Taraxasterol mitigates Con A-induced hepatitis in mice by suppressing interleukin-2 expression and its signaling in T lymphocytes.

Discovery of anti-inflammatory drugs that can suppress T lymphocyte activation and proliferation by inhibiting TCR/CD3 and IL-2/IL-2R signaling is still needed in clinic, though rapamycin and other related reagents have made great success. Taraxasterol (TAS) is an active ingredient of dandelion, an anti-inflammatory medicinal herb with low in vivo toxicity that has long been used in China. Yet the action mechanism of TAS on lymphocytes remains elusive. The anti-inflammatory effects of TAS were evaluated in C57BL/6 mouse primary lymphocytes stimulated with concanavalin A (Con A) in vitro and in mouse model of Con A-induced acute hepatitis in vivo. Our results showed that TAS significantly suppressed Con A-induced acute hepatitis in a mouse model, reducing the hepatic necrosis areas, the release of aminotransferases, and the production of IL-2 and other inflammatory cytokines. Supporting this, in vitro study also showed that TAS reduced the production of IL-2 and the expression of IL-2 receptor subunit α (CD25) upon the stimulation of Con A, which was likely mediated by suppressing NF-κB activation. The downstream pathways of IL-2/IL-2R signaling, including the activation of PI3K/PDK1/mTOR, STAT3 and STAT5, were also suppressed by TAS. Consistently, Con A-induced T cell proliferation was also inhibited by TAS in vitro. Our data indicate that TAS can suppress both T lymphocyte activation and cell proliferation by down-regulating IL-2 expression and its signaling pathway thereby ameliorating Con A-induced acute hepatitis, highlighting TAS as a potential drug candidate for treating inflammatory diseases including autoimmune hepatitis.

[Anti-inflammatory target prediction of Gentianae Radix et Rhizoma based on network pharmacology and construction of a bioassay method for its quality control].

Based on the heat-clearing and detoxifying effects of Gentianae Radix et Rhizoma, the network pharmacology is mainly used to predict the potential targets of Gentianae Radix et Rhizoma for anti-inflammatory activity and to perform the experimental verification. A method for detecting the biological potency of Gentianae Radix et Rhizoma based on verifiable targets has been established to provide a reference for improving the quality evaluation and control standards of Gentianae Radix et Rhizoma. High performance liquid chromatography can be used to construct chemical fingerprints of different batches of Gentianae Radix et Rhizoma. Constructing a component-target-disease network of Gentianae Radix et Rhizoma for its anti-inflammatory activity was applied to screen potential anti-inflammatory components and related targets of Gentianae Radix et Rhizoma, and to verify the target of Gentianae Radix et Rhizoma by using biological evaluation methods. Detecting the biological potency of different batches of Gentianae Radix et Rhizoma extracts was used to inhibit COX-2 enzyme activity based the verifiable target cyclooxygenase-2(COX-2). The results showed that different batches of Gentianae Radix et Rhizoma accorded with the pharmacopoeia testing regulations, and the chemical fingerprints have a high similarity(similarity>0.93), suggesting that there is no significant difference in the characteristics of the chemical components. Based on network pharmacology predictions, 18 candidate targets were found to have potential direct interactions with the ingredients in Gentianae Radix et Rhizoma. Among them, the most important target is COX-2. Based on the experimental verification of recombinant human COX-2 protease activity inhibition, Gentianae Radix et Rhizoma can inhibit the COX-2 enzyme activity in a dose-dependent manner. It can function with a low concentration(0.75 mg·mL~(-1)), which preliminarily confirmed the accuracy of network pharmacology prediction. The biological potency detection method of Gentianae Radix et Rhizoma based on COX-2 inhibitory activity was optimized and established. The qualitative response parallel line method was used to calculate the biological potency of anti-inflammatory activity, which ranged from 23.04 to 46.60 U·mg~(-1). For network pharmacology prediction, it can screen and clarify the possible targets of traditional Chinese medicine rapidly, which can guide the establishment of a biological evaluation method for the quality of medicinal materials with related activities. Compared with chemical fingerprints, the biological potency testing can better detect quality fluctuations of traditional Chinese medicine.

Inhibition of NLRP3 Inflammasome Activation and Pyroptosis in Macrophages by Taraxasterol Is Associated With Its Regulation on mTOR Signaling.

Taraxasterol (TAS) is an active ingredient of Dandelion (Taraxacum mongolicum Hand. -Mazz.), a medicinal plant that has long been used in China for treatment of inflammatory disorders. But the underlying mechanism for its therapeutic effects on inflammatory disorders is not completely clear. Inflammasome activation is a critical step of innate immune response to infection and aseptic inflammation. Among the various types of inflammasome sensors that has been reported, NLR family pyrin domain containing 3 (NLRP3) is implicated in various inflammatory diseases and therefore has been most extensively studied. In this study, we aimed to explore whether TAS could influence NLPR3 inflammasome activation in macrophages. The results showed that TAS dose-dependently suppressed the activation of caspase-1 in lipopolysaccharide (LPS)-primed murine primary macrophages upon nigericin treatment, resulting in reduced mature interleukin-1ß (IL-1ß) release and gasdermin D (GSDMD) cleavage. TAS greatly reduced ASC speck formation upon the stimulation of nigericin or extracellular ATP. Consistent with reduced cleavage of GSDMD, nigericin-induced pyroptosis was alleviated by TAS. Interestingly, TAS time-dependently suppressed the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and mTORC2 signaling induced by LPS priming. Like TAS, both INK-128 (inhibiting both mTORC1 and mTORC2) and rapamycin (inhibiting mTORC1 only) also inhibited NLRP3 inflammasome activation, though their effects on mTOR signaling were different. Moreover, TAS treatment alleviated mitochondrial damage by nigericin and improved mouse survival from bacterial infection, accompanied by reduced IL-1ß levels in vivo. Collectively, by inhibiting the NLRP3 inflammasome activation, TAS displayed anti-inflammatory effects likely through regulation of the mTOR signaling in macrophages, highlighting a potential action mechanism for the anti-inflammatory activity of Dandelion in treating inflammation-related disorders, which warrants further clinical investigation.

Berberine augments hypertrophy of colonic patches in mice with intraperitoneal bacterial infection.

Colonic patches, the counterparts of Peyer's patches in the small intestine, are dynamically regulated lymphoid tissues in the colon that have an important role in defensing against microbial infections. Berberine is an isoquinoline alkaloid extracted from medicinal herbs including Rhizoma coptidis and has long been used for the treatment of infectious gastroenteritis, but its impact on the colonic lymphoid tissues (such as colonic patches) is unknown. In this study, we aimed to investigate whether berberine had any influences on the colonic patches in mice with bacterial infection. The results showed that oral berberine administration in bacterial infected mice substantially enhanced the hypertrophy of colonic patches, which usually possessed the features of two large B-cell follicles with a separate T-cell area. Moreover, the colonic patches displayed follicular dendritic cell networks within the B-cell follicles, indicative of mature colonic patches containing germinal centers. Concomitant with enlarged colonic patches, the cultured colon of infected mice treated with berberine secreted significantly higher levels of interleukin-1ß (IL-1ß), IL-6, TNF-α, and CCL-2, while NLRP3 inhibitor MMC950 or knockout of NLRP3 gene abrogated berberine-induced hypertrophy of colonic patches, suggesting the involvement of the NLRP3 signaling pathway in this process. Functionally, oral administration of berberine ameliorated liver inflammation and improved formed feces in the colon. Altogether, these results indicated that berberine was able to augment the hypertrophy of colonic patches in mice with bacterial infection probably through enhancing local inflammatory responses in the colon.

Impact of Shenfu injection on a composite of organ dysfunction development in critically ill patients with coronavirus disease 2019 (COVID-19): A structured summary of a study protocol for a randomized controlled trial.

OBJECTIVES: This study aims to determine the protection provided by Shenfu injection (a traditional Chinese medicine) against development of organ dysfunction in critically ill patients with coronavirus disease 2019 (COVID-19). TRIAL DESIGN: This study is a multicenter, randomized, controlled, open-label, two-arm ratio 1:1, parallel group clinical trial. PARTICIPANTS: The patients, who are aged from 18 to 75 years old, with a confirmed or suspected diagnosis of severe or critical COVID-19, will be consecutively recruited in the study, according to the guideline on diagnosis and treatment of COVID-19 (the 7th version) issued by National Health Commission of the People's Republic of China. Exclusion criteria include pregnant and breastfeeding women, atopy or allergies to Shenfu Injection (SFI), severe underlying disease (malignant tumor with multiple metastases, uncontrolled hemopathy, cachexia, severe malnutrition, HIV), active bleeding, obstructive pneumonia caused by lung tumor, severe pulmonary interstitial fibrosis, alveolar proteinosis and allergic alveolitis, continuous use of immunosuppressive drugs in last 6 months, organ transplantation, expected death within 48 hours, the patients considered unsuitable for this study by researchers. The study is conducted in 11 ICUs of designated hospitals for COVID-19, located in 5 cities of China. INTERVENTION AND COMPARATOR: The enrolled patients will randomly receive 100 ml SFI (study group) or identical volume of saline (control group) twice a day for seven consecutive days. Patients in the both groups will be given usual care and the necessary supportive therapies as recommended by the latest edition of the management guidelines for COVID-19 (the 7th version so far). MAIN OUTCOMES: The primary endpoint is a composite of newly developed or exacerbated organ dysfunction. This is defined as an increase in the sequential organ failure assessment (SOFA) score of two or more, indicating sepsis and involvement of at least one organ. The SOFA score will be measured for the 14 days after enrolment from the baseline (the score at randomization). The secondary endpoints are shown below: • SOFA score in total • Pneumonia severity index score • Dosage of vasoactive drugs • Ventilation free days within 28 days • Length of stay in intensive care unit • Total hospital costs to treat the patient • 28-day mortality • The incidence of adverse drug events related to SFI RANDOMISATION: The block randomization codes were generated by SAS V.9.1 for allocation of participants in this study. The ratio of random distribution is 1:1. The sealed envelope method is used for allocation concealment. BLINDING (MASKING): The patients and statistical personnel analyzing study data are both blinded. The blinding of group assignment is not adopted for the medical staff. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): This study is expected to recruit 300 patients with COVID-19, (150 in each group). TRIAL STATUS: Protocol version 2.0, February 15, 2020. Patient recruitment started on February 25, and will end on August 31, 2020. TRIAL REGISTRATION: Chinese Clinical Trial Registry: ChiCTR2000030043. Registered February 21, 2020, http://www.chictr.org.cn/showprojen.aspx?proj=49866 FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this letter serves as a summary of the key elements of the full protocol.

Piperine metabolically regulates peritoneal resident macrophages to potentiate their functions against bacterial infection.

Pepper, a daily-used seasoning for promoting appetite, is widely used in folk medicine for treating gastrointestinal diseases. Piperine is the major alkaloid in pepper and possesses a wide range of pharmacological activities. However, the mechanism for linking metabolic and medicinal activities of piperine remains unknown. Here we report that piperine robustly boosts mTORC1 activity by recruiting more system L1 amino acid transporter (SLC7A5/SLC3A2) to the cell membrane, thus promoting amino acid metabolism. Piperine-induced increase of mTORC1 activity in resident peritoneal macrophages (pMΦs) is correlated with enhanced production of IL-6 and TNF-α upon LPS stimulation. Such an enhancement of cytokine production could be abrogated by inhibitors of the mTOR signaling pathway, indicating mTOR's action in this process. Moreover, piperine treatment protected resident pMΦs from bacterium-induced apoptosis and disappearance, and increased their bacterial phagocytic ability. Consequently, piperine administration conferred mice resistance against bacterial infection and even sepsis. Our data highlight that piperine has the capacity to metabolically reprogram peritoneal resident macrophages to fortify their innate functions against bacterial infection.

[Changing laws of serum high mobility group box 1 protein in septic rats and the intervention effect of xuebijing].

OBJECTIVE: To investigate the changing laws of serum high mobility group box 1 protein (HMGB1) in septic rats and intervention effect of Xuebijing on it. METHODS: Lipopolysaccharide (LPS) (5 mg/kg BW) was intravenously injected into the tail vein of healthy male Wistar rats to prepare the sepsis rat model. In Experiment 1: 50 Wistar rats were randomly divided into three groups, i.e., the normal group (A, n=10); the LPS model group (B, n=10), the LPS +Xuebijing treatment group (C, n=30). Rats in the C group were further divided into three subgroups, i.e., 2 h before LPS injection (group C1), 2 h after LPS injection (group C2), and 8 h after LPS injection (group C3), 10 in each group. Blood samples were collected from the caudal vein to detect serum HMGB1 levels by Western blot at 4, 12, 24, 48, and 72 h after LPS injection. Experiment 2: 30 Wistar rats were equally divided into the LPS model group (D) and the LPS + Xuebijing treatment group (E), 15 in each group. They were treated as rats in the B group and the C1 group respectively. Five rats were sacrificed at 12, 24, and 48 h after LPS injection in the two groups. Blood as well as the tissue samples were harvested to measure such indices as ALT, AST, Cr, and BUN, as well as pathological changes of liver, lung, and kidney. RESULTS: (1) Compared with the A group, serum HMGB1 levels were higher at various time points in the B group (P < 0.05). Compared with the B group, serum HMGB1 levels at 12,24,48, and 72 h decreased in the C1, C2, and C3 groups. Besides, the decrease was more obvious at 24 h and 48 h.The decrement in the C3 group was less than that in the C1 and C2 groups (P < 0.05). (2) In the D group, ALT, AST, Cr, and BUN were significantly higher than those in the A group and reached the peak at 24 h (P < 0.05). Compared with the E group, AST, Cr, and BUN at 24 and 48 h, and ALT at each time point decreased significantly in the E group (P < 0.05). (3)The results of pathological section of liver, lung, and kidney showed local congestion and hemorrhage, cell edema/necrosis/degeneration, infiltration of inflammatory cells, damage of characteristic structures and so on; particularly serious lesion occurred at 24 and 48 h in the D group. The microscopic lesion was obviously alleviated in the E group than in the D group at corresponding time points. CONCLUSIONS: The serum HMGB1 levels increased in septic rats, with late occurrence of peak value and longer duration of the high value. HMGB1 played an important role in excessive inflammatory response and multiple organ dysfunction. Xuebijing could reduce the serum levels of HMGB1, improve biochemical parameters, and attenuate severe inflammatory response of liver, lung, and kidney tissues in septic rats. Besides, the earlier use, the better effect obtained.

Xuebijing Protects Rats from Sepsis Challenged with Acinetobacter baumannii by Promoting Annexin A1 Expression and Inhibiting Proinflammatory Cytokines Secretion.

Xuebijing (XBJ) injection is a herbal medicine that has been widely used in the treatment of sepsis in China; however, its role in the development and progression of Acinetobacter baumannii sepsis and the underlying mechanisms remain uninvestigated. In the present study, fifty-four male Wistar rats were randomly assigned to normal-control group, sepsis-control group, and sepsis + XBJ group, each containing three subgroups of different treatment time periods (6, 12, and 24 hrs following injection, resp.). The sepsis model was established by intraperitoneal injection of A. baumannii ATCC 19606. For XBJ treatment, 4 mL/kg XBJ was administrated simultaneously by intravenous injection through caudal vein every 12 hrs. All animals demonstrated ill state, obvious intestinal dysfunction, histopathological lung damages, and overactive inflammatory responses after A. baumannii infection, and these events could be partially reversed by XBJ treatment from the beginning of infection. XBJ induced an increase in the expression of anti-inflammatory mediator annexin A1; however, two proinflammatory cytokines, interleukin-8 (IL-8) and tumor necrosis factor- α (TNF- α ), were decreased at the each monitored time point. These findings suggested that XBJ via its cytokine-mediated anti-inflammatory effects might have a potential role in preventing the progression of A. baumannii infection to sepsis by early administration.

[The effect of Xuebijing on liver translationally controlled tumor protein expression in Acinetobacter baumannii sepsis rats].

OBJECTIVE: To study the effect of Xuebijing on liver expressing translationally controlled tumor protein (TCTP) in Acinetobacter baumannii sepsis rats. METHODS: Among 42 healthy adult male Wistar rats of clean grade, 6 rats were randomly selected as the control group, others were randomly divided into two groups by the method of random digits table: sepsis group(n=18), Xuebijing group(n=18). Sepsis model was established through intraperitoneal injecting Acinetobacter baumannii suspension, and the Xuebijing injection was administrated through caudal vein 30 minutes later in Xuebijing group. After making model for 6, 12 and 24 hours, 6 rats were randomly selected from sepsis group and Xuebijing group, and then the rats were sacrificed, liver tissue samples were extracted for hematoxylin and eosin (HE) staining. Pathological changes of the liver were observed, and immunohistochemical analysis of liver tissue TCTP expression positive cells and the expression of TCTP in liver cells were detected by Western blotting method. RESULTS: HE staining of liver indicated that it was inflammatory injured in sepsis group, and inflammation decreased in Xuebijing group. Immunohistochemistry results showed that, compared with the control group, TCTP positive cells expression score at 6, 12 and 24 hours in sepsis group were significantly increased (7.33±0.82, 10.67±1.21, 7.67±1.21 vs. 2.50±1.05, all P<0.05). Compared with sepsis group, liver tissue TCTP positive cells expression score at 6, 12 and 24 hours in Xuebijing group (5.83±0.75, 7.50±1.05, 5.67±1.37) were significantly decreased (all P<0.05). Western blotting results showed that, compared with the control group, TCTP expression at 6, 12 and 24 hours in sepsis group were significantly increased (1.94±0.59, 3.20±0.72, 1.96±0.55 vs. 0.93±0.24, all P<0.05); compared with sepsis group, TCTP expression at 6, 12 and 24 hours in Xuebijing group (1.38±0.36, 2.03±0.49, 1.30±0.30) were significantly decreased (all P<0.05). CONCLUSIONS: Xuebijing can reduce inflammatory injury in liver of rats with Acinetobacter baumannii sepsis, and its mechanism may be associated with reduced hepatic cells expressed TCTP.

Chemical composition and antioxidant activity of the essential oil of endemic Viola tianshanica.

The composition and in vitro antioxidant activities of the essential oil and methanol extract of the aerial parts of Viola tianshanica were evaluated in this research. GC-MS analysis of the essential oil resulted in the identification of 15 constituents, representing 89.67% of the oil. The major compounds detected in the essential oil were dibutyl phthalate (15.19%), hexadecanoate methyl (8.65%), n-hexadecanoic acid (3.07%) and 2,3-pentanedione (2.62%). Essential oil and methanol extract were tested for their antioxidant activities using 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging and ß-carotene linoleic acid assay. In addition, the total phenol of essential oil, polar subfraction and non-polar subfraction were determined.

Aesculetin-induced apoptosis through a ROS-mediated mitochondrial dysfunction pathway in human cervical cancer cells.

Aesculetin (1) is an important coumarin found in various plant materials. It has been shown to have antiproliferative effects on several types of human cancer cells, but its effect on cervical cancer cells in vitro is unknown. In this study, we investigated that the cytotoxic effect of 1 on a non-cancer cell line (293) was smaller than on a tumor cell line (HeLa). This is the first report showing the possible mechanism of antiproliferative effect of 1 for the prevention of cervical cancer in cell culture models. It was found that 1 inhibited cell viability by inducing apoptosis, as evidenced by the formation of apoptotic bodies, generation of reactive oxygen species (ROS), and the accumulation of cells in the sub-G1 phase. Treatment with compound 1 decreased the cell growth in a dose-dependent manner with an IC(50) value of 37.8 microM. Aesculetin-induced apoptosis was correlated with mitochondrial dysfunction (DeltaPsi(m)), leading to the release of cytochrome c from the mitochondria to the cytosol, as well as the proteolytic activation of caspases in HeLa cells. These results indicate that 1 induces apoptosis in HeLa cells through a ROS-mediated mitochondrial dysfunction pathway.

Molecular cloning, expression profiling, and yeast complementation of 19 beta-tubulin cDNAs from developing cotton ovules.

Microtubules are a major structural component of the cytoskeleton and participate in cell division, intracellular transport, and cell morphogenesis. In the present study, 795 cotton tubulin expressed sequence tags were analysed and 19 beta-tubulin genes (TUB) cloned from a cotton cDNA library. Among the group, 12 cotton TUBs (GhTUBs) are reported for the first time here. Transcription profiling revealed that nine GhTUBs were highly expressed in elongating fibre cells as compared with fuzzless-lintless mutant ovules. Treating cultured wild-type cotton ovules with exogenous phytohormones showed that individual genes can be induced by different agents. Gibberellin induced expression of GhTUB1 and GhTUB3, ethylene induced expression of GhTUB5, GhTUB9, and GhTUB12, brassinosteroids induced expression of GhTUB1, GhTUB3, GhTUB9, and GhTUB12, and lignoceric acid induced expression of GhTUB1, GhTUB3, and GhTUB12. When GhTUBs were transformed into the Saccharomyces cerevisiae inviable mutant, tub2, which is deficient in beta-tubulin, one ovule-specific and eight of nine fibre-preferential GhTUBs rescued this lethality. This study suggests that the proteins encoded by cotton GhTUBs are involved during cotton fibre development.

[Study on selective breeding of medicinal Chrysanthemum morifolium].

OBJECTIVE: To investigate botanical characters and yield of four cultivars of Chrysanthemum morifolium for further study on their genetic diversity and selective breeding. METHOD: The characters were observed and yield was investigated by field randomized block and analysis of variance. RESULT AND CONCLUSION: The botanical characters are difference among four cultivars; the amount of single flower head is the main factor influencing on the output of Chrysanthemum morifolium (r = 0.925); the yield of "Hongxinju" and "Xiaobaiju" are remarkably higher than that of "Dabaiju" and "Changbanju".

[Salt-tolerance evaluation of seedlings of medicinal Chrysanthemum morifolium cultivar].

OBJECTIVE: To evaluate salt tolerance of seedlings of 4 medicinal C. morifolium cultivars to be transplanted, and to expand the planting area. METHOD: Seedlings were cultivated in hoagland nutrient solution containing various concentrations of NaCl for 30 days. The height, dry weight and chlorophyll content were investigated. Identification index mainly in relative growth rate, the evaluation of NaCl effects on the growth, K+, Na+ and Cl- distribution in seedlings were surveyed. RESULT AND CONCLUSION: The salt tolerance was difference among four cultivars of C. morifolium. The salt tolerance of "Dabaiju" and "Changbanju" was weak, while "Hongxinju" and "Xiaobaiju" was strong. "Hongxinju" and "Xiaobaiju" may be planted in salinte soil area.

[Comparative study on pesticide and heavy metal residuals of four cultivars of Chrysanthemum morifolium].

OBJECTIVE: To study organochlorine pesticide and heavy metal residuals of Chrysanthemum morifolium. METHOD: The contents of organochlorine pesticide residuals was determined by chromatography, Cu, Pb, Cd was determined by AAS, and As, Hg was determined by AFS. RESULT AND CONCLUSION: The contents of organochlorine pesticide and heavy metals residuals are up to "Green Trade Standards of Importing & Exporting Medicinal plants & Preprations" (MOFTEC, 2001), but the contents are different among four cultivars clearly.

[Comparative study on internal quality of four cultivars of Chrysanthemum morifolium].

OBJECTIVE: To study the genetic diversity of C. morifolium on the chemical constituents. METHOD: Chemical constituents of four cultivars cultivated with the same conditions were compared in three types of index: chlorogenic acid, flavonoid and volatile oil. RESULT AND CONCLUSION: With different cultivars and processing methods, the contents of chlorogenic acid, flavonoid and volatile oil extracted from C. morifolium vary great extent.