Panax species are mostly valuable medicinal plants. While some species' seeds are sensitive to dehydration, the dehydration tolerance of seeds from other Panax species remains unclear. The phospholipase D(PLD) gene plays an important role in plant responses to dehydration stress. However, the characteristics of the PLD gene family and their mechanisms of response to dehydration stress in seeds of Panax species with different dehydration tolerances are not well understood. This study used seeds from eight Panax species to measure the germination rates and PLD activity after dehydration and to analyze the correlation between dehydration tolerance and seed traits. Bioinformatics analysis was also conducted to characterize the PnPLD and PvPLD gene families and to evaluate their expression patterns under dehydration stress. The dehydration tolerance of Panax seeds was ranked from high to low as follows: P. ginseng, P. zingiberensis, P. quinquefolius, P. vietnamensis var. fuscidiscus, P. japonicus var. angustifolius, P. japonicus, P. notoginseng, and P. stipuleanatus. A significant negative correlation was found between dehydration tolerance and seed shape(three-dimensional variance), with flatter seeds exhibiting stronger dehydration tolerance(r=-0.792). Eighteen and nineteen PLD members were identified in P. notoginseng and P. vietnamensis var. fuscidiscus, respectively. These members were classified into five isoforms: α, β, γ, δ, and ζ. The gene structures, subcellular localization, physicochemical properties, and other characteristics of PnPLD and PvPLD were similar. Both promoters contained regulatory elements associated with plant growth and development, hormone responses, and both abiotic and biotic stress. During dehydration, the PLD enzyme activity in P. notoginseng seeds gradually increased as the water content decreased, whereas in P. vietnamensis var. fuscidiscus, PLD activity first decreased and then increased. The expression of PLDα and PLDδ in P. notoginseng seeds initially increased and then decreased, whereas in P. vietnamensis var. fuscidiscus, the expression of PLDα and PLDδ consistently decreased. In conclusion, the dehydration tolerance of Panax seeds showed a significant negative correlation with seed shape. The dehydration tolerance in P. vietnamensis var. fuscidiscus and dehydration sensitivity of P. notoginseng seeds may be related to differences in PLD enzyme activity and the expression of PLDα and PLDδ genes. This study provided the first systematic comparison of dehydration tolerance in Panax seeds and analyzed the causes of tolerance differences and the optimal water content for long-term storage at ultra-low temperatures, thus providing a theoretical basis for the short-term and ultra-low temperature long-term storage of medicinal plant seeds with varying dehydration tolerances.
Seeds/metabolism* ; Panax/physiology* ; Plant Proteins/metabolism* ; Gene Expression Regulation, Plant ; Phospholipase D/metabolism* ; Plants, Medicinal/enzymology* ; Germination ; Multigene Family ; Water/metabolism* ; Dehydration ; Phylogeny

This study investigated the therapeutic effects and mechanisms of Modified Yiyi Fuzi Baijiang Powder on ulcerative colitis(UC) in rats from the perspective of dampness. SD rats were randomly allocated into six groups(n=10): control, model, mesalazine, and Modified Yiyi Fuzi Baijiang Powder at low(3.96 g·kg~(-1)·d~(-1)), medium(7.92 g·kg~(-1)·d~(-1)), and high(15.84 g·kg~(-1)·d~(-1)) doses. UC was induced in all groups except the control by administration with 3% dextran sulfate sodium(DSS) solution for 7 days. The disease activity index(DAI) was recorded, and the colon tissue was collected for analysis. Histopathological changes were assessed by hematoxylin-eosin staining. Serum levels of D-lactic acid(D-LA) and diamine oxidase(DAO) were measured by ELISA. Immunohistochemistry and PCR were employed to evaluate the expression of aquaporins(AQP3, AQP4) and tight junction proteins [zonula occludens-1(ZO-1) and occludin] at both protein and mRNA levels. Compared with the control group, the model group showed an increased DAI scores(P<0.05), intestinal mucosal damage, elevated serum levels of DAO and D-LA(P<0.05), and decreased expression of AQP3, AQP4, ZO-1, and occludin(P<0.05). Treatment with Modified Yiyi Fuzi Baijiang Powder reduced the DAI scores(P<0.05), lowered the serum levels of D-LA and DAO(P<0.05), and upregulated the expression of AQP3, AQP4, ZO-1, and occludin at both protein and mRNA levels compared with the model group. These findings suggest that Modified Yiyi Fuzi Baijiang Powder exerts therapeutic effects on UC by reducing the intestinal mucosal permeability, promoting colonic mucosal repair, and regulating abnormal intestinal water metabolism, which may involve the upregulation of AQP3 and AQP4 expression.
Animals ; Colitis, Ulcerative/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Intestinal Mucosa/metabolism* ; Male ; Aquaporin 3/metabolism* ; Aquaporin 4/metabolism* ; Permeability/drug effects* ; Humans ; Powders ; Intestinal Barrier Function

This study adopted a three-dimensional "effect-dose-mechanism" evaluation system to screen the optimal regimen of Yuxuebi Tablets(YXB) combined with ibuprofen(IBU) for chronic musculoskeletal pain(CMP) intervention and elucidate its pharmacological mechanism, so as to provide a scientific basis for the clinical application of the regimen. The experiments were conducted using 8-week-old ICR mice, which were randomly divided into sham operation(sham) group, model(CFA) group, IBU group, YXB group, stasis paralysis tablets combined with ibuprofen low-dose group(IBU-L-YXB), stasis paralysis combined with ibuprofen high-dose group(IBU-H-YXB), stasis paralysis tablets combined with ibuprofen high-dose with ibuprofen discontinuation on the 10th day of administration(IBU-10-YXB), and stasis paralysis tablets combined with ibuprofen high-dose with ibuprofen halving on the 10th day of administration(IBU-1/2-YXB) group. An animal model was established using the CFA plantar injection method. On D0(the second day post-modeling), the success of model establishment was assessed, followed by continuous drug administration for 18 consecutive days from D1 to D18. During this period, mechanical pain threshold was measured by the Von Frey test; thermal hyperalgesia was detected by the hot plate test, and depression-like behavior was observed by the tail suspension test. After treatment, peripheral blood was collected from all groups for complete blood biochemical analysis, and the injected feet of the sham, CFA, IBU, YXB, IBU-YXB, and IBU-10-YXB groups were subjected to oxylipin metabolomics analysis. Immunofluorescence double staining was further performed to detect the co-expression of key oxylipin metabolic enzymes(COX2, LTA4H, and 5/12/15-LOX) and macrophage marker CD68 in the sham, CFA, IBU, and YXB-L/M/H groups. Subsequently, confirmatory analysis of positive indicators was conducted in the sham, CFA, IBU, YXB, IBU-YXB, and IBU-10-YXB groups. On D6(acute phase), mechanical pain sensitivity data showed that compared with the CFA group, only the three combination groups(IBU-YXB, IBU-10-YXB, and IBU-1/2-YXB) exhibited significantly increased paw withdrawal thresholds. On D17(chronic phase), only the IBU-10-YXB group showed a mechanical pain threshold significantly higher than all other monotherapy and combination groups. On D17, thermal pain data showed that compared with the CFA group, all groups except IBU-1/2-YXB had significantly prolonged paw withdrawal latency. On D18, tail suspension data showed that compared with the CFA group, the YXB, IBU-YXB, and IBU-10-YXB groups had significantly reduced immobility time. In summary, IBU-10-YXB stably improved the core symptoms of acute and chronic inflammatory pain. Complete blood count data showed that compared with the sham group, the CFA group had significantly increased mean platelet volume(MPV), while compared with the CFA group, the IBU-YXB and IBU-10-YXB groups had significantly reduced MPV. Moreover, the platelet distribution width(PDW) of the IBU-10-YXB group was further reduced compared with the CFA group. These data suggest that the IBU-10-YXB combination regimen has superior effects on inflammation and blood circulation improvement compared with other treatment groups. At the mechanistic level, each treatment group differentially regulated pro-inflammatory and pro-resolving oxylipin(SPM). Specifically, compared with the CFA group, the IBU and IBU-YXB groups significantly inhibited the synthesis of the prostaglandin family downstream of COX2, reducing pro-inflammatory oxylipins PGD2 and 6-keto-PGF1α but inhibiting PGE1 and PGE2, which played positive roles in peripheral circulation, vasodilation, and inflammation resolution. Compared with the CFA group, the YXB group tended to inhibit the pro-inflammatory oxylipin LTB4 downstream of LTA4H and increase SPMs such as LXA4. The IBU-10-YXB group bidirectionally regulated pro-inflammatory oxylipins and SPMs. Compared with IBU, IBU-10-YXB significantly inhibited the pro-inflammatory mediator 5-HETE. Meanwhile, IBU-10-YXB broadly upregulated SPMs, as evidenced by significant upregulation of LXA4 compared with the CFA group, significant upregulation of LXA5 compared with the IBU and IBU-YXB groups, significant upregulation of RvD1 compared with the CFA group and all other treatment groups, and significant upregulation of RvD5 compared with the sham group. Immunofluorescence double staining results were as follows: compared with the CFA group, the IBU group specifically inhibited the oxylipin metabolic enzyme COX2. In the YXB group, COX2, LTA4H, and 5/12-LOX were significantly inhibited. Within the optimal analgesic dose range, YXB's inhibitory effects on COX2 and LTA4H were dose-dependent, while its inhibitory effects on 5/12-LOX were inversely dose-dependent. The two combination groups(IBU-YXB and IBU-10-YXB) inhibited COX2 and LTA4H without significantly affecting 5-LOX, while IBU-10-YXB further significantly inhibited 12-LOX. These results suggest that the IBU-10-YXB combination regimen effectively maintains stable inhibition of COX2, LTA4H, and 12-LOX while enhancing 5-LOX expression. This combinatorial strategy effectively suppresses pro-inflammatory oxylipins and promotes SPM biosynthesis, overcoming IBU's analgesic ceiling effect and its blockade of pain resolution pathways while compensating for YXB's inability to effectively intervene in acute pain and inflammation. Therefore, it achieves more stable anti-inflammatory, analgesic, and antidepressant effects.
Animals ; Ibuprofen/administration & dosage* ; Mice ; Mice, Inbred ICR ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Musculoskeletal Pain/immunology* ; Tablets ; Humans ; Chronic Pain/metabolism* ; Drug Therapy, Combination ; Disease Models, Animal

Hepatocellular carcinoma (HCC) expresses abundant glycolytic enzymes and displays comprehensive glucose metabolism reprogramming. Aldolase A (ALDOA) plays a prominent role in glycolysis; however, little is known about its role in HCC development. In the present study, we aim to explore how ALDOA is involved in HCC proliferation. HCC proliferation was markedly suppressed both in vitro and in vivo following ALDOA knockout, which is consistent with ALDOA overexpression encouraging HCC proliferation. Mechanistically, ALDOA knockout partially limits the glycolytic flux in HCC cells. Meanwhile, ALDOA translocated to nuclei and directly interacted with c-Jun to facilitate its Thr93 phosphorylation by P21-activated protein kinase; ALDOA knockout markedly diminished c-Jun Thr93 phosphorylation and then dampened c-Jun transcription function. A crucial site Y364 mutation in ALDOA disrupted its interaction with c-Jun, and Y364S ALDOA expression failed to rescue cell proliferation in ALDOA deletion cells. In HCC patients, the expression level of ALDOA was correlated with the phosphorylation level of c-Jun (Thr93) and poor prognosis. Remarkably, hepatic ALDOA was significantly upregulated in the promotion and progression stages of diethylnitrosamine-induced HCC models, and the knockdown of A ldoa strikingly decreased HCC development in vivo. Our study demonstrated that ALDOA is a vital driver for HCC development by activating c-Jun-mediated oncogene transcription, opening additional avenues for anti-cancer therapies.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation, joint destruction, and functional impairment. Angiogenesis plays a key role in the pathological progression of RA with dysfunction of endothelial cells to promote synovial inflammation, sustain pannus formation, subsequently leading to joint damage. Colquhounia Root Tablets (CRT), a Chinese patent drug, has shown a satisfying clinical efficacy in treating RA, while the underlying mechanism by which CRT inhibits RA-associated angiogenesis remains unclear. In this study, we applied a research approach combining transcriptomic data analysis, bio-network mapping, and in vivo and in vitro experiments to explore the molecular mechanisms of CRT in suppressing angiogenesis in RA. Animal welfare and experimental procedures follow the regulations of the Animal Ethics Committee of Institute of Basic Theory for Chinese Medicine, China Academy of Chinese Medical Sciences (ratification number: IBTCMCACMS21-2307-06). Network analysis identified that key genes such as nucleotide-binding oligomerization domain-containing protein 2 (NOD2), SMAD family member 3 (SMAD3), and vascular endothelial growth factor A (VEGFA) significantly enriched in pathways related to NOD-like receptor signaling and VEGF signaling, indicating that CRT may inhibit angiogenesis by regulating vascular endothelial cell function with modulating angiogenesis-related pathways. In vivo data showed that CRT significantly reduced the positive expression of CD31 and VEGF in the ankle joint of adjuvant-induced arthritis (AIA) rats. In vitro data further confirmed that CRT effectively inhibited VEGF-induced migration, invasion, and tube formation in HUVECs, while significantly reduced the expression of angiogenesis-related factors VEGF/CD31/Ang-1, as well as the positive expression of VEGF and CD31 in HUVECs. Furthermore, CRT markedly decreased the protein expression of NOD2, VEGFA, and SMAD3. In conclusion, these findings indicate that CRT may inhibit the RA-related angiogenesis by targeting the NOD2/SMAD3/VEGF signaling axis to improve endothelial cell function, enriching the scientific connotation of CRT in inhibiting pathological angiogenesis in RA and also offer new insights for clinical prevention and treatment of RA.

AIM: To analyze the clinical utility and value of the ultra-wide-field swept-source optical coherence tomography angiography(UWF-SS-OCTA)technique in changes of blood flow density and thickness in the central and peripheral regions of the retina and choroid in patients with nonproliferative diabetic retinopathy(NPDR)with or without diabetic kidney disease(DKD).METHODS: Cross-sectional study. Totally 50 cases(50 eyes)of diabetes mellitus(DM)that visited our hospital between June 2023 and June 2024 were included. They were divided into three groups: NPDR combined with DKD group(DKD group, n=20), NPDR without DKD group(NDKD group, n=20), and DM without retinopathy group(DM group, n=10, which served as control). In order to investigate the impact of DKD on ocular microangiopathy in NPDR patients, the retina and choroid within 24 mm×20 mm of the scan were separated into central and peripheral areas using the 3×3 nine-grid partition option that comes with UWF-SS-OCTA, and the parameters were then quantitatively assessed.RESULTS:The central and peripheral blood flow density of the choroidal capillary layer(CCP)was statistically significant between the DM group and the DKD group(t=3.93, P=0.0003; t=3.34, P=0.0016), and between the NDKD group and the DKD group(t=-3.06, P=0.003; t=-2.55, P=0.013), but there was no statistically significant difference between the DM group and the NDKD group(t=1.44, P=0.157; t=1.26, P=0.21). The mid-large choroidal vessel(MLCV)showed a progressive decline in central and peripheral blood flow density in the DM, NDKD, and DKD groups(F=13.74, 19.03, all P<0.0001). The DM, NDKD, and DKD groups saw a progressive decrease in central and peripheral choroidal thickness(CT; F=10.72, P=0.0001; F=13.12, P<0.001).CONCLUSION:CCP, MLCV, and CT can be used as visual indicators to identify impaired renal function in patients with NPDR. UWF-SS-OCTA can support the development of precise and noninvasive monitoring and treatment technology for diabetic ocular microangiopathy, while also offering a scientific foundation for the joint management of DR and DKD.

ObjectiveTo investigate the effect and mechanism of Sishenwan in restoring the intestinal barrier function in the rat model of diarrhea-predominant irritable bowel syndrome （IBS-D） due to spleen-kidney Yang deficiency based on intestinal flora and short-chain fatty acids. MethodsAfter the delivery of 10 SPF-grade pregnant rats， 4 male suckling rats were kept in each litter for the experiment. The male suckling rats were randomly allocated into blank， model， low-dose （3.51 g·kg^-1） Sishenwan， high-dose （7.02 g·kg^-1） Sishenwan， and Peifeikang （0.54 g·kg^-1） groups， with 8 rats in each group. The blank group was fed conventionally， and the other groups were subjected to mother-child separation and Sennae Folium gavage （1 g·mL^-1， 10 mL·kg^-1） for the modeling of IBS-D due to spleen-kidney Yang deficiency. After the modeling was completed， the rats in Sishenwan groups were administrated with the corresponding dose of Sishenwan decoction by gavage， and the Peifeikang group with bifidobacterium triple live powder+normal saline suspension. The blank and model groups were treated with an equal volume of normal saline by gavage. The general conditions and fecal characteristics of rats were observed. After 2 weeks of administration， the rats were anesthetized for sample collection. The pathological changes of the colon tissue in rats were observed by hematoxylin-eosin staining. Enzyme-linked immunosorbent assay was employed to measure the levels of transforming growth factor-beta （TGF-β）， interleukin-10 （IL-10）， and interleukin-22 （IL-22）. Immumohistochemical staining （IHC） was performed to detect the positive expression of zonula occludens-1 （ZO-1） and occludin in the colon tissue. Western blot was employed to determine the protein levels of ZO-1 and occludin in the colon tissue of rats， and 16S rRNA gene sequencing was performed for intestinal flora. Gas chromatography-mass spectrometry was employed to determine the content of short-chain fatty acids （SCFAs） in the cecum contents of rats. ResultsThe colon tissue in the blank group presented a clear structure， neat glands， and no inflammatory cell infiltration. In the model group， the colon tissue showcased a disorganized structure， irregular arrangement of glands， and inflammatory cell infiltration. Compared with the model group， the low-dose and high-dose Sishenwan groups and the Peifeikang group exhibited an intact colon tissue structure， regular arrangement of glands， and reduced inflammatory cell infiltration. Compared with the blank group， the modeling lowered the levels of TGF-β， IL-10， and IL-22 in the serum （P<0.01）， down-regulated the protein levels of ZO-1 and occludin in the colon tissue （P<0.01）， and decreased the content of acetic acid and propionic acid and increased the content of butyric acid in cecum contents （P<0.05）. Compared with the model group， low-dose and high-dose Sishenwan raised the levels of TGF-β， IL-10， and IL-22 in the serum （P<0.05， P<0.01）， and Peifeikang elevated the levels of TGF-β and IL-10 in the serum （P<0.01）. High-dose Sishenwan and Peifeikang up-regulated the protein levels of ZO-1 and occludin （P<0.05， P<0.01）， increased the content of acetic acid and propionic acid in cecum contents （P<0.05）， and decreased the content of butyric acid （P<0.05）. The 16S rRNA gene sequencing results showed that the intestinal flora structure of the model group changed compared with that of the blank group. Compared with the model group， Sishenwan and Peifeikang increased the relative abundance of Lachnospiraceae， Muribaculaceae， Akkermansiaceae， Ligilactobacillus， UBA3282， Akkermansia， and Corynebacterium while reducing the relative abundance of Oscillospiraceae， Desulfovibrionaceae， Lactobacillus， Romboutsia， and Desulfovibrio. They can restore the intestinal flora structure similar to that in the blank group. ConclusionSishenwan can alleviate diarrhea symptoms and colonic mucosal inflammation， increase the expression of tight junction proteins in the colonic mucosa， and strengthen the intestinal barrier in IBS-D rats with the syndrome of spleen-kidney Yang deficiency. The mechanism of action may be related to optimizing the structure and balance of intestinal flora and regulating the SCFAs， and the effect of high-dose Sishenwan is obvious.

ObjectiveTo investigate the effect and mechanism of Sishenwan in restoring the intestinal barrier function in the rat model of diarrhea-predominant irritable bowel syndrome （IBS-D） due to spleen-kidney Yang deficiency based on intestinal flora and short-chain fatty acids. MethodsAfter the delivery of 10 SPF-grade pregnant rats， 4 male suckling rats were kept in each litter for the experiment. The male suckling rats were randomly allocated into blank， model， low-dose （3.51 g·kg^-1） Sishenwan， high-dose （7.02 g·kg^-1） Sishenwan， and Peifeikang （0.54 g·kg^-1） groups， with 8 rats in each group. The blank group was fed conventionally， and the other groups were subjected to mother-child separation and Sennae Folium gavage （1 g·mL^-1， 10 mL·kg^-1） for the modeling of IBS-D due to spleen-kidney Yang deficiency. After the modeling was completed， the rats in Sishenwan groups were administrated with the corresponding dose of Sishenwan decoction by gavage， and the Peifeikang group with bifidobacterium triple live powder+normal saline suspension. The blank and model groups were treated with an equal volume of normal saline by gavage. The general conditions and fecal characteristics of rats were observed. After 2 weeks of administration， the rats were anesthetized for sample collection. The pathological changes of the colon tissue in rats were observed by hematoxylin-eosin staining. Enzyme-linked immunosorbent assay was employed to measure the levels of transforming growth factor-beta （TGF-β）， interleukin-10 （IL-10）， and interleukin-22 （IL-22）. Immumohistochemical staining （IHC） was performed to detect the positive expression of zonula occludens-1 （ZO-1） and occludin in the colon tissue. Western blot was employed to determine the protein levels of ZO-1 and occludin in the colon tissue of rats， and 16S rRNA gene sequencing was performed for intestinal flora. Gas chromatography-mass spectrometry was employed to determine the content of short-chain fatty acids （SCFAs） in the cecum contents of rats. ResultsThe colon tissue in the blank group presented a clear structure， neat glands， and no inflammatory cell infiltration. In the model group， the colon tissue showcased a disorganized structure， irregular arrangement of glands， and inflammatory cell infiltration. Compared with the model group， the low-dose and high-dose Sishenwan groups and the Peifeikang group exhibited an intact colon tissue structure， regular arrangement of glands， and reduced inflammatory cell infiltration. Compared with the blank group， the modeling lowered the levels of TGF-β， IL-10， and IL-22 in the serum （P<0.01）， down-regulated the protein levels of ZO-1 and occludin in the colon tissue （P<0.01）， and decreased the content of acetic acid and propionic acid and increased the content of butyric acid in cecum contents （P<0.05）. Compared with the model group， low-dose and high-dose Sishenwan raised the levels of TGF-β， IL-10， and IL-22 in the serum （P<0.05， P<0.01）， and Peifeikang elevated the levels of TGF-β and IL-10 in the serum （P<0.01）. High-dose Sishenwan and Peifeikang up-regulated the protein levels of ZO-1 and occludin （P<0.05， P<0.01）， increased the content of acetic acid and propionic acid in cecum contents （P<0.05）， and decreased the content of butyric acid （P<0.05）. The 16S rRNA gene sequencing results showed that the intestinal flora structure of the model group changed compared with that of the blank group. Compared with the model group， Sishenwan and Peifeikang increased the relative abundance of Lachnospiraceae， Muribaculaceae， Akkermansiaceae， Ligilactobacillus， UBA3282， Akkermansia， and Corynebacterium while reducing the relative abundance of Oscillospiraceae， Desulfovibrionaceae， Lactobacillus， Romboutsia， and Desulfovibrio. They can restore the intestinal flora structure similar to that in the blank group. ConclusionSishenwan can alleviate diarrhea symptoms and colonic mucosal inflammation， increase the expression of tight junction proteins in the colonic mucosa， and strengthen the intestinal barrier in IBS-D rats with the syndrome of spleen-kidney Yang deficiency. The mechanism of action may be related to optimizing the structure and balance of intestinal flora and regulating the SCFAs， and the effect of high-dose Sishenwan is obvious.