Diazepam inhibits LPS-induced pyroptosis and inflammation and alleviates pulmonary fibrosis in mice by regulating the let-7a-5p/MYD88 axis.

BACKGROUND AND OBJECTIVE: Pulmonary fibrosis caused by lung injury is accompanied by varying degrees of inflammation, and diazepam can reduce the levels of inflammatory factors. Therefore, the purpose of this study was to determine whether diazepam can inhibit inflammation and ameliorate pulmonary fibrosis by regulating the let-7a-5p/myeloid differentiation factor 88 (MYD88) axis. METHODS: Lipopolysaccharide (LPS) was used to induce cell pyroptosis in an animal model of pulmonary fibrosis. After treatment with diazepam, changes in cell proliferation and apoptosis were observed, and the occurrence of inflammation and pulmonary fibrosis in the mice was detected. RESULTS: The results showed that LPS can successfully induce cell pyroptosis and inflammatory responses and cause lung fibrosis in mice. Diazepam inhibits the expression of pyroptosis-related factors and inflammatory factors; moreover, it attenuates the occurrence of pulmonary fibrosis in mice. Mechanistically, diazepam can upregulate the expression of let-7a-5p, inhibit the expression of MYD88, and reduce inflammation and inhibit pulmonary fibrosis by regulating the let-7a-5p/MYD88 axis. CONCLUSION: Our findings indicated that diazepam can inhibit LPS-induced pyroptosis and inflammatory responses and alleviate pulmonary fibrosis in mice by regulating the let-7a-5p/MYD88 axis.

[Discrete element simulation study of mixing process of Guizhi Fuling Capsules: parameter calibration].

The mixing process is a critical link in the formation of oral solid preparations of traditional Chinese medicine. This paper took the extract powder of Guizhi Fuling Capsules and Paeonol powder as research objects. The angle of repose, loose packing density, and particle size of the two powders were measured to calibrate discrete element simulation parameters for the mixing process. The discrete element method was used to calibrate the simulated solid density of Paeonol powder and extract powder of Guizhi Fuling Capsules based on the Hertz-Mindlin with JKR V2 contact model and particle scaling. The Plackett-Burman experimental design was used to screen out the critical contact parameters that had a significant effect on the simulation of the angle of repose. The regression model between the critical contact parameters and the simulated angle of repose was established by the Box-Behnken experimental design, and the critical contact parameters of each powder were optimized based on the regression model. The best combination of critical contact parameters of the extract powder of Guizhi Fuling Capsules was found to be 0.51 for particle-particle static friction coefficient, 0.31 for particle-particle rolling friction coefficient, and 0.64 for particle-stainless steel static friction coefficient. For Paeonol powder, the best combination of critical contact parameters was 0.4 for particle-particle static friction coefficient and 0.19 for particle-particle rolling friction coefficient. The best combination of contact parameters between Paeonol powder and extract powder of Guizhi Fuling Capsules was 0.27 for collision recovery coefficient, 0.49 for static friction coefficient, and 0.38 for rolling friction coefficient. The verification results show that the relative error between the simulated value and the measured value of the angle of repose of the two single powders is less than 1%, while the relative error between the simulated value and the measured value of the angle of repose of the mixed powder with a mass ratio of 1∶1 is less than 4%. These research results provide reliable physical property simulation data for the mixed simulation experiment of extract powder of Guizhi Fuling Capsules and Paeonol powder.

[Research progress and maturity assessment of continuous manufacturing of traditional Chinese medicine].

The pharmaceutical manufacturing model is gradually changing from intermittent manufacturing to continuous manufacturing and intelligent manufacturing. This paper briefly reviewed the supervision and research progress in continuous pharmaceutical manufacturing in China and abroad and described the definition and advantages of continuous pharmaceutical manufacturing. The continuous manufacturing of traditional Chinese medicine(TCM) at the current stage was summarized in the following three terms: the enhancement of the continuity of intermittent manufacturing operations, the integration of continuous equipment to improve physical continuity between units, and the application of advanced process control strategies to improve process continuity. To achieve continuous manufacturing of TCM, the corresponding key technologies, such as material property characterization, process modeling and simulation, process analysis technology, and system integration, were analyzed from the process and equipment, respectively. It was proposed that the continuous manufacturing equipment system should have the characteristics of high speed, high response, and high reliability, "three high(H~3)" for short. Considering the characteristics and current situation of TCM manufacturing, based on the two dimensions of product quality control and production efficiency, a maturity assessment model for continuous manufacturing of TCM, consisting of operation continuity, equipment continuity, process continuity, and quality control continuity, was proposed to provide references for the application of continuous manufacturing technology for TCM. The implementation of continuous manufacturing or the application of key continuous manufacturing technologies in TCM can help to systematically integrate advanced pharmaceutical technology elements and promote the uniformity of TCM quality and the improvement of production efficiency.

[Manufacturing classification system for oral solid dosage forms of traditional Chinese medicines (â ¡): classification of tablets disintegration behavior].

In this paper, 50 batches of representative traditional Chinese medicine tablets were selected and the disintegration time was examined with the method in Chinese Pharmacopoeia. The disintegration time and disintegration phenomenon were recorded, and the dissolution behaviors of water-soluble and ultraviolet-absorbent components during the disintegration process of tablets were characterized by self-control method. The results revealed that coating type and raw material type influenced the disintegration time of tablets. It was found that only 4% of traditional Chinese medicine tablets had obvious fragmentation during the disintegration process, while 96% of traditional Chinese medicine tablets showed gradual dissolution or dispersion. Furthermore, according to the disintegration speed, disintegration phenomenon, and whether the cumulative dissolution of measured components was > 90% at complete disintegration, a disintegration behavior classification system(DBCS) was created for the regular-release traditional Chinese medicine tablets. As a result, the disintegration behaviors of 50 batches of traditional Chinese medicine tablets were classified into four categories, i.e. ⅠA_2, ⅠB_1, ⅡB_1, and ⅡB_2. traditional Chinese medicine tablets(Class I) with disintegration time ≤ 30 min were defined to be rapid in disintegration, which can be the objective of optimization or improvement of Chinese herbal extract(semi extract) tablets. Different drug release models were used to fit the dissolution curve of traditional Chinese medicine tablets with gradual dissolution or dispersion phenomenon(i.e. Type B tablets). The results showed that the dissolution curves of water-soluble components in the disintegration process conformed to the zero order kinetics and the Ritger-Peppas model. It could be inferred that the disintegration mechanisms of type B tablets were a combination of dissolution controlled and swelling controlled mechanisms. This study contributes to understanding the disintegration behavior of traditional Chinese medicine tablets, and provides a reference for the design and improvement of disintegration performance of traditional Chinese medicine tablets.

[Patent application of intelligent quality control technology in traditional Chinese medicine production process: a review].

In the new stage for intelligent manufacturing of traditional Chinese medicine(TCM) from pilot demonstration to in-depth application and comprehensive promotion, how to raise the degree of intelligence for the process quality control system has become the bottleneck of the development of TCM production process control technology. This article has sorted out 226 TCM intelligent manufacturing projects that have been approved by the national and provincial governments since the implementation of the "Made in China 2025" plan and 145 related pharmaceutical enterprises. Then, the patents applied by these pharmaceutical enterprises were thoroughly retrieved, and 135 patents in terms of intelligent quality control technology in the production process were found. The technical details about intelligent quality control at both the unit levels such as cultivation, processing of crude herbs, preparation pretreatment, pharmaceutical preparations, and the production workshop level were reviewed from three aspects, i.e., intelligent quality sensing, intelligent process cognition, and intelligent process control. The results showed that intelligent quality control technologies have been preliminarily applied to the whole process of TCM production. The intelligence control of the extraction and concentration processes and the intelligent sensing of critical quality attributes are currently the focus of pharmaceutical enterprises. However, there is a lack of process cognitive patent technology for the TCM manufacturing process, which fails to meet the requirements of closed-loop integration of intelligent sensing and intelligent control technologies. It is suggested that in the future, with the help of artificial intelligence and machine learning methods, the process cognitive bottleneck of TCM production can be overcome, and the holistic quality formation mechanisms of TCM products can be elucidated. Moreover, key technologies for system integration and intelligent equipment are expected to be innovated and accelerated to enhance the quality uniformity and manufacturing reliability of TCM.

Effect of multiple factors on the adsorption of Cd in an alluvial soil from Xiba, China.

The effects of organic matter, free Fe oxides and Mn oxides in an alluvial soil on adsorption of Cd were studied through selective chemical extraction and adsorption experiments. Compared to untreated soil, after H2O2 treatment for removal of organic matter and NH2OH·HCl treatment for removal Mn oxides, the distribution coefficient (Kd) decreased by a maximum of 25.2% and 64.1%, respectively. After dithionite-citrate-bicarbonate treatment for removal of free Fe oxides, Kd increased by 1670.2%. After increasing the solution pH from 2 to 3, Kd increased by 2842.1%, whereas after increasing the solution pH from 3 to 7, the adsorption tended stabilize. As the ionic strength increased from 0.001 M to 0.1 M NaNO3, Kd gradually decreased, whereas at the same ionic strength, Kd decreased as the initial concentration of Cd increased. The effects of different background electrolytes on Cd adsorption was as follows: Ca2+ > Mg2+ > K+ > Na+ for cations and Cl- ≈ SO42- > NO3- for anions. The adsorption capacity of Cd increased as the increased of temperature, and it's a spontaneous endothermic process. The pseudo second-order rate model described the process of Cd adsorption well.

Diazepam inhibited lipopolysaccharide (LPS)-induced pyroptotic cell death and alleviated pulmonary fibrosis in mice by specifically activating GABAA receptor α4-subunit.

Diazepam could regulate immune system and inflammation, which might be a potential therapeutic agent for pulmonary fibrosis in clinic. This study showed that diazepam reversed LPS-induced inhibition of cell proliferation and promotion of cell apoptosis. Of note, LPS specifically induced Caspase-11 dependent cell pyroptosis, which were significantly attenuated by diazepam or pyroptosis inhibitor necrosulfonamide (NSA) treatment. In addition, α4- and α5-subunits of GABAARs were highly expressed in human bronchial 16HBE cells, human pulmonary epithelial cells (BEAS-2B) and pulmonary epithelial cells isolated from mice (mPECs). Further results showed that only knock-down of α4-GABAARs abrogated the effects of diazepam on LPS induced cell pyroptosis, apoptosis and proliferation. Similiarly, either diazepam or NSA treatment could alleviate development of LPS induced inflammatory reactions and pulmonary fibrosis in mice, which were abrogated by synergistically knocking down α4-GABAARs. Taken together, diazepam alleviated LPS-induced cell pyroptosis and development of pulmonary fibrosis in mice by activating α4-GABAARs.

[Comparative studies on photosynthetic characteristics of Notopterygium incisum and N. forbesii].

OBJECTIVE: To compare the photosynthetic characteristics of Notopterygium incisum and N. forbesii in order to provide basic data for introduction and cultivation of the two wild medicinal species. METHOD: The light-response, CO2-response and Chlorophy II fluorescence parameters of leaves at the booting stages between N. incisum and N. forbesii, were analyzed in situ by Li-6400 Portable Photosynthesis system under natural conditions. RESULT: 1) The light saturation point (LSP) was 1539 micromol x m(-2) x s(-1) for N. incisum and 1464 micromol x m(-2) x s(-1) for N. forbesii, the maximum net photosynthetic rate (Pmax) was 22.95 micromol x m(-2) x s(-1) for N. incisum and 19.65 micromol x m(-2) x s(-1) for N. forbesii, the apparent quantum yield (AQY) was 0.0509 for N. incisum and 0.0470 for N. forbesii, LSP, AQY and Pmax of N. incisum were significantly higher than those of N. forbesii; the light compensation point (LCP) was 17.92 micromol x m(-2) x s(-1) for N. incisum and 26.69 micromol x m(-2) x s(-1) for N. forbesii, LCP of N. incisum was significantly lower than that of N. forbesii. 2) The carbondioxide compensation point (CCP) were 33.41 micromol x mol(-1) for N. incisum and 37.82 micromol x mol(-1) for N. forbesii, the carbon dioxide saturation point (CSP) were 988 micromol x mol(-1) for N. incisum and 1150 micromol x mol(-1) for N. forbesii, CCP and CSP of N. incisum were significantly lower than N. forbesii; the carboxylation efficiency (CE) were 0.0591 for N. incisum and 0.0459 for N. forbesii; the maximum rate of RuBP regeneration (Jmax) were 28.18 micromol x m(-2) x s(-1) for N. incisum and 25.32 micromol x m(-2) x s(-1) for N. forbesii; the light respiration rate (Rd) were 1.971 micromol x m(-2) x s(-1) for N. incisum and 1.736 micromol x m(-2) x s(-1) for N. forbesii, CE, Jmax and Rd of N. incisum were higher than those of N. forbesii. 3) The primary light energy conversion of PS II (Fv/Fm) was 0.8213 for N. incisum and 0.8257 for N. forbesii, wihich didn't showed significant difference, between N. incisum and N. forbesii there was no photoinhibition. CONCLUSION: Both N. incisum and N. forbesii were C3 type plant, could perfectly acclimate to light condition. However, the weak light of N. incisum was absorbed significantly higher than that of N. forbesii, strong photosynthesis ability causes assimilation products accumulation of N. incisum obviously to be higher than that of N. forbesii.

Investigation of transcriptional gene silencing and mechanism induced by shRNAs targeted to RUNX3 in vitro.

AIM: To investigate transcriptional gene silencing induced by short hairpin RNAs (shRNAs) that target gene prompter regions of RUNX3 gene, and whether shRNAs homologous to DNA sequences may serve as initiators for methylation. METHODS: According to the principle of RNAi design, pSilencer3.1-H1-shRNA/RUNX3 expression vector was constructed, The recombinant plasmid shRNA was transfected into human stomach carcinoma cell line SGC7901 with Lipofectamine 2000. Then, the positive cell clones were screened by G418. The mRNA and protein expression level of RUNX3 in the stable transfected cell line SGC7901 were determined by RT-PCR, Western blotting and immunocytochemistry. Characteristics of the cell lines including SGC7901, pSilencer3.1-H1/SGC7901 and pSilencer3.1-H1-shRNA/RUNX3/SGC7901 were analyzed with growth curves, clone formation rate and cell-cycle distribution. The activated level of RUNX3 was examined after treatment with the different density of 5'-aza-2'-deoxycytidine (5-Aza-CdR) by using semi-quantitative RT-PCR and Western blotting. RESULTS: In the cell line SGC7901 transfected with pSilencer3.1-H1-shRNA/RUNX3, mRNA and protein expression of the RUNX3 gene was lost identified by RT-PCR, Western blotting and immunocytochemistry assay. The growth of pSilencer3.1-H1-shRNA/ RUNX3/SGC7901 cells without expression of RUNX3 was the fastest (P < 0.05), its rate of clone formation was the highest (P < 0.01), and the cell distribution in G(0)/G(1) and S/M phases was lowest and highest, respectively (P < 0.05), compared with that of the transfected pSilencer3.1-H1 and non-transfected cells. Through RT-PCR and Western blot assay, inactivated RUNX3 could not be reactivated by 5-Aza-CdR. CONCLUSION: We found that, although shRNAs targeted to gene prompter regions of RUNX3 could effectively induce transcriptional repression with chromatic changes characteristic of inaction promoters, this was independent of DNA methylation, and the presence of RNA-dependent transcriptional silencing showed that RNA-directed DNA methylation might be an existing gene regulatory mechanism relative to the methylated in humans.