Simultaneous determination of twenty-nine active compounds in fuzhengjiedu granules by HPLC-QQQ-MS/MS.

As an empirical medicine of traditional Chinese medicine, Fuzhengjiedu Granules have shown an effect against COVID-19 in clinical and inflammatory animal models. It is formulated with eight herbs, including Aconiti Lateralis Radix Praeparata, Zingiberis Rhizoma, Glycyrrhizae Radix Et Rhizoma, Lonicerae Japonicae Flos, Gleditsiae Spina, Fici Radix, Pogostemonis Herba, and Citri Reticulatae Pericarpium. This study established a high-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) method by simultaneously determining 29 active compounds in the granules with significant content differences. Separation by gradient elution using acetonitrile and water (0.1% formic acid) as mobile phases was performed on a Waters Acquilty UPLC T3 column (2.1 mm × 100 mm, 1.7 µm). A triple quadrupole mass spectrometer, operating in positive and negative ionization modes, was used for multiple reaction monitoring to detect the 29 compounds. All calibration curves showed good linear regression (r2 > 0.998). RSDs of precision, reproducibility, and stability of active compounds were all lower than 5.0%. The recovery rates were 95.4-104.9%, with RSDs< 5.0%. This method was successfully used to analyze the samples, and the results showed that 26 representative active components from 8 herbs were detected in the granules. While aconitine, mesaconitine, and hypaconitine were not detected, indicating that the existing samples were safe. The granules had the maximum and minimum content of hesperidin (27.3 ± 0.375 mg/g) and benzoylaconine (38.2 ± 0.759 ng/g). To conclude, a fast, accurate, sensitive, and reliable HPLC-QQQ-MS/MS method was established, which can simultaneously detect 29 active compounds that have a considerable difference in the content of Fuzhengjiedu Granules. This study can be used to control the quality and safety of Fuzhengjiedu Granules and provide a basis and guarantee for further experimental research and clinical application.

Response of salinity gradient power generation to inflow mode and temperature difference by reverse electrodialysis.

Sustainable utilization has been becoming the core idea of concentrated seawater disposal, which makes the harvest of salinity gradient power based on reverse electrodialysis (RED) become one of the important ways. As the important factors affecting RED performance, different flow orientations along the membrane and solution temperature have been studied in the previous researches. However, there are still some details that need to be clarified. In this study, the inflow mode was further detailed investigated. The results showed that after eliminating the interference of bubbles in the counter-current, the co-current was still better than the counter-current; when the solution of HCC (high concentration compartment) and LCC (low concentration compartment) was circulated for 3 h, the concentration of concentrated seawater discharge liquid was reduced by 6.93%, which was conducive to reducing the negative impact on the marine ecological environment. Meanwhile, the response of salinity gradient power generation to temperature difference was that high temperature had a positive effect on power density, and the order was both the HCC and LCC (0.44 W m-2) > LCC (0.42 W m-2) > HCC (0.39 W m-2). Although the RED performance was more sensitive to the temperature rise of LCC, the positive temperature difference between HCC and LCC is a more practical advantage because the temperature of concentrated seawater in HCC is usually high. These new observations could provide supports for the industrial development of RED in generating electricity economically and reducing the negative environmental impact of concentrated seawater.

Recovery of a patient with severe COVID-19 by acupuncture and Chinese herbal medicine adjuvant to standard care.

There is currently no drug or therapy that can cure the coronavirus disease 2019 (COVID-19), which is highly contagious and can be life-threatening in severe cases. Therefore, seeking potential effective therapies is an urgent task. An older female at the Leishenshan Hospital in Wuhan, China, with a severe case of COVID-19 with significant shortness of breath and decrease in peripheral oxygen saturation (SpO2), was treated using manual acupuncture and Chinese herbal medicine granule formula Fuzheng Rescue Lung with Xuebijing Injection in addition to standard care. The patient's breath rate, SpO2, heart rate, ratio of neutrophil/lymphocyte (NLR), ratio of monocyte/lymphocyte (MLR), C-reactive protein (CRP), and chest computed tomography were monitored. Acupuncture significantly improved the patient's breathing function, increased SpO2, and decreased her heart rate. Chinese herbal medicine might make the effect of acupuncture more stable; the use of herbal medicine also seemed to accelerate the absorption of lung infection lesions when its dosage was increased. The combination of acupuncture and herbs decreased NLR from 14.14 to 5.83, MLR from 1.15 to 0.33 and CRP from 15.25 to 6.01 mg/L. These results indicate that acupuncture and Chinese herbal medicine, as adjuvants to standard care, might achieve better results in treating severe cases of COVID-19.

Shegan-Mahuang Decoction ameliorates asthmatic airway hyperresponsiveness by downregulating Th2/Th17 cells but upregulating CD4+FoxP3+ Tregs.

ETHNOPHARMACOLOGICAL RELEVANCE: Shegan-Mahuang Decoction (SMD), also named Yakammaoto or Shegan-Mahuang Tang, is a classic formula of traditional Chinese medicine with nine herbs, including Asarum sieboldii Miq., Aster tataricus L.f., Ephedra sinica Stapf, Belamcanda chinensis (L.) Redouté, Pinellia ternata (Thunb.) Breit., Schisandra chinensis (Turcz.) Baill., Tussilago farfara L., Zingiber officinale Roscoe, and Ziziphus jujuba Mill. SMD was originally discovered by Zhang Zhongjing in Eastern Han dynasty. It has been widely used as traditional medicine to treat flu-like symptoms in China and Japan for around twenty centuries. It was also utilized for the treatment of the early stage of acute asthma. However, the immune mechanisms underlying its therapeutic effects remain unknown. AIM OF THE STUDY: This study was set to investigate the effects of SMD on asthmatic airway hyperresponsiveness and its impacts on adaptive immunity in a mouse model of asthma. MATERIALS AND METHODS: The HPLC fingerprint profile of the water extract of SMD recorded 22 peaks, including those equivalent to guanosine, chlorogenic acid, tectoridin, 6-gingerol and wuweizisu B, as described previously (Yen et al., 2014). Airway hyperresponsiveness was assessed by measuring the airway resistance. Cellular infiltration was measured via H&E staining and immunochemistry while gene expression was analyzed using real-time RT-PCR. Treg frequency was determined through flow analysis whereas cytokine production in the supernatant was evaluated using ELISA. Finally, mTOR and NF-kB signalings were analyzed via Western blotting. RESULTS: We found that SMD largely corrected the imbalance of Th cell subsets in asthmatic mice with a significant inhibition of Th2 and Th17 cytokine production, thereby reducing asthmatic airway hyperresponsiveness. Moreover, lung function tests showed that SMD reduced airway hyperresponsiveness while immunohistochemical analyses demonstrated that SMD attenuated pulmonary infiltration of CD3+ and CD4+ T cells. Further, we observed a significant increase in the proportion of CD4+Foxp3+ Tregs in SMD-treated asthmatic mice. We also found that SMD downregulated gene expression of GATA3 and ROR-γt in murine lung tissue. In addition, both mTOR- and NF-kB-related protein expressions were reduced in the lung tissue of SMD-treated mice. SMD inhibited Th2/Th17 cytokine production by CD4+ T cells and also their mTOR activity in vitro. CONCLUSIONS: Our findings demonstrate that SMD attenuates asthmatic airway hyperresponsiveness by hindering Th2/Th17 differentiation, promoting CD4+FoxP3+ Treg generation and suppressing mTOR and NF-kB activities.

A Novel Immunosuppressant, Luteolin, Modulates Alloimmunity and Suppresses Murine Allograft Rejection.

An allograft is rejected in the absence of any immunosuppressive treatment because of vigorous alloimmunity and thus requires extensive immunosuppression for its survival. Although there are many conventional immunosuppressants for clinical use, it is necessary to seek alternatives to existing drugs, especially in case of transplant patients with complicated conditions. Luteolin, a natural ingredient, exists in many plants. It exhibits multiple biological and pharmacological effects, including anti-inflammatory properties. In particular, luteolin has been shown to upregulate CD4+CD25+ regulatory T cells (Tregs) in the context of airway inflammation. However, it remains unknown whether luteolin regulates alloimmune responses. In this study, we demonstrated that luteolin significantly prolonged murine skin allograft survival, ameliorated cellular infiltration, and downregulated proinflammatory cytokine gene expression in skin allografts. Furthermore, luteolin increased the percentage of CD4+Foxp3+ Tregs while reducing frequency of mature dendritic cells and CD44highCD62Llow effector CD4+/CD8+ T cells posttransplantation. It also suppressed the proliferation of T cells and their production of cytokines IFN-γ and IL-17A in vitro while increasing IL-10 level in the supernatant. Moreover, luteolin promoted CD4+Foxp3+ Treg generation from CD4+CD25- T cells in vitro. Depleting Tregs largely, although not totally, reversed luteolin-mediated extension of allograft survival. More importantly, luteolin inhibited AKT/mTOR signaling in T cells. Thus, for the first time, to our knowledge, we found that luteolin is an emerging immunosuppressant as an mTOR inhibitor in allotransplantation. This finding could be important for the suppression of human allograft rejection, although it remains to be determined whether luteolin has an advantage over other conventional immunosuppressants in suppression of allograft rejection.

Chinese herbal medicine for the treatment of cough variant asthma: a study protocol for a double-blind randomized controlled trial.

BACKGROUND: Cough variant asthma (CVA) is one of the leading causes of chronic coughing. The main treatment is currently anti-inflammatory medication. However, the coughing may return or be aggravated and lung function may deteriorate once the anti-inflammatory treatment is stopped. The effect of Chinese herbal medicine (CHM) on chronic coughing is remarkable, but high-quality evidence supporting its effectiveness is still lacking. This trial aims to evaluate the safety and efficacy, especially the long-term efficacy, of CHM plus anti-inflammatory medications for the treatment of CVA. METHODS/DESIGN: A randomized placebo-controlled double-blind trial will be conducted. It will consist of a 3-month intervention followed by a 6-month follow-up period. The target sample size is 60 patients with CVA who are between 18 and 70 years old. The eligible subjects will be allocated randomly into the experimental or control group in a ratio of 1:1. Patients in the experimental group will take CHM granules (4.9 g twice daily), while patients in the control group will be given a matched placebo. An administration of salmeterol/fluticasone propionate combination for 12 weeks will be the basic therapy for the two groups. The primary outcome is the cough visual analog scales (CVAS). The secondary outcomes include quality of life, rate of symptom relapse, lung function, and blood tests. A safety assessment will also be performed during the trial. DISCUSSION: The evidence gathered by the trial will be a valuable addition to informing treatment options for patients with CVA. TRIAL REGISTRATION: http://www.chictr.org.cn , ID: ChiCTR-IOR-16009148. Registered on 3 September 2016.

Anti-inflammatory effects of rosmarinic acid-4-O-ß-D-glucoside in reducing acute lung injury in mice infected with influenza virus.

Rosmarinic acid-4-O-ß-D-glucoside (RAG) is a dicaffeoyl phenolic compound isolated from Sarcandra glabra (Thunb.) Nakai. Preliminary studies show that RAG has significant anti-inflammatory properties and can alleviate ear swelling in mice and the paw swelling in rats. Here, the anti-influenza effects of RAG were investigated in mice infected with A/FM/1/47 H1N1 virus. The survival rate and body weight were observed, the lung edema, virus copies, inflammatory cytokines (including IL-4, IL-5, TNF-α and IFN-γ) and oxidative damage indexes (including SOD, MDA, NO, and CAT) were measured. Moreover, immune cell recruitment in alveoli was measured with white blood cells and differential counts. Therapeutic RAG concentrations substantially improve the symptoms, mitigate body weight loss and alleviate lung edema induced by virus, thus improve survival protection effects. Furthermore, RAG was shown to regulate influenza virus-induced inflammatory cytokine expression, specifically by downregulating the Th1 cell cytokines IFN-γ, TNF-α and upregulating the Th2 cell cytokines IL-4, IL-5. Cell migration and infiltration were also diminished after RAG administration.

[Recognition of Similarities and Differences--a New Thinking Paradigm of Critically Care Integrative Medicine].

"Wise men could recognize similarities, but the fool only recognizes differences" in Su-wen, which expounded clinical thinking methods of Chinese medicine (CM). "To recognize similarities and differences simultaneously" is of important clinical significance in understanding the laws of diseases. CM pays much attention to recognize similarities, while modern medicine emphasizes the differences observed. In order to develop integrative medicine (IM), similarities recognition and differences identification must be combined together to innovate new thinking methods of IM.

[Atypical teratoid/rhabdoid tumors of central nervous system in childhood: a clinical and histopathologic study of 6 cases].

OBJECTIVE: To study the clinicopathologic features, immunohistochemical findings, diagnosis and differential diagnosis of atypical teratoid/rhabdoid tumors (AT/RT) of central nervous system in childhood. METHODS: The clinicopathologic data, morphologic features and immunophenotypes were reviewed in 6 cases of AT/RT. EnVision method was applied. Antibodies include cytokeratin (CK), epithelial membrane antigen (EMA), vimentin, smooth muscle actin (SMA), muscle specific actin (MSA), glial fibrinary acid protein (GFAP), desmin, placental alkaline phosphatase (PLAP) and INI1. RESULTS: Five of the six cases of AT/RT occurred in infancy and early childhood. Histologically, the predominant component was rhabdoid cells. Cytoplasmic inclusions were present in all cases. Primitive neuroectodermal tumor (PNET) component was also identified in 5 of the 6 cases studied. Immunohistochemically, the tumor cells were positive for cytokeratin, epithelial membrane antigen and vimentin. The staining for INI1, desmin and PLAP was negative. Smooth muscle actin was expressed in 2 cases and glial fibrillary acidic protein in 5 cases. The proliferative index as demonstrated by Ki-67 staining was high. CONCLUSIONS: AT/RT is not a particularly uncommon malignancy in childhood. The histologic hallmark is the presence of rhabdoid cells with cytoplasmic inclusions. The tumor cells are positive for cytokeratin, epithelial membrane antigen and vimentin, and negative for INI1. Differential diagnosis includes PNET, medulloblastoma and medullomyoblastoma.

[Correlation between typing of peripheral neuroblastic tumors and prognosis: a clinicopathologic study of 135 cases].

OBJECTIVE: To study the clinicopathologic characteristics of peripheral neuroblastic tumors and to investigate the prognostic significance of International Neuroblastoma Pathology Classification (INPC). METHODS: One hundred and thirty-five cases of peripheral neuroblastic tumors encountered in Shanghai Children's Medical Center were enrolled into the study. All the cases were classified according to INPC and International Neuroblastoma Staging System (INSS). The follow-up data were analyzed. RESULTS: The consensus diagnoses of the 135 cases were as follows: 80 cases (59.2%) of neuroblastoma, 24 cases (17.8%) of ganglioneuroblastoma, intermixed, 17 cases (12.6%) of ganglioneuroma and 14 cases (10.4%) of ganglioneuroblastoma, nodular. The cases were subdivided into 2 subgroups: favorable histology (number = 90, 66.7%) and unfavorable histology (number = 45, 33.3%). According to INSS, the number of cases in stages I, II, III and IV was 22 (16.3%), 24 (17.8%), 34 (25.2%) and 55 (40.7%), respectively. The survival of peripheral neuroblastic tumors correlated with histologic diagnosis, INPC and INSS (P < 0.05). CONCLUSION: Diagnostic categorization of peripheral neuroblastic tumors according to INPC is of prognostic value.

Glial fibrillary acidic protein expression is an indicator of teratoma maturation in children.

BACKGROUND: The present diagnosis of teratomas is limited to visual examination of their tissues. For the sake of treatment, teratomas are graded according to degrees of nerve tissue maturation. Mature fetal nerve tissue contains the astrocyte-specific intermediate filament protein, the glial fibrillary acidic protein (GFAP). This study aimed to investigate GFAP expressions in the nerve tissue of immature and mature teratomas, and to evaluate if GFAP is indicative of teratoma maturation in pediatric patients. METHODS: Nerve tissue specimens were collected from immature (10 children) and mature teratomas (45 children). Nerve tissue specimens as a control group were taken from 33 children with neuroblastoma. GFAP expression of the specimens was studied by immunohistochemical and semi-quantitative analyses. RESULTS: GFAP expression was low in the nerve tissue of immature teratomas and high in that of mature ones. A semi-quantitative analysis confirmed statistically significant difference between the GFAP expressions of immature and mature teratomas (P=0.0001). CONCLUSION: GFAP is highly expressed in the nerve tissue of mature teratomas and low in that of immature ones, suggesting that the GFAP expression is a meaningful indicator of teratoma maturation. It is helpful for pathologists to diagnose and classify teratomas.

Leydig cell transplantation restores androgen production in surgically castrated prepubertal rats.

Prepubertal testicular dysfunction and the subsequent development of hypogonadism affects an estimated one in 200 children worldwide. As the testosterone levels are dynamic during development and puberty, traditional hormone treatment regimens are often inadequate, thereby leaving associated physiological conditions unresolved. Therefore, we have investigated the potential therapeutic effect of mature Leydig cell transplantation for the treatment of prepubertal primary hypogonadism through the use of a surgically induced hypogonadistic rat model system. In the experiment, Leydig cells were surgically isolated from mature Sprague-Dawley rats and transplanted into prepubertal recipients. Serum testosterone levels and microscopic analysis of the stained testicular interstitium were compared with sham-treated controls, as well as with castrated and intact rats during sexual development. At 4 weeks post-implantation, serum testosterone was detectable in Leydig cell recipients, but not in surgical controls, and progressively increased as a function of time until reaching levels comparable with sexually mature males at 12 weeks post-implantation. Histological analysis revealed a high rate of Leydig cell survival as well as steroidogenic secretory activity. Therefore, we conclude that mature Leydig cell transplantation in prepubertal hypogonadism recipients has therapeutic potential in rats and merits further investigation for clinical application.

[Unusual expression and molecular mechanisms of E-cadherin, beta-catenin in correlation with clinicopathologic parameters in neuroblastoma].

OBJECTIVE: To study the expression of E-cadherin and beta-catenin in neuroblastomas of various degrees of differentiation, and to investigate their molecular mechanisms in correlation with clinicopathologic parameters. METHODS: Immunohistochemistry EnVision method was used to detect E-cadherin and beta-catenin expression in 90 paraffin-embedded tissue samples of neuroblastomas. The methylation status of CpG islands of E-cadherin promoter was investigated by MSP in 7 fresh tissue and 24 paraffin-embedded tissue samples. The mutation status of exon 3 of beta-catenin gene was studied by PCR in 7 fresh tissue samples. Statistical analysis of the data was performed by SPSS software. RESULTS: E-cadherin and beta-catenin were abnormally expressed in neuroblastomas in general. The expression of beta-catenin in well-differentiated neuroblastoms was markedly higher (47/70, 67.1%) than that of the poorly differentiated tumors (8/20, 40.0%). There was a markedly decreased expression of both genes in tumors with lymph node metastasis than those without. Demethylation was seen in some regions of the promoter of E-cadherin in 31 cases of nuroblatomas. PCR of the exon 3 of beta-catenin followed by DNA sequencing demonstrated rearrangements and mutations in 7 cases, including 2 cases harboring identical point mutation at gene position 27184, leading to a T-->A alteration. CONCLUSIONS: The abnormal over-expression of E-cadherin in neuroblastomas is independent of the methylation status of their promoter sequences. The abnormal expression of beta-catenin may be related to mutational changes at exon 3 of the gene.

[Metanephric stromal tumor: a pathologic study of two cases].

OBJECTIVE: To study the clinicopathologic features of metanephric stromal tumor (MST), with emphasis on diagnostic criteria. METHODS: The clinicopathologic findings in 2 cases of MST were analyzed and the literature of this entity was reviewed. RESULTS: Cases of MST were unilateral and mostly centered in renal medulla. The tumor was separated from adjacent renal tissue by sharp and scalloped borders. Entrapped tubules and glomeruli were commonly seen within the lesion. The tumor cells were spindle to stellate in shape and arranged in a nodular pattern. On low power examination, alternating areas of high and low tumor cellularity were noted. Characteristically, there were onion skin-like concentric cuffs of tumor cells around entrapped tubules. The small intratumoral vasculatures showed irregular thickening ("angiodysplasia"). Immunohistochemical study demonstrated that the tumor cells diffusely expressed CD34. CONCLUSIONS: Which the tumor cells around the entrapped renal tubules and blood vessels imparts a nodular appearance, as well as the tumor cells labbed for CD34 are the highly characteristic pathologic findings of MST.

[Expression of KAI1/CD82 in neuroblastoma and its correlation to prognosis].

BACKGROUND & OBJECTIVE: KAI1/CD82 was recently detected as a tumor metastasis suppressor gene. Its silencing contributes to progression and infiltration of some tumors. Our study was designed to investigate the expression of KAI1/CD82 in neuroblastoma, and explore its correlation to clinicopathologic characteristics and prognosis of patients with neuroblastoma. METHODS: The EnVision immunohistochemistry was used to detect the expression of KAI1/CD82 in 90 specimens of neuroblastoma (28 specimens of ganglioneuroblastoma and 62 specimens of neuroblastoma). Clinical data and follow-up data of the 90 patients were analyzed. RESULTS: Positive rate of KAI1/CD82 was significantly higher in ganglioneuroblastoma than in neuroblastoma (39.3% vs. 14.5%, P=0.014). Its expression was negatively correlated to clinical stage of neuroblastoma (P=0.003). CONCLUSIONS: The change of KAI1/CD82 expression is an early event in tumorigenesis of neuroblastoma. Its down-regulation may be considered as a potential indicator to judge the differentiation and metastasis of neuroblastoma, which can serve as one of the combined indexes to clinical assessment of prognosis.

[Eosinophilic cystitis in children:a report of six cases and review of literature].

OBJECTIVE: Eosinophilic cystitis is a very rare tumor-like inflammatory disease especially in children. It can involve any portion of the bladder and interfere with its function. Here we present 6 pediatric cases and review the literature. METHODS: Six children, with mean age of 8.3 years, were referred to our clinic. Their major complaints included voiding urgency, frequency, dysuria, enuresis, intermittent hematuria and abdominal pain. Ultrasound, CT scan and voiding cystourethrogram (VCUG) showed diffuse thickening of bladder wall and a filling-defect lesion. They all suggested the possibility of bladder tumor. The final diagnosis of eosinophilic cystitis was made by cystoscopic tissue biopsy or open surgery. Patients underwent bladder autoaugmentation, mass resection or mere oral administration of corticosteroid and antibiotics respectively. RESULTS: 5 among 6 patients were followed up for 5 - 28 months. Their voiding complaints and image changes gradually disappeared. Up to now, there is no relapse. CONCLUSIONS: The clinical presentation of eosinophilic cystitis includes irritative voiding symptoms, hematuria and abdominal pain. Tumor-like changes could be found in the imagination examinations. A biopsy is mandatory to establish the diagnosis. Treatment in general varies from the removal of the antigenic stimulus, if identifiable, to administration of corticosteroid and antibiotics. When patients fail to respond to the medication treatment, the opening operation could be required.