The mechanism study of inhibition effect of prepared Radix Rehmanniainon combined with Radix Astragali osteoporosis through PI3K-AKT signaling pathway.

PURPOSE: To observe the mechanism of prepared Radix Rehmanniainon combined with Radix Astragali in treating osteoporosis. METHODS: Osteoporosis rat model was established by bilateral ovariectomy combined with low-calcium diet feeding. Bone mineral density was measured by bone densitometer. Bone metabolism markers in serum were detected by enzyme linked immunosorbent assay (ELISA), bone tissue structure was observed by hematoxylin-eosin staining, and the effect of prepared Radix Rehmanniainon combined with Radix Astragali on PI3K-AKT signaling pathway was investigated by immunohistochemistry and reverse transcription polymerase chain reaction. RESULTS: Compared with the model group, the bone tissue structure and imbalance of bone metabolism were improved, and the bone mineral density was significantly increased in the prepared Radix Rehmanniainon combined with Radix Astragali groups. After intervention with prepared Radix Rehmanniainon combined with Radix Astragali, the positive expression of PIK3CA and Akt1 in rat bone tissue was enhanced, and the expression levels of Akt1 mRNA were significantly increased. CONCLUSIONS: Prepared Radix Rehmanniainon combined with Radix Astragali may treat osteoporosis by activating PI3K/AKT pathway.

First report of Pectobacterium carotovorum and Pectobacterium brasiliense causing bacterial soft rot of bok choy in Taiwan.

Bok choy (Brassica rapa var. chinensis) is one of the most popular leafy green vegetables in Asia (Wang et al. 2019; Zhang et al. 2014). In May 2022, disease resembling bacterial soft rot was observed in a commercial greenhouse located in Xiluo, Yunlin County, Taiwan. Affected plants exhibited maceration, primarily close to the base of the plants (Fig. S1). Almost all bok choy plants (about 1,800 plants in total) on site were symptomatic. Macerated tissues were collected from six plants. The samples were homogenized in 10 mM MgCl2 and bacteria were isolated on nutrient agar (NA) by streak plating. After 1 day of culturing at 28°C, creamy-white, round colonies were consistently grown on all the plates, and six strains (Br1 to Br6) were obtained; each isolated from a different plant. The strains were able to ferment glucose and induced maceration on potato tuber slices (Schaad et al. 2001) but could not produce indigoidine on NGM medium (NA added with glycerol and MnCl2; Lee and Yu 2006). The DNA samples of these strains were tested with Pectobacterium-specific primers Y1 and Y2 (Darrasse et al. 1994) and all samples produced the expected amplicon. To identify the isolated pathogens, 1,592-bp sequences concatenated from fragments of the leuS (452 bp), dnaX (492 bp), and recA (648 bp) genes (GenBank accession nos. OP360013-OP360021) were obtained for each strain as previously described (Portier et al. 2019). Three genotypes were detected, the sequences of strains Br1, Br2, Br4, and Br5 were identical, while strains Br3 and Br6 each belong to a different genotype. The sequence identity between Br3 and Br6 was 98.2%. The concatenated sequences (dnaX-leuS-recA), along with those of type strains from known Pectobacterium species, were subjected to maximum likelihood analysis. The reconstructed trees showed that strains Br1, Br2, Br4, and Br5 grouped with P. carotovorum CFBP2046T (Fig. S2); the sequence identity between the isolated strains and the type strain was 98.7%. Strains Br3 and Br6 clustered with P. brasiliense CFBP6617T (Fig S2); the sequence identity between CFBP6617T and Br3 and Br6 were 97.5% and 98.4%, respectively. The six strains were inoculated onto 55-day-old bok choy plants using previously described prick inoculation methods (Wei et al. 2019). Autoclaved toothpicks, each carrying 9.3 x 106- 5.6 x 107 cfu of bacteria, were used to inoculate the base of plant leaves. All six strains were tested, and each strain had three replicates. Plants in the control group were stabbed with bacteria-free toothpicks. The plants were enclosed in clear plastic bags during the assay to maintain humidity and kept in a growth chamber (27/25°C day/night; 14-h photoperiod). After 1 d, all inoculated plants produced soft rot symptoms resembling those observed in the sampling site. No noticeable differences were observed among symptoms produced by different strains. The controls were symptomless. One strain was re-isolated from each treatment group and their identity were confirmed by sequencing the dnaX gene. All re-isolated strains shared the same sequences with those of the original strains tested. This is the first report of P. brasiliense and P. carotovorum causing bacterial soft rot of bok choy in Taiwan. Importantly, the findings showed that different Pectobacterium species and genotypes could induce symptoms on a crop in the same facility at the same time, highlighting the potential complexity of interactions among different soft rot bacteria in the environment.

The mechanism study of inhibition effect of prepared Radix Rehmanniainon combined with Radix Astragali osteoporosis through PI3K-AKT signaling pathway

Purpose: To observe the mechanism of prepared Radix Rehmanniainon combined with Radix Astragali in treating osteoporosis. Methods: Osteoporosis rat model was established by bilateral ovariectomy combined with low-calcium diet feeding. Bone mineral density was measured by bone densitometer. Bone metabolism markers in serum were detected by enzyme linked immunosorbent assay (ELISA), bone tissue structure was observed by hematoxylin-eosin staining, and the effect of prepared Radix Rehmanniainon combined with Radix Astragali on PI3K-AKT signaling pathway was investigated by immunohistochemistry and reverse transcription polymerase chain reaction. Results: Compared with the model group, the bone tissue structure and imbalance of bone metabolism were improved, and the bone mineral density was significantly increased in the prepared Radix Rehmanniainon combined with Radix Astragali groups. After intervention with prepared Radix Rehmanniainon combined with Radix Astragali, the positive expression of PIK3CA and Akt1 in rat bone tissue was enhanced, and the expression levels of Akt1 mRNA were significantly increased. Conclusions: Prepared Radix Rehmanniainon combined with Radix Astragali may treat osteoporosis by activating PI3K/AKT pathway.

Effect of iguratimod on diclofenac metabolism by CYP2C9 in rats and human recombinant CYP2C9 yeast cells

Iguratimod (IGU, also known as T-614), a novel disease modifying antirheumatic drug intended to cure patients with rheumatoid arthritis (RA). The purpose of this study is to evaluate the effect of IGU on the pharmacokinetics of CYP2C9 probe drug diclofenac and its metabolite 4′-hydroxy diclofenac in vivo and in vitro. In in vivo experiments, 24 rats were randomly assigned to three groups consisting of the control group (Normal saline), low dose IGU group (10 mg/kg) and high dose IGU group (30 mg/kg). Blood samples were collected from orbital sinuses vein before 1 hour and serial times of giving diclofenac (15 mg/kg) to all the rats. Plasma concentration of diclofenac and its metabolite 4´-hydroxy diclofenac were assayed by high performance liquid chromatography. Pharmacokinetic parameters were assessed by Winnonlin 6.4 pharmacokinetic software. Moreover, in vitro studies were performed in recombinant human CYP2C9 yeast cell system. IGU at low dose showed no significant differences in the pharmacokinetic parameters of diclofenac and 4-hydroxy diclofenac in vivo when compared with control group (p>0.005). However, at the high dose of IGU, the pharmacokinetic parameters of 4´-hydroxy metabolite of diclofenac increase in half-life (T1/2) and mean area under the curve (AUC0→24), while a decrease in mean clearance (CL, mL/h/kg) and volume of distribution Vz (mL/kg). In addition, in in vitro study, high doses of IGU reduces the metabolism rate of diclofenac. IGU at high dose significantly increase the pharmacokinetics parameters of 4´-hydroxy diclofenac in rats. Additionally, it also showed the potent inhibitory effect on diclofenac metabolism in recombinant human CYP2C9 yeast cells.

Association between driving pressure and development of postoperative pulmonary complications in patients undergoing mechanical ventilation for general anaesthesia: a meta-analysis of individual patient data.

BACKGROUND: Protective mechanical ventilation strategies using low tidal volume or high levels of positive end-expiratory pressure (PEEP) improve outcomes for patients who have had surgery. The role of the driving pressure, which is the difference between the plateau pressure and the level of positive end-expiratory pressure is not known. We investigated the association of tidal volume, the level of PEEP, and driving pressure during intraoperative ventilation with the development of postoperative pulmonary complications. METHODS: We did a meta-analysis of individual patient data from randomised controlled trials of protective ventilation during general anesthaesia for surgery published up to July 30, 2015. The main outcome was development of postoperative pulmonary complications (postoperative lung injury, pulmonary infection, or barotrauma). FINDINGS: We included data from 17 randomised controlled trials, including 2250 patients. Multivariate analysis suggested that driving pressure was associated with the development of postoperative pulmonary complications (odds ratio [OR] for one unit increase of driving pressure 1·16, 95% CI 1·13-1·19; p<0·0001), whereas we detected no association for tidal volume (1·05, 0·98-1·13; p=0·179). PEEP did not have a large enough effect in univariate analysis to warrant inclusion in the multivariate analysis. In a mediator analysis, driving pressure was the only significant mediator of the effects of protective ventilation on development of pulmonary complications (p=0·027). In two studies that compared low with high PEEP during low tidal volume ventilation, an increase in the level of PEEP that resulted in an increase in driving pressure was associated with more postoperative pulmonary complications (OR 3·11, 95% CI 1·39-6·96; p=0·006). INTERPRETATION: In patients having surgery, intraoperative high driving pressure and changes in the level of PEEP that result in an increase of driving pressure are associated with more postoperative pulmonary complications. However, a randomised controlled trial comparing ventilation based on driving pressure with usual care is needed to confirm these findings. FUNDING: None.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells.

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.

Simultaneous application of BrdU and WST-1 measurements for detection of the proliferation and viability of airway smooth muscle cells

BACKGROUND: BrdU is a commonly used reagent in cell proliferation assays, and WST-1 measurement is widely used to detect cell viability. However, no previous study has formally reported the combination of the two assays, which may be used to detect the proliferation and viability simultaneously. In this study, we examined the effect of adding BrdU 2 h prior to the WST-1 assay and tried to test the possibility of the combined detection using rat airway smooth muscle cells. RESULTS: The WST-1 measurements obtained from the combined detection were consistent with those obtained from the separate detection, which suggested that the addition of BrdU 2 h prior to the WST-1 analysis did not affect the WST-1 results. The BrdU measurements obtained from the combined detection also demonstrated the same trend as that obtained from the separate detection, and dosages of 200, 400 and 800 ng/ml testing reagent significantly inhibited the proliferation of rat airway smooth muscle cells. CONCLUSIONS: Our study suggests that the BrdU and WST-1 measurements can be applied simultaneously without mutual interference, which may increase the efficacy and consistency of these measurements to a certain extent.