-butyl-8,9-diol (3) by modern spectroscopy methods (1D NMR, 2D NMR, UV, IR and MS). 1-3 are all new compounds. Compounds 1-3 were screened for their anti-renal fibrosis activities in vitro, and none of them showed obvious effect.

Objective To investigate the effects of leptin on hypothalamic neuron activity and lipid metabolism in adipose tissue of obese mice.Methods 10 leptin-deficient obese(ob/ob)mice with homozygous mutation of leptin gene and 10 wild-type(WT)mice born in the same litter were randomly divided into control group and leptin treatment group.The activity of pro-opiomelanocortin(POMC)neurons and tyrosine hydroxylase(TH+)neurons,the morphological changes of adipose tissue and the expression of lipid-related genes were analyzed by immunofluorescent staining,HE staining and Real-time PCR.Results Compared with the WT mice,the ob/ob mice showed decreased activity of POMC neurons and TH+neurons and larger cell diameter in adipose tissue and liver tissue.In addition,the expressions of heat-related genes uncoupling protein 1(UCP1),cytochrome c oxidase subunit 8B(Cox8b)and cell death-inducing DNA fragmentation factor,alpha subunit-like effector A(Cidea)in subcutaneous white fat in ob/ob mice decreased significantly,and the expressions of lipid synthesis-related genes sterol regulatory element binding transcription factor 1(Srebp1)and fatty acid synthase(Fas)increased significantly.After treated with leptin,the activities of POMC and TH+neurons were increased,and the cellular diameter and the degree of vacuolar degeneration were reduced in the adipose tissue and liver.Further analyses showed that the expressions of thermogenesis-related genes and lipolysis-related genes were increased,but expressions of lipid synthesis-related genes were reduced in brown adipose tissue.Conclusion Leptin treatment could prevent the increasing of obesity in ob/ob mice,which is associated with increased lipolysis and reduced lipid synthesis through activation of hypothalamic POMC neurons and peripheral adipose tissue sympathetic nervous system.

The present study observed the regulatory effect of total flavonoids of Ziziphora clinopodioides on autophagy and the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathways in ApoE~(-/-) mice and explored the mechanism of total flavonoids of Z. clinopodioides against atherosclerosis(AS). ApoE~(-/-) mice were fed on a high-fat diet for eight weeks to induce an AS model. The model mice were randomly divided into a model group, a positive control group, and low-, medium-and high-dose groups of total flavonoids of Z. clinopodioides, while C57BL/6J mice fed on a common diet were assigned to the blank group. The serum and aorta samples were collected after intragastric administration for 12 weeks, and the serum levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein-cholesterol(LDL-C), and high density lipoprotein-cholesterol(HDL-C) were detected by an automatic biochemical analyzer. The serum expression levels of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), matrix metalloproteinase-2(MMP-2), and matrix metalloprotei-nase-9(MMP-9) were detected by enzyme-linked immunosorbent assay(ELISA). Oil red O staining was used to observe the aortic plaque area in mice. Hematoxylin-eosin(HE) staining was used to observe the aortic plaque and pathological changes in mice. The expression of P62 and LC3 in the aorta was detected by the immunofluorescence method. The protein expression of LC3Ⅱ/Ⅰ, Beclin-1, P62, p-PI3K, p-Akt, and p-mTOR in the aorta of mice was detected by Western blot. The results showed that compared with the blank group, the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2 and MMP-9 in the model group were significantly increased(P<0.01 or P<0.05), the content of HDL-C was decreased(P<0.05), intra-aortic plaque area was enlarged(P<0.01), the expression of LC3 in the aorta was significantly down-regulated, P62 expression was up-regulated(P<0.01 or P<0.05), the expressions of LC3Ⅱ/Ⅰ and Beclin-1 in the aortic lysate were significantly down-regulated, and the expressions of p-PI3K, p-Akt, p-mTOR and P62 were significantly increased(P<0.01). The medium-and high-dose groups of total flavonoids of Z. clinopodioides could reduce the serum levels of TC, TG, LDL-C, ICAM-1, VCAM-1, MMP-2, and MMP-9 in AS model mice(P<0.01 or P<0.05), and increase the content of HDL-C(P<0.01 or P<0.05). The aortic plaque area of mice after middle and high doses of total flavonoids of Z. clinopodioides was significantly reduced(P<0.01), the content of foam cells decrease, and the narrowing of the lumen decreased. The total flavonoids of Z. clinopodioides significantly increased the expression of LC3 in the aorta and the expression of LC3Ⅱ/Ⅰ and Beclin-1 in the lysate, and decreased the expression of P62 in the aorta and the expression of p-PI3K, p-Akt, p-mTOR and P62 in the lysate(P<0.01 or P<0.05). The results showed that the total flavonoids of Z. clinopodioides could improve the content of blood lipids and inflammatory factors, and reduce the generation of foam cells and plaques in aortic tissue, and the mechanism may be related to the regulation of PI3K/Akt/mTOR signaling pathway.
Animals ; Mice ; Apolipoproteins E ; Atherosclerosis/genetics* ; Beclin-1 ; Cholesterol, LDL ; Intercellular Adhesion Molecule-1 ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Plaque, Atherosclerotic ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/genetics* ; Vascular Cell Adhesion Molecule-1/genetics*

OBJECTIVE: To observe the effect of acupotomy on the morphology and ultrastructure of rectus femoris muscle in rabbits with knee osteoarthritis and to reveal the possible therapeutic mechanism involved in the effect of acupotomology on the treatment of knee osteoarthritis(KOA). METHODS: Twenty-four male New Zealand rabbits aged 6 months and weighed (2.0±0.5) kg were randomly divided into blank group, model group and acupotomy group, 8 rabbits in each group. KOA model was established by modified Videman method with left hind limb extended plaster immobility for 6 weeks. In acupotomy group, the transfascial focal points of quadriceps femoris muscle were released by acupotomy under the guidance of Jingjin theory for 4 times and once a week, and the treatment points include Hedingci, Binwaixia, Binneixia. Blank group and model group were fed normally without intervention. One week after the end of the intervention, the pennation angle(PA), muscle thickness(MT), cross-sectional area(CSA) and strain ratio(SR) of rectus femoris were measured by ultrasound. HE staining was used to observe the changes of the tissue morphology, the number of muscle fibers and the average area of muscle fibers. The myofibril of rectus femoris, sarcomere and myofilament were observed by transmission electron microscope. RESULTS: The PA of rectus femoris muscle in the blank group was (9.05±0.21)°. The MT was(1.09±0.09) cm and the CSA was(1.30±0.01) cm². The PA of rectus femoris muscle in the model group was (3.06±0.15)°. The MT was (0.71±0.02) cm and the CSA was(0.77±0.02) cm². The PA of rectus femoris muscle in the acupotomy group was (6.94±0.28)°. The MT was (0.80±0.05) cm and the CSA was(0.94±0.03) cm². The muscle PA, MT and CSA of rectus femoris in the model group were significantly smaller than those in the blank group (P<0.05). Those in acupotomy group were significantly increased compared with those in model group (P<0.05). The SR of rectus femoris muscle was 1.19±0.02 in the blank group, 3.50±0.05 in the model group and 1.99±0.07 in the acupotomy group. The elastic SR of the model group was significantly higher than that of the blank group (P<0.05). These in acupotomy group was significantly lower than that in model group(P<0.05). The results of HE staining showed:in blank group, the fascicles of rectus femoris were arranged neatly, the number of beam of muscle fibers within the fixed visual field was 94.38±3.50 and the average CSA was(0.75±0.22) mm². In model group, the fascicles of rectus femoris with different sizes were disorganized with a small amount of inflammatory cell infiltration, the number of beam of muscle fibers within the fixed visual field was 196.63±2.62 and the average CSA was(0.26±0.03) mm². Compared to the blank group, a significant increase in the number of muscle fibers in the fixed field in the model group (P<0.05) and the average CSA decreased significantly(P<0.05). In acupotomy group, the rectus femoris fascicles in the acupotomy group tended to be arranged in a more orderly manner, with the inflammatory cells decreased, the number of beam of muscle fibers within the fixed visual field was 132.88±4.61 and the average CSA was(0.70±0.07) mm². Compared to the model group, a significant decrease in the number of muscle fibers in the fixed field in the model group(P<0.05) and the average CSA increased significantly(P<0.05). The results of transmission electron microscope showed:compared with the blank group, the overall arrangement of the myofibrils of the rectus femoris in the model group was less structured. There was fracture between the muscle fibers and the sarcomere, the myofilaments were disordered, and the fracture of the Z line was discontinuous. Compared with the model group, the myofibrillar texture of rectus femoris in acupotomy group was clearer, and the Z line was more continuous. CONCLUSION Based on the jingjin theory, the release of quadriceps femoris by acupotomy can effectively improve the morphology and structure of rectus femoris, and promote the repair and reconstruction of chronic skeletal muscle injury in rabbits with KOA, which may be one of the mechanisms of acupotomy in the treatment of KOA.
Acupuncture Therapy ; Animals ; Humans ; Male ; Muscle, Skeletal ; Osteoarthritis, Knee/therapy* ; Quadriceps Muscle ; Rabbits ; Ultrasonography

OBJECTIVE: To explore the ultrasonic anatomical characteristics and needle-knife insertion approach of common tendon lesions in knee osteoarthritis (KOA), so as to provide the references for accurate release of KOA by needle- knife along tendon lesions based on meridian sinew theory. METHODS: Sixty patients with one-knee KOA were selected. High-frequency musculoskeletal ultrasound was used to collect sonograms at the anatomical positions of "Hedingci" "Binwaixia" "Binneixia" and "Yinlingshang". The anatomic levels were marked on the sonograms. The anatomic levels and sonographic features of lesions were compared and analyzed, and the relevant data of needle-knife simulation approach was measured. RESULTS: The "Hedingci" lesions were mainly located at the attachment of quadriceps tendon to patella and suprapatellar bursa. The "Binwaixia" and "Binneixia" lesions were mainly located at the attachment of retinaculum patellae laterale and retinaculum patellae mediale to patella and infrapatellar fat pad. The "Yinlingshang" lesions were mainly located at the attachment of goose foot tendon to medial tibial condyle and bursa of goose foot. With "Hedingci" as an example, when the needle-knife entry point was 1 cm above the patella, the attachment of quadriceps tendon to patella was released, and the average depth of needle-knife was (3.60±0.10) cm, and the needle body was perpendicular to the skin. The average depth of needle-knife for releasing suprapatellar bursa was (2.35±0.17) cm, and the needle body was 45° towards head. CONCLUSION The musculoskeletal ultrasound could clearly show the local detailed anatomical level, ultrasonic characteristics and anatomical level of common tendon lesions of KOA, and could improve the accuracy of needle-knife along tendon lesions with non-direct vision, which has important reference value for needle-knife medical standardization and standardized operation.
Humans ; Knee Joint/surgery* ; Meridians ; Osteoarthritis, Knee/surgery* ; Patella ; Tendons/surgery* ; Ultrasonography

Objective: Several COVID-19 patients have overlapping comorbidities. The independent role of each component contributing to the risk of COVID-19 is unknown, and how some non-cardiometabolic comorbidities affect the risk of COVID-19 remains unclear. Methods: A retrospective follow-up design was adopted. A total of 1,160 laboratory-confirmed patients were enrolled from nine provinces in China. Data on comorbidities were obtained from the patients' medical records. Multivariable logistic regression models were used to estimate the odds ratio ( Results: Overall, 158 (13.6%) patients were diagnosed with severe illness and 32 (2.7%) had unfavorable outcomes. Hypertension (2.87, 1.30-6.32), type 2 diabetes (T2DM) (3.57, 2.32-5.49), cardiovascular disease (CVD) (3.78, 1.81-7.89), fatty liver disease (7.53, 1.96-28.96), hyperlipidemia (2.15, 1.26-3.67), other lung diseases (6.00, 3.01-11.96), and electrolyte imbalance (10.40, 3.00-26.10) were independently linked to increased odds of being severely ill. T2DM (6.07, 2.89-12.75), CVD (8.47, 6.03-11.89), and electrolyte imbalance (19.44, 11.47-32.96) were also strong predictors of unfavorable outcomes. Women with comorbidities were more likely to have severe disease on admission (5.46, 3.25-9.19), while men with comorbidities were more likely to have unfavorable treatment outcomes (6.58, 1.46-29.64) within two weeks. Conclusion Besides hypertension, diabetes, and CVD, fatty liver disease, hyperlipidemia, other lung diseases, and electrolyte imbalance were independent risk factors for COVID-19 severity and poor treatment outcome. Women with comorbidities were more likely to have severe disease, while men with comorbidities were more likely to have unfavorable treatment outcomes.
Adult ; Aged ; COVID-19/virology* ; China/epidemiology* ; Comorbidity ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Severity of Illness Index ; Treatment Outcome

Objective: To investigate the mechanism of Zuoguiwan in treating ovariectomy-induced osteoporosis rats by receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) signaling pathway mediated by β₂-adrenergic receptor (β₂AR). Method: Forty Sprague-Dawley female rats were randomly divided into Sham-operated group (Sham) and four ovariectomized (OVX) subgroups. Rats in Sham and OVX groups were treated with 17β-estradiol (50 μg·kg^-1·d^-1), and low and high-dose ZGW (2.3,4.6 g·kg^-1 lyophilized powder) for 3 months, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum markers of bone turnover. Micro-CT was used to evaluate and measure trabecular bone microarchitecture and bone mineral density (BMD) of the right distal femur. Western blot analysis and Real-time PCR were used to measure mRNA and protein expressions of β₂AR, OPG and RANKL. Result: After 12 weeks of treatment with Zuoguiwan, the level of serum β-cross-linked c-telopeptide of type Ι collagen (β-CTX) (P<0.01) was lower, while the level of serum bone-specific alkaline phosphatase (BALP) was higher (P<0.01) than those in the OVX group. Moreover, it could prevent the OVX-induced bone loss, and alleviate the trabecular bone microarchitecture of distal femur. Furthermore, Zuoguiwan could up-regulate the mRNA and protein expressions of OPG in tibia of the Zuoguiwan groups(P<0.01), reduce the mRNA and protein expressions of β₂AR in the hypothalamus (P<0.01), and down-regulated the mRNA and protein expressions of RANKL (P<0.05) in the tibia, compared with those in the OVX group. Conclusion: The mechanism of Zuoguiwan in alleviating BMD and trabecular bone microarchitecture in ovariectomy-induced osteoporosis rats might be related to the regulation of RANKL/OPG Pathway mediated by β₂AR.

Zuoguiwan is a classic prescription for replenishing vital essence, tonifying kidney-Yin and nourishing the bone marrow. Zuoguiwan is one of the effective prescriptions for the prevention and treatment of osteoporosis (OP), which reflects the thought of Reinforcing Yang from Yin. The OP animal model simulates the pathological state and pathogenesis of OP in human, which is an important means to research the pathogenesis of OP and verify the effect of drugs. In this paper, two kinds of animal models and characteristics of Zuoguiwan in the prevention and treatment of osteoporosis were discussed in details. They are the primary osteoporosis animal models, including ovariectomized animal models and spontaneous elderly osteoporosis animal models, and the secondary osteoporosis models, including glucocorticoid-induced animal models, cyclophosphamide-induced animal models and subtotal nephrectomy animal models. The evaluation methods of Zuoguiwan in preventing and treating OP, including bone absorption markers and bone formation markers analyzed by enzyme-linked immunosorbent assay (ELISA), bone mineral density detected with dual-energy X-ray, the number of trabeculae, trabecular segregation, trabecular thickness, bone volume/tissue volume ratio and bone surface/volume ratio analyzed using micro-CT, bone pathological morphology observed by hematoxylin-eosin staining, bone biomechanical properties, such as the maximum load force based on biomechanical test. In order to provide scientific reference for the basic and clinical research of Zuoguiwan, the OP animal models and the pharmacodynamic effect of Zuoguiwan are evaluated comprehensively with five different and objective evaluation methods. However, the animal model of OP needs to be further optimized to highlight the pathogenesis and syndrome characteristics of Zuoguiwan in the treatment of OP.

Background: Macrophage accumulation in the vascular wall is a hallmark of atherosclerosis. Studies showed that shifting of oxidized lipids-induced inflammatory macrophages towards an anti-inflammatory phenotype by promoting oxidative metabolism attenuated atherosclerosis progression. Therefore, this study aimed to investigate whether metformin, which has ameliorated atherosclerosis in animal models and clinical trials, modulated oxidized low-density lipoprotein (Ox-LDL) induced inflammatory status in macrophages by regulating cellular oxidative metabolism. Methods: Murine raw264.7 macrophages were incubated with Ox-LDL (50 μg/mL) in the presence or absence of metformin (15 μmol/L) for 24 h. Real-time polymerase chain reaction was used to quantify the transcription of classically activated (M1) proinflammatory and alternatively activated (M2) anti-inflammatory markers and mitochondrial DNA copy numbers. Cellular reactive oxygen species (ROS) production and mitochondrial membrane potential were detected by immunofluorescence. Cellular adenosine triphosphate (ATP) synthesis, glucose uptake, and lactic acid production were measured by commercial kit and normalized to cellular lysates. Western blotting analysis was performed to detect the expression of mitochondrial fusion/fission related proteins, enzymes mediating lipid metabolism and signaling pathway of glucose transport. Differences between groups were analyzed using one-way analysis of variance. Results: Metformin improved Ox-LDL-impaired anti-inflammatory phenotype in raw264.7 macrophages as shown by up-regulated transcription of anti-inflammatory markers including interleukin 10 (0.76 ± 0.04 vs. 0.94 ± 0.01, P = 0.003) and Resistin-like molecule alpha (0.67 ± 0.08 vs. 1.78 ± 0.34, P = 0.030). Conversely, Ox-LDL-diminished phosphorylation of Akt was upregulated by metformin treatment (0.47 ± 0.05 vs. 1.02 ± 0.08, P = 0.040), associated with an improvement of mitochondrial function, characterized by decreased ROS generation (2.50 ± 0.07 vs. 2.15 ± 0.04, P = 0.040), increased lipid oxidation, and elevated cellular ATP production (0.026 ± 0.001 vs. 0.035 ± 0.003, P = 0.020). Moreover, metformin-mediated Akt activation increased Akt substrate of 160 kDa (AS160) phosphorylation (0.51 ± 0.04 vs. 1.03 ± 0.03, P = 0.0041), promoted membrane translocation of glucose transporter 1, and increased glucose influx into the cells (4.78 ± 0.04 vs. 5.47 ± 0.01, P < 0.001). Conclusion This study suggested that targeting macrophage metabolism with new or existing drugs had therapeutic potential for the prevention and treatment of diabetes-accelerated atherosclerosis.