1.Effect of Hei Xiaoyaosan on Neuroinflammation and NLRP3/Caspase-1/GSDMD Signaling Pathway in APP/PS1 Mice
Jun ZHOU ; Mingcheng LI ; Yujie LYU ; Zhipeng MENG ; Yunyun HU ; Huping WANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(9):124-133
ObjectiveTo observe the effects of Hei Xiaoyaosan on the learning and memory abilities of Alzheimer's disease model mice (APP/PS1 mice), and to explore its mechanism through the inflammatory cascade mediated by nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3)/cysteine aspartate-specific protease (Caspase-1)/gasdermin D (GSDMD) signaling pathway. MethodsSPF-grade 4-month-old APP/PS1 mice were randomly divided into the model group, MCC950 group, and Hei Xiaoyaosan high-, medium-, and low-dose groups. C57BL/6J mice were used as the blank group. After 7 days of adaptive feeding, mice in each group were intervened. The Hei Xiaoyaosan high-, medium-, and low-dose groups were given corresponding doses by gavage (25.79, 12.90, 6.45 g·kg-1·d-1), the MCC950 group was intraperitoneally injected with 10 mg·kg-1·2 d-1, and the blank group received the same volume of physiological saline by gavage. After 90 days of intervention, the learning and memory abilities were assessed using the Y maze and Morris water maze tests. The structural changes of hippocampal neurons were observed by hematoxylin-eosin (HE) staining. The expression of amyloid precursor protein (APP) in the hippocampal CA3 region was detected by immunohistochemistry. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of interleukin (IL)-10, IL-18, and IL-1β in the hippocampus. Western blot was applied to detect the protein expression of NLRP3, Caspase-1, GSDMD, and GSDMD-N in the hippocampus. Immunofluorescence was used to detect the co-localization of GSDMD-N and ionized calcium-binding adapter molecule-1 (Iba-1) in the hippocampus. Results① In the Y maze test, compared with the blank group, the spontaneous alternation rate of the model group was significantly reduced (P<0.01). Compared with the model group, the spontaneous alternation rate in the Hei Xiaoyaosan high- and low-dose groups was significantly increased (P<0.01). ② In the Morris water maze test, during the 1-4 days of the location navigation test, the escape latency time of mice decreased with the extension of training time. On day 4, compared with the blank group, the model group showed a significantly increased escape latency (P<0.05). Compared with the model group, the MCC950 group and the Hei Xiaoyaosan low-dose group showed significantly reduced escape latency (P<0.05). In the spatial exploration experiment, compared with the blank group, the number of platform crossings in the model group was significantly reduced (P<0.01). Compared with the model group, the Hei Xiaoyaosan low-dose group showed significantly increased platform crossings (P<0.05). ③ HE staining showed that, compared with the blank group, the hippocampal CA3 cells of the model group were damaged, arranged loosely and irregularly, swollen, with unclear boundaries, and the nuclei were pyknotic and deeply stained. MCC950 and all doses of Hei Xiaoyaosan improved the hippocampal CA3 cell damage in APP/PS1 mice to varying degrees. ④ Immunohistochemical results indicated that, compared with the blank group, the expression of APP in the hippocampal CA3 region was significantly increased in the model group (P<0.01). MCC950 and all doses of Hei Xiaoyaosan could reduce the expression of APP in the hippocampal CA3 region of APP/PS1 mice (P<0.01). ⑤ ELISA results showed that the levels of IL-18 and IL-1β in the hippocampus of mice in the model group were significantly increased, and IL-10 levels were significantly reduced (P<0.01). Compared with the model group, the IL-18 levels in the MCC950 group and the Hei Xiaoyaosan medium- and low-dose groups were significantly reduced (P<0.01). IL-1β levels in the hippocampus of the MCC950 group and Hei Xiaoyaosan high-, medium-, and low-dose groups were significantly decreased (P<0.01). The IL-10 levels in the hippocampus of the MCC950 group and the Hei Xiaoyaosan medium- and low-dose groups were increased (P<0.05, P<0.01). ⑥ Western blot results showed that compared with the blank group, the protein levels of NLRP3, Caspase-1, GSDMD, and GSDMD-N in the hippocampus of the model group were significantly elevated (P<0.01). Compared with the model group, the content of NLRP3 and Caspase-1 in the hippocampus of the treated groups was decreased (P<0.05, P<0.01). The content of GSDMD in the hippocampus of the Hei Xiaoyaosan high-, medium-, and low-dose groups was reduced (P<0.05, P<0.01), and the content of GSDMD-N in the hippocampus of the Hei Xiaoyaosan medium- and low-dose groups was decreased (P<0.05, P<0.01). ⑦ Immunofluorescence results showed that, compared with the blank group, the co-expression of GSDMD-N and Iba-1 in the hippocampus of the model group was significantly increased (P<0.01). Compared with the model group, the co-expression of GSDMD-N and Iba-1 in the treated groups was significantly reduced (P<0.01). ConclusionHei Xiaoyaosan may regulate the NLRP3/Caspase-1/GSDMD signaling pathway to affect the release of inflammatory factors, alleviate neuroinflammation,improve hippocampal histopathological changes,and improve learning and memory deficits,thus providing potential therapeutic benefits for Alzheimer's disease.
2.Modified Lianpoyin Formula Treats Hp-associated Gastritis by Regulating Mitochondrial Autophagy and NLRP3 Inflammasome Signaling Pathway
Siyi ZHANG ; Haopeng DANG ; Wenliang LYU ; Wentao ZHOU ; Wei GUO ; Lin LIU ; Lan ZENG ; Yujie SUN ; Luming LIANG ; Yi ZHAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):178-187
ObjectiveTo explore the effect of modified Lianpoyin formula (LPYJWF) in the treatment of Helicobacter pylori (Hp)-associated gastric mucosal damage based on mitochondrial autophagy and NLRP3 inflammasome signaling pathway. MethodsA total of 60 eight-week-old Balb/c male mice were assigned via the random number table method into control, model, high-dose LPYJWF (LPYJWF-H, 27.3 g·kg-1·d-1), medium-dose LPYJWF (LPYJWF-M, 13.65 g·kg-1·d-1), low-dose LPYJWF (LPYJWF-L, 6.83 g·kg-1·d-1), and quadruple therapy groups. Except the control group, other groups were modeled for Hp infection. Mice were administrated with LPYJWF at corresponding doses by gavage. Quadruple therapy group was given omeprazole (6.06 mg·kg-1·d-1) + amoxicillin (303 mg·kg-1·d-1) + clarithromycin (151.67 mg·kg-1·d-1) + colloidal pectin capsules (30.3 mg·kg-1·d-1) by gavage. The control group was given an equal volume of 0.9% NaCl for 14 days. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of gastric mucosa, and Warthin-Starry (W-S) silver staining was used to detect Hp colonization. Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of the gastric tissue, and immunofluorescence co-localization assay was adopted to detect the expression of mitochondrial transcription factor A (TFAM) and translocase of the outer mitochondrial membrane member 20 (TOMM20). The water-soluble tetrazolium salt method and thiobarbituric acid method were used to determine the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), respectively, in the gastric tissue. Western blot was employed to measure the protein levels of PTEN-induced kinase 1 (PINK1), Parkin, p62, microtubule-associated protein 1 light chain 3 (LC3), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), interleukin-1β (IL-1β), and interleukin-18 (IL-18). Real-time quantitative PCR was employed to assess the mRNA levels of PINK1, Parkin, p62, and LC3. ResultsCompared with the control group, the model group presented obvious gastric mucosal damage, colonization of a large number of Hp, severe mitochondrial damage, vacuolated structures due to excessive autophagy, reduced TOMM20 and TFAM co-expression in the gastric mucosal tissue, and reduced SOD and increased MDA (P<0.01). In addition, the gastric tissue in the model group showed up-regulated protein and mRNA levels of PINK1, Parkin, and LC3 and down-regulated protein and mRNA levels of p62 (P<0.01, as well as increased expression of inflammasome-associated proteins NLRP3, ASC, IL-1β, and IL-18 (P<0.01). Compared with the model group, the LPYJWF and quadruple therapy groups showed alleviated pathological damage of gastric mucosa, reduced Hp colonization, mitigated mitochondrial damage, and increased co-expression of TOMM20 and TFAM. The SOD level was elevated in the LPYJWF-L group (P<0.01), and the MDA levels became lowered in the LPYJWF and quadruple therapy groups (P<0.05, P<0.01). Furthermore, the LPYJWF and quadruple therapy groups showed down-regulated mRNA levels of PINK1, Parkin, and LC3 and protein levels of PINK1 and Parkin, and up-regulated mRNA level of p62 (P<0.01). The LPYJWF-M, LPYJWF-H, and quadruple therapy groups showcased down-regulated LC3 Ⅱ/LC3 Ⅰ level (P<0.05, P<0.01) and up-regulated protein level of p62 (P<0.01). The expression of inflammasome-associated proteins NLRP3, ASC, IL-1β, and IL-18 were reduced in the LPYJWF and quadruple therapy groups (P<0.05, P<0.01). ConclusionLPYJWF ameliorates gastric mucosal damage and exerts mucosa-protective effects in Hp-infected mice, which may be related to the inhibition of excessive mitochondrial autophagy, thereby inhibiting the activation of the NLRP3 inflammasome pathway.
3.Modified Lianpoyin Formula Treats Hp-associated Gastritis by Regulating Mitochondrial Autophagy and NLRP3 Inflammasome Signaling Pathway
Siyi ZHANG ; Haopeng DANG ; Wenliang LYU ; Wentao ZHOU ; Wei GUO ; Lin LIU ; Lan ZENG ; Yujie SUN ; Luming LIANG ; Yi ZHAO
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(21):178-187
ObjectiveTo explore the effect of modified Lianpoyin formula (LPYJWF) in the treatment of Helicobacter pylori (Hp)-associated gastric mucosal damage based on mitochondrial autophagy and NLRP3 inflammasome signaling pathway. MethodsA total of 60 eight-week-old Balb/c male mice were assigned via the random number table method into control, model, high-dose LPYJWF (LPYJWF-H, 27.3 g·kg-1·d-1), medium-dose LPYJWF (LPYJWF-M, 13.65 g·kg-1·d-1), low-dose LPYJWF (LPYJWF-L, 6.83 g·kg-1·d-1), and quadruple therapy groups. Except the control group, other groups were modeled for Hp infection. Mice were administrated with LPYJWF at corresponding doses by gavage. Quadruple therapy group was given omeprazole (6.06 mg·kg-1·d-1) + amoxicillin (303 mg·kg-1·d-1) + clarithromycin (151.67 mg·kg-1·d-1) + colloidal pectin capsules (30.3 mg·kg-1·d-1) by gavage. The control group was given an equal volume of 0.9% NaCl for 14 days. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of gastric mucosa, and Warthin-Starry (W-S) silver staining was used to detect Hp colonization. Transmission electron microscopy was employed to observe the mitochondrial ultrastructure of the gastric tissue, and immunofluorescence co-localization assay was adopted to detect the expression of mitochondrial transcription factor A (TFAM) and translocase of the outer mitochondrial membrane member 20 (TOMM20). The water-soluble tetrazolium salt method and thiobarbituric acid method were used to determine the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), respectively, in the gastric tissue. Western blot was employed to measure the protein levels of PTEN-induced kinase 1 (PINK1), Parkin, p62, microtubule-associated protein 1 light chain 3 (LC3), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), interleukin-1β (IL-1β), and interleukin-18 (IL-18). Real-time quantitative PCR was employed to assess the mRNA levels of PINK1, Parkin, p62, and LC3. ResultsCompared with the control group, the model group presented obvious gastric mucosal damage, colonization of a large number of Hp, severe mitochondrial damage, vacuolated structures due to excessive autophagy, reduced TOMM20 and TFAM co-expression in the gastric mucosal tissue, and reduced SOD and increased MDA (P<0.01). In addition, the gastric tissue in the model group showed up-regulated protein and mRNA levels of PINK1, Parkin, and LC3 and down-regulated protein and mRNA levels of p62 (P<0.01, as well as increased expression of inflammasome-associated proteins NLRP3, ASC, IL-1β, and IL-18 (P<0.01). Compared with the model group, the LPYJWF and quadruple therapy groups showed alleviated pathological damage of gastric mucosa, reduced Hp colonization, mitigated mitochondrial damage, and increased co-expression of TOMM20 and TFAM. The SOD level was elevated in the LPYJWF-L group (P<0.01), and the MDA levels became lowered in the LPYJWF and quadruple therapy groups (P<0.05, P<0.01). Furthermore, the LPYJWF and quadruple therapy groups showed down-regulated mRNA levels of PINK1, Parkin, and LC3 and protein levels of PINK1 and Parkin, and up-regulated mRNA level of p62 (P<0.01). The LPYJWF-M, LPYJWF-H, and quadruple therapy groups showcased down-regulated LC3 Ⅱ/LC3 Ⅰ level (P<0.05, P<0.01) and up-regulated protein level of p62 (P<0.01). The expression of inflammasome-associated proteins NLRP3, ASC, IL-1β, and IL-18 were reduced in the LPYJWF and quadruple therapy groups (P<0.05, P<0.01). ConclusionLPYJWF ameliorates gastric mucosal damage and exerts mucosa-protective effects in Hp-infected mice, which may be related to the inhibition of excessive mitochondrial autophagy, thereby inhibiting the activation of the NLRP3 inflammasome pathway.
4.The Development and Application of Chatbots in Healthcare: From Traditional Methods to Large Language Models
Zixing WANG ; Le QI ; Xiaodan LIAN ; Ziheng ZHOU ; Aiwei MENG ; Xintong WU ; Xiaoyuan GAO ; Yujie YANG ; Yiyang LIU ; Wei ZHAO ; Xiaolin DIAO
Medical Journal of Peking Union Medical College Hospital 2025;16(5):1170-1178
With the rapid advancement of artificial intelligence technology, chatbots have shown great potential in the healthcare sector. From personalized health advice to chronic disease management and psychological support, chatbots have demonstrated significant advantages in improving the efficiency and quality of healthcare services. As the scope of their applications expands, the relationship between technological complexity and practical application scenarios has become increasingly intertwined, necessitating a more comprehensive evaluation of both aspects. This paper, from the perspective of he althcare applications, systematically reviews the technological pathways and development of chatbots in the medical field, providing an in-depth analysis of their performance across various medical scenarios. It thoroughly examines the advantages and limitations of chatbots, aiming to offer theoretical support for future research and propose feasible recommendations for the broader adoption of chatbot technologies in healthcare.
5.Analysis of bile acid profile among patients with intrahepatic cholestasis of pregnancy
HU Yujie ; SHI Xinyan ; SHEN Yonghai ; ZHOU Yayuan ; CHEN Yu
Journal of Preventive Medicine 2025;37(7):727-731
Objective:
To analyze the differences in bile acid profiles during different pregnancy durations of patients with intrahepatic cholestasis of pregnancy (ICP), so as to provide a reference for early prevention and treatment of ICP and optimization of maternal-infant health outcomes.
Methods:
Pregnant women who underwent routine prenatal examinations and delivered at Hangzhou Obstetrics and Gynecology Hospital from 2021 to 2023 were selected as study subjects. According to the ICP guidelines (2020), pregnant women were categorized into normal group, mild ICP group, and moderate/severe ICP group. Age, parity, and gravidity were collected through the obstetric electronic medical record system, liver function indicators and seven bile acid levels were collected through the hospital's laboratory information system. Differences in bile acid profiles across pregnancy durations among the three groups were compared.
Results:
A total of 238 pregnant women were enrolled, including 57 cases (23.95%) in the normal group, 136 cases (57.14%) in the mild ICP group, and 45 cases (18.91%) in the moderate/severe ICP group. There were statistically significant differences between the three groups in total bile acid (TBA), cholic acid (CA), chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), glycocholic acid (GCA), taurocholic acid (TCA) levels (all P<0.05). Compared with the normal group, CA, GCDCA and GCA, and TCA were higher in the mild and moderate/severe ICP groups; compared with the mild ICP group, GCA was higher in the moderate/severe ICP group (all P<0.05). Significant differences were observed in the levels of GCDCA, GCA, and TCA among three groups pregnant women in the early, mid, and late pregnancy (all P<0.05). Compared with the normal group, mild ICP group had higher GCDCA and GCA in the early and mid pregnancy; moderate/severe ICP group had higher TCA in the early pregnancy and higher GCDCA and GCA in the late pregnancy. Compared with the mild ICP group, mild ICP group had higher TCA in the early pregnancy and the moderate/severe ICP group had higher GCA in the late pregnancy.
Conclusions
GCDCA, GCA, and TCA levels remain higher in ICP patients than in normal pregnant women across all pregnancy durations. Personalized perinatal management plans can be developed based on bile acid profile dynamics to optimize maternal-fetal outcomes.
6.Impact of milk and egg supplementation on body composition and bone mineral density of rural primary school students in Yunnan Province
Chinese Journal of School Health 2025;46(10):1401-1405
Objective:
To investigate the impact of milk and egg supplementation on body composition and bone mineral density of rural primary school students in Yunnan Province, so as to provide a reference for developing targeted nutritional intervention strategies.
Methods:
In December 2023, a cluster sampling method was adopted to select students from grades one to three in four primary schools each from Jinggu and Shidian countys of Yunnan Province, as the intervention group (662 students). Additionally, two boarding primary schools were selected from each county based on the principle of matching scale and student numbers as the control group (455 students). Starting from April 2023, the intervention group received 200 mL milk and 50 g eggs during the break on school days for 8 months, while the control group maintained their usual diet behavior. Body composition was measured by using bioelectrical impedance analysis, and distal radial bone mineral density was assessed via dual energy X-ray absorptiometry in April and December 2023. The intervention effects were analyzed by using a difference in-differences approach.
Results:
The final measurements of body fat percentage, skeletal muscle mass and fat free mass of the intervention group and the control group of primary school students were significantly higher than the baseline values, and the net effect of milk and egg intervention on these body composition indicators was not statistically significant ( P >0.05, both before and after adjustment). In contrast, bone mineral density increased significantly by 0.02 g/cm 2 in the intervention group. The net intervention effect on bone mineral density was statistically significant ( β=0.02, 95%CI =0.00-0.04), and remained significant after model adjustment ( β=0.02, 95%CI =0.00-0.04) (both P < 0.05). Subgroup analysis showed statistically significant effects of the intervention among girls ( β=0.02, 95%CI =0.00-0.04), day students ( β=0.04, 95%CI =0.01-0.07), and students with normal nutritional status ( β=0.02, 95%CI =0.00-0.04) (all P <0.05). No significant effect of milk and egg supplementation was observed on body composition indicators (all P <0.05).
Conclusions
Milk and egg supplementation can improve bone mineral density among rural primary school students in Yunnan Province. It is recommended that rural school aged children should increase intake of milk and eggs to support growth and development.
7.Study on the chemical constituents of the active parts of Piper wallichii
Ling ZENG ; Yujie HU ; Ling LI ; Xiaojian GONG ; Chanyuan ZHOU ; Dongsheng FAN
China Pharmacy 2025;36(21):2632-2637
OBJECTIVE To analyze the chemical constituents of the active parts of Piper wallichii. METHODS The petroleum ether-extract fraction was prepared from the methanol extract of P. wallichii. Separation and purification were performed using semi-preparative high-performance liquid chromatography. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS Nineteen compounds were isolated from the petroleum ether-extract fraction from the methanol extract of P. wallichii, identified as 3-acetoxybenzyl benzoate (1), 2-acetoxybenzyl benzoate (2), 2-methoxybenzyl benzoate (3), 3-methoxybenzyl benzoate (4), 4-hydroxy-3-methoxybenzyl benzoate (5), 3-hydroxybenzyl benzoate(6), benzyl benzoate (7), ganschisandrine (8), lancifolin A (9), (7R,8R,3′R)-7-acetoxy-3′,4′-dimethoxy-3,4-methylenedioxy-6′-oxo- Δ1′,4′,8′-8.3′-lignan (10), (7S,8R,3′S)-Δ8′-3′,6′-dihydro-3′-methoxy-3,4-methylenedioxy-6′-oxo-8.3′,7.O.4′-lignan (11), (7R, 8R,3′S)-Δ8′-3′,6′-dihydro-3′-methoxy-3,4-methylenedioxy-6′-oxo-8.3′,7.O.4′-lignan (12), isodihydrofutoquinol A (13), licarin A (14), licarin B (15), 2-(2′,5′-dimethoxyphenyl)-3,4- dimethyl-5-(3″,4″-dimethoxyphenyl)- tetrahydrofuran (16), galgravin (17), velutin (18), and piyunin A (19). CONCLUSIONS Compound 1 is a new benzyl benzoate compound. Compounds 3-5, 8 and 9 are isolated from the Piper genus for the first time, while compounds 2, 6, 10-13 and 15-19 are isolated from P. wallichii for the first time.
8.Mechanism of catalpol regulating Th17 cell differentiation by interfering PKM2/LDHA expression
Yu GE ; Xue CHEN ; Furong WANG ; Yujie BAO ; Peng DING ; Lingling ZHOU
China Pharmacy 2024;35(1):15-20
OBJECTIVE To investigate the mechanism of catalpol affecting the differentiation of helper T cell 17 (Th17) by interfering the expressions of pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). METHODS The naive CD4+ T cells were selected from the spleen of C57BL/6 mice, and were differentiated into Th17 cells by adding directional differentiation stimulants for 72 hours. At the same time, the cells were treated with 0 (directed control), 20, 40 and 80 μg/mL catalpol. The flow cytometry was used to detect the proportion of Th17 cell differentiation in cells; the colorimetric method was adopted to detect the levels of pyruvate and lactate in cell culture supernatant; mRNA expressions of retinoid-related orphan nuclear receptor gamma t (RORγt), PKM2 and LDHA were detected by qRT-PCR method; Western blot was used to detect the expression levels of PKM2, LDHA, signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 (p-STAT3) proteins in cells. RESULTS Compared with the directed control group, after 72 hours of treatment with 20, 40, 80 μg/mL catalpol, the differentiation ratio of Th17 cells were decreased by 6.74%, 8.41%, 9.24%, and the levels of pyruvate and lactate in the cell culture supernatant, the mRNA expressions of PKM2, LDHA and RORγt as well as the protein expressions of PKM2 and LDHA and the phosphorylation of STAT3 were significantly reduced (P<0.05). CONCLUSIONS Catalpol can reduce the glycolysis level by down-regulating the expressions of PKM2 and LDHA, thereby inhibiting the differentiation of Th17 cells.
9.Application of data mining algorithms in research on traditional Chinese medicine formula
Huizhi LI ; Xiaoling ZHOU ; Yujie YANG ; Xinyou ZHANG
China Pharmacy 2024;35(1):112-118
In recent years, data mining algorithms have been widely employed in scientific research within the field of traditional Chinese medicine (TCM). The data mining algorithms are used to effectively handle and analyze the complex data in TCM formulas, providing a rational explanation for the mechanism of action. This method has proven particularly useful in uncovering patterns of compatibility and frequent combinations of herbs in TCM, thereby enhancing the reliability and accuracy of clinical diagnosis, target screening, and the study of new drugs. This paper reviews and analyzes 147 papers on TCM formula research that utilize data mining algorithms. The results indicate that data mining algorithms play a unique advantage in six sub- areas, including the study on the mechanism of action in TCM formula, the dose-efficacy of TCM formulas, the identification of core drugs pairs/groups, mining the relationships among “formulas-drug-symptom”, the discovery of new formulas, and mining the compatibility law. Notably, association rules and clustering algorithms are the most representative.
10.Effect of Dendrobium officinale leaf fermentation fluid on a mouse model of alcoholic hepatitis and its mechanism of action
Xingnian ZHOU ; Yuhong LIU ; Yujie QIN ; Quan ZHANG ; Mingliang CHENG ; Hong LI
Journal of Clinical Hepatology 2024;40(2):335-342
ObjectiveTo investigate the intervention mechanism of Dendrobium officinale leaf fermentation fluid in mice with alcoholic hepatitis. MethodsA total of 70 healthy male C57BL/6J mice, aged 6-8 weeks, were randomly divided into normal group, model group, liquid feed control group, silybin group, and low-, middle-, and high-dose Dendrobium officinale leaf fermentation fluid groups, with 10 mice in each group. The mice in the normal group were given normal diet, and those in the other groups were given Lieber-DeCarli classic liquid diet for 8 weeks to induce alcoholic hepatitis. During modeling, the mice in the low-, middle-, and high-dose Dendrobium officinale leaf fermentation fluid groups were given Dendrobium liquid manufactured by Warmen Pharmaceutical, and the mice in all the other groups were given pure water; the mice in the normal group, the model group, and the liquid feed control group were given normal saline by gavage, those in the silybin group were given silybin 0.25 mL/10 g by gavage, and those in the low-, middle-, and high-dose Dendrobium officinale leaf fermentation fluid groups were given Dendrobium officinale leaf fermentation fluid at a dose of 0.125 mL/10 g, 0.250 mL/10 g, and 0.375 mL/10 g, respectively, by gavage, once a day. At week 8, chloral hydrate was injected intraperitoneally for anesthesia, and blood samples were collected from the eyeball. After serum was separated, the biochemical method was used to measure the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT); HE staining and oil red staining were used to observe liver histopathology and lipid accumulation in mice; multiplex Luminex assay was used to measure the serum levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and CCL2; quantitative real-time PCR, Western blot, and immunofluorescence assay were used to measure the protein expression levels of NLRP3, caspase-1, caspase-11, gasdermin D (GSDMD), N-terminal gasdermin D (GSDMD-N) in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the normal group, the model group had significant increases in the serum levels of AST, ALT, IL-6, IL-1β, TNF-α, and CCL2 (all P<0.05), and compared with the model group, the high-dose Dendrobium officinale leaf fermentation fluid group had significant reductions in the serum levels of AST, ALT, IL-6, IL-1β, TNF-α, and CCL2 (all P<0.05). HE staining showed that the model group had disordered structure of hepatic lobules, with a large number of steatosis vacuoles and massive cell necrosis, and compared with the model group, the high-dose Dendrobium officinale leaf fermentation fluid group had alleviation of liver histopathological injury, intact structure of most hepatic lobules, and a small amount of inflammatory cell infiltration. Oil red staining showed that the model group had accumulation of large and small lipid droplets in the liver and a significant increase in liver fat content, and compared with the model group, the high-dose Dendrobium officinale leaf fermentation fluid group had significant alleviation of hepatic steatosis, with the presence of sporadic small lipid droplets. Immunofluorescence assay of liver tissue showed that compared with the normal group, the model group had a significant increase in the ratio of GSDMD-positive staining area in hepatocyte cytoplasm (P<0.001), and compared with the model group, the high-dose Dendrobium officinale leaf fermentation fluid group had a significant reduction in such ratio in hepatocyte cytoplasm (P<0.001). Quantitative real-time PCR showed that compared with the normal group, the model group had significant increases in the protein expression levels of NLRP3, caspase-1, caspase-11, GSDMD, GSDMD-N, interleukin-18 (IL-18), and IL-1β in liver tissue (all P<0.05), and compared with the model group, the high-dose Dendrobium officinale leaf fermentation fluid group had significant reductions in the protein expression levels of NLRP3, caspase-1, caspase-11, GSDMD, GSDMD-N, IL-18, and IL-1 (all P<0.05). Compared with the model group, the high-dose Dendrobium officinale leaf fermentation fluid group had significant reductions in the protein expression levels of caspase-1 and caspase-11 (both P<0.05), with a relative expression level of caspase-1 of 1.757 (reduced by 26.6% compared with the model group) and a relative expression level of caspase-11 of 0.455 (reduced by 70.3% compared with the model group), suggesting that caspase-11 showed a greater reduction than caspase-1. ConclusionDendrobium officinale leaf fermentation fluid can alleviate alcoholic hepatitis in mice, possibly by inhibiting the non-classical cell pyroptosis pathway mediated by caspase-11.


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