Network pharmacology analysis and clinical efficacy of the traditional Chinese medicine Bu-Shen-Jian-Pi. Part 3: Alleviation of hypoxia, muscle-wasting, and modulation of redox functions in amyotrophic lateral sclerosis.

OBJECTIVE: The aim of this clinical study is to obtain evidence for the clinical efficacy of Bu-Shen-Jian-Pi formula (BSJP), a traditional Chinese medicine, used for the treatment of amyotrophic lateral sclerosis, a relatively rare, progressive and usually fatal disease possibly associated with alterations in tissue redox status, hypoxia, and muscular injury. BACKGROUND: The active agents in BSJP formula† causing apoptosis, modulation of redox changes, and alterations in the immune status have been studied previously by us using cell cultures. The findings from these investigations have been incorporated into pharmacology databases employed in our analysis of BSJP using network pharmacology analysis/artifical intelligence. This information has been used here in the design of the investigation and to optimize evaluation of the clinical efficacy and usefulness of this herbal medicine, as far as possible using evidence-based medicine criteria. MATERIALS AND METHODS: The design of the study was a randomized multi-center, controlled clinical trial in 127 patients with confirmed diagnoses of amyotrophic lateral sclerosis. Patients and investigator were double-blinded. Clinical efficacy was determined using the Amyotrophic Lateral Sclerosis Symptom Score in Integrative Treatment Scale (ALS-SSIT) and the Amyotrophic Lateral Sclerosis Rating Scale-Revised (ALSFRS-R), together with tests of limb muscle strength using the manual muscle test (MMT), forced vital capacity (FVC), and clinical chemistry laboratory tests over a 20-week observation period. RESULTS: The scores of ALS-SSIT in the BSJP group increased significantly (22%) after treatment. The ALSFRS-R score in the BSJP group decreased significantly after treatment (19%). The rate of decrease in muscle function (MMT score) in most BSJP patients was lower than that in the control group, where the differences in the scores for the trapezius and triceps brachii were statistically significant compared to the control group. The fall in FVC in the BJSP group was significantly slower than in the control group. There were no marked differences observed in the frequency of side effects. Serum vitamin D3 levels in the BSJP group showed greater increases compared to the control group. CONCLUSION: BSJP treatment reduced the rate of progression of amyotrophic lateral sclerosis according to the ALS-SSITS and ALSFRS scores and significantly reduced the rate of deterioration in muscle function in the limbs of amyotrophic lateral sclerosis patients. The modes of action of BSJP in treating amyotrophic lateral sclerosis are probably diverse and multi targeted, some of which may involve regulation of serum vitamin D3 and alleviation of the impairments in liver and kidney function.

The role of differentially expressed miR-660 in peripheral blood lymphocytes of patients with pulmonary tuberculosis.

OBJECTIVE: This study aimed to investigate the significance of miRNA expression levels in peripheral blood lymphocytes of patients clinically diagnosed with pulmonary tuberculosis. METHOD: Pulmonary tuberculosis-related datasets in the Gene Expression Omnibus (GEO) database were analyzed, and DE-miRNAs were screened for Gene Ontology (GO) analysis and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment to construct a DE-miRNA-DE-mRNA network. The peripheral blood lymphocytes of 10 patients with pulmonary tuberculosis, 10 patients with rifampicin-resistant tuberculosis and 10 healthy volunteers were selected for validation of RNA expression levels. qRT-PCR was done to verify the expression of DE-miRNA, and western blotting was done to check the expression levels of genes of associated pathways. RESULTS: Differential expression of miR-660 was found in pulmonary tuberculosis through data analysis and literature mining. The differential expression was also confirmed by qRT-PCR in samples from patients and healthy controls. The expression of miR-660 was significantly upregulated (p < 0.01) in patients with pulmonary tuberculosis and rifampicin-resistant pulmonary tuberculosis compared with the healthy controls. According to western blotting results, the expression levels of P-NF-κB and AKT in patients with pulmonary tuberculosis and NF-κB, P-NF-κB, AKT and p-AKT in patients with rifampicin-resistant tuberculosis were significantly upregulated (p < 0.01). CONCLUSION: The high expression levels of miR-660 may activate the AKT/NF-κB signalling pathway and has the potential to serve as a potential biomarker for the diagnosis of pulmonary tuberculosis.

Morphogenesis, ultrastructure, and chemical profiling of trichomes in Artemisia argyi H. Lév. & Vaniot (Asteraceae).

MAIN CONCLUSION: Glandular trichomes of Artemisia argyi H. Lév. & Vaniot are the key tissues for the production of flavonoid and terpenoid metabolites. Artemisia argyi H. Lév. & Vaniot is an herbaceous perennial plant that has been widely used in traditional medicine for thousands of years. Glandular trichomes (GTs) and nonglandular trichomes (NGTs) have been reported on the leaf surface of A. argyi. The aim of this study was to elucidate the morphogenetic process and to analyze the metabolites of trichomes in A. argyi. The morphogenesis of GTs and NGTs was characterized using light, scanning, and transmission electron microscopy. The constituents of GTs were analyzed using laser microdissection combined with gas and liquid chromatography-mass spectrometry. Five developmental stages of two types of GTs and four developmental stages of one type of NGTs were observed. Two types of mature GT and one type of NGT were composed of 10, 5, and 4-6 cells, respectively. A large storage cavity was detected between the cuticle and cell walls in the first type of mature GT. Large nuclei, nucleoli, and mitochondria were observed in the basal and intermediate cells of the second type of GT. In addition, large vacuoles were observed in the basal and apical cells, and large nuclei were observed in the middle cells of NGTs. One monoterpene and seven flavonoids were identified in GTs of A. argyi. We suggest that GTs are the key tissues for the production of bioactive metabolites in A. argyi. This study provides an important theoretical basis and technical approach for clarifying the regulatory mechanisms for trichome development and bioactive metabolite biosynthesis in A. argyi.

Lung adenocarcinoma-related TNF-α-dependent inflammation upregulates MHC-II on alveolar type II cells through CXCR-2 to contribute to Treg expansion.

MHC-II on alveolar type-II (AT-II) cells is associated with immune tolerance in an inflammatory microenvironment. Recently, we found TNF-α upregulated MHC-II in AT-II in vitro. In this study, we explored whether TNF-α-mediated inflammation upregulates MHC-II on AT-II cells to trigger Treg expansion in inflammation-driven lung adenocarcinoma (IDLA). Using urethane-induced mice IDLA model, we found that IDLA cells mainly arise from AT-II cells, which are the major source of MHC-II. Blocking urethane-induced inflammation by TNF-α neutralization inhibited tumorigenesis and reversed MHC-II upregulation on tumor cells of AT-II cellular origin in IDLA. MHC-II-dependent AT-II cells were isolated from IDLA-induced Treg expansion. In human LA samples, we found high expression of MHC-II in tumor cells of AT-II cellular origin, which was correlated with increased Foxp3+ T cells infiltration as well as CXCR-2 expression. CXCR-2 and MHC-II colocalization was observed in inflamed lung tissue and IDLA cells of AT-II cellular origin. Furthermore, at the pro-IDLA inflammatory stage, TNF-α-neutralization or CXCR-2 deficiency inhibited the upregulation of MHC-II on AT-II cells in inflamed lung tissue. Thus, tumor cells of AT-II cellular origin contribute to Treg expansion in an MHC-II-dependent manner in TNF-α-mediated IDLA. At the pro-tumor inflammatory stage, TNF-α-dependent lung inflammation plays an important role in MHC-II upregulation on AT-II cells.

A long-wavelength activable AIEgen fluorescent probe for HClO and cell apoptosis imaging.

Hypochlorous acid (HClO) is an important bactericide, and adjusting the content of HClO helps to improve the host's innate immunity and resist microbial invasion. Aggregation-induced luminescence (AIE) is the opposite of aggregation-induced quenching (ACQ). Compounds with AIE properties emit weakly in a dispersed state in solution and they can emit strong fluorescence in an aggregated state. In this article, we proposed a new AIE fluorescent probe QM-ClO based on the quinoline-malononitrile (QM) fluorophore and dimethylthiocarbamate (DMTC) to detect HClO. The probe QM-ClO showed a fast response time, a low detection limit of 30.8 nM and a large Stokes shift (190 nm). Carbonyl cyanide metachlorophenyl-hydrazone (CCCP) was used to induce cell apoptosis, and then an increase in the HClO content was observed in the cell. It is proved that cell apoptosis can lead to the increase of the HClO content in the cell. This probe provides an effective tool for studying apoptosis-related diseases.

Imaging Hg2+-Induced Oxidative Stress by NIR Molecular Probe with "Dual-Key-and-Lock" Strategy.

Mercury (Hg) is considered an extremely toxic heavy metal which is extremely harmful to both the human body and environment. In addition, Hg2+-induced oxidative stress also exerts a crucial role to play in pathophysiological mechanisms of mercury toxicity. Thus, efficient and specific fluorescent probes for imaging Hg2+-induced oxidative stress are necessary. In the present study, we rationally design a novel Hg2+-activated and ICT-based NIR emission fluorescent probe NIR-HO for sequentially monitoring the ONOO- level with a "dual-key-and-lock" strategy. The probe NIR-HO showed rapid response and excellent specificity and sensitivity for the detection of Hg2+ and ONOO- in vitro. Cell imaging demonstrated that Hg2+-induced oxidative stress was involved in ONOO- upregulation. Also, GSH, NAC, and EDTA were employed as excellent detoxifying drugs against Hg2+-induced toxicity. Moreover, the probe NIR-HO was successfully used for imaging Hg2+ and ONOO- in vivo. In brief, NIR-HO provides a simple and powerful approach which can be used to image Hg2+-induced oxidative stress in the pathological environment.

TNF-α-dependent lung inflammation upregulates superoxide dismutase-2 to promote tumor cell proliferation in lung adenocarcinoma.

Manganese superoxide dismutase (SOD-2), an important primary antioxidant enzyme located in mitochondria, plays a critical role in tumor progression. Reportedly, the proinflammatory cytokine, tumor necrosis factor (TNF)-α, can increase SOD-2 expression in a human lung adenocarcinoma cell line in vitro, indicating that TNF-α-mediated inflammation may regulate SOD-2 expression, which may be related to cancer promotion. Using a urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mice model, we investigated whether and how TNF-α-mediated inflammation upregulated SOD-2 expression in lung adenocarcinoma. Our results showed that SOD-2 was mostly expressed on surfactant protein-C+ AT-II cells (alveolar type II cell) and tumor cells in IDLA mice, which were surrounded by CD68+ macrophages. Blocking TNF-α-dependent inflammation downregulated SOD-2 expression in inflamed lung tissues at the protumor stage and also inhibited SOD-2 expression in tumor cells in the IDLA model. In human lung adenocarcinoma, both the number of infiltrating CD68+ macrophages and TNF-α expression correlated positively with SOD-2 expression, which is related to lymph node metastasis and TNM stage. We collected the conditioned medium from lipopolysaccharide-activated phorbol myristate acetate-induced THP1 (M1) cells to stimulate A549 and H1299 cells and observed that THP1-M1 upregulated SOD-2 by secreting TNF-α. Blocking SOD-2 expression significantly inhibited TNF-α-induced cell proliferation in A549 and H1299 cells in vitro. Thus, TNF-α-mediated lung inflammation can upregulate SOD-2 expression in lung adenocarcinoma, and macrophages contribute to SOD-2 upregulation by secreting TNF-α.

A FRET-based ratiometric fluorescent probe to detect cysteine metabolism in mitochondria.

As an important biothiol in living cells, cysteine is closely related to oxidative damage in living organisms. Sulfite from cysteine metabolism in living cells plays a crucial role in maintaining homeostasis in an organism, and the unbalance of sulfite in vivo would lead to multiple diseases. Thus the development of a new fluorescent probe for cysteine metabolism is needed urgently in mitochondria which are the main place of cysteine metabolism. Herein we construct a novel targeting mitochondria fluorescent probe CP-K based on the FRET mechanism to visualize sulfite in living MCF-7 cells. Probe CP-K displays a large Stokes shift of 150 nm, a low detection limit (26.3 nM) and "naked eye" detection after the addition of HSO3-. Importantly, it is appropriate for imaging the endogenous sulfite from cysteine metabolism in living cells.

[Cloning and prokaryotic expression analysis of squalene synthase CpSQS1 and CpSQS2 from Crataegus pinnatifida].

In order to understand the structural characteristics of squalene synthase genes in the triterpenoids biosynthesis pathway of Crataegus pinnatifida, the squalene synthase genes of C. pinnatifida was cloned and analyzed by bioinformatics and prokaryotic expression. Two squalene synthase genes CpSQS1 and CpSQS2 were cloned from C. pinnatifida fruit by RT-PCR. The ORF length of CpSQS1 and CpSQS2 were 1 239 bp and 1 233 bp respectively, encoding 412 aa and 410 aa respectively. CpSQS1 and CpSQS2 were predicted to be stable acidic proteins by online tools. The secondary structure was mainly composed of α-helix structure, and the tertiary structure was predicted by homology modeling. Structural functional domain analysis showed that 35-367 aa of CpSQS1 and CpSQS2 cDNA containing conserved trans-isoprenyl pyrophosphate synthase domains. Transmembrane domain analysis predicted that two transmembrane domains were founded in CpSQS1 and CpSQS2. The squalene synthase amino sequence of C. pinnatifida had higher homology with the known SQS of Salvia miltiorrhiza and Glycyrrhiza glabra. Phylogenetic tree analysis showed that CpSQS1 and CpSQS2 were clustered into one branch of MdSQS1 and MdSQS2, which were consistent with the phylogenetic rule. Prokaryotic expression vector pGEX-4 T-1-CpSQS1 and pGEX-4 T-1-CpSQS2 were transformed into Escherichia coli Transetta(DE3) for induction, and the target protein was successfully expressed at 65 kDa. The expression levels of CpSQS2 were significantly higher than that of CpSQS1 in three different developmental stages of C. pinnatifida. In this study, the full-length cDNA sequences of C. pinnatifida SQS1 and SQS2 were cloned and analyzed for the first time, which provided the foundation for further study on the metabolic pathway of C. pinnatifida triterpenoids.

[Evolution of commercial specification and experimental identification terms of Gastrodiae Rhizoma].

Gastrodia Rhizoma, as a precious Chinese materia medica, has attracted the attention of Chinese materia medica experts in the past dynasties for the commercial specification and experimental identification, and has gradually formed a wealth of terms concerning commercial specification and experimental identification. Through combing the literatures of successive dynasties, this paper discussed the change of the commercial specification of the Gastrodiae Rhizoma and formation of its identifying terms. It has found that the Gastrodiae Rhizoma mainly came from the dried rhizomes of the Gastrodia elata f.elata before the Qing Dynasty. Since the Qing Dynasty, G. elata in Yunnan and Guizhou gradually arose and become one of sources of mainstream commodities. After that, G. elata f. glauca and G. elata f. elata were becoming the main sources of Gastrodiae Rhizoma. Before the large-scale cultivation of G. elata in the 1970 s, there is only wild G. elata over the country. In terms of commercial specification, they were often classified into Chunma and Dongma according to their harvest time. With the successful promotion of cultivation technology and the endangered wild resources of G. elata, the Dongma became the mainstream in the market. The adulterants of G. elata increased significantly in the 1960 s and 1970 s, in this period, the terms of experimental identification for G. elata also increased obviously. Experimental identification is distinctive in different times, therefore, studying experimental identification of medicinal materials helps to promote the development of the Chinese materia medica.

Atmospheric correction for retrieving ground brightness temperature at commonly-used passive microwave frequencies.

An analysis of the atmospheric impact on ground brightness temperature (Tg) is performed for numerous land surface types at commonly-used frequencies (i.e., 1.4 GHz, 6.93 GHz, 10.65 GHz, 18.7 GHz, 23.8 GHz, 36.5 GHz and 89.0 GHz). The results indicate that the atmosphere has a negligible impact on Tg at 1.4 GHz for land surfaces with emissivities greater than 0.7, at 6.93 GHz for land surfaces with emissivities greater than 0.8, and at 10.65 GHz for land surfaces with emissivities greater than 0.9 if a root mean square error (RMSE) less than 1 K is desired. To remove the atmospheric effect on Tg, a generalized atmospheric correction method is proposed by parameterizing the atmospheric transmittance τ and upwelling atmospheric brightness temperature Tba↑. Better accuracies with Tg RMSEs less than 1 K are achieved at 1.4 GHz, 6.93 GHz, 10.65 GHz, 18.7 GHz and 36.5 GHz, and worse accuracies with RMSEs of 1.34 K and 4.35 K are obtained at 23.8 GHz and 89.0 GHz, respectively. Additionally, a simplified atmospheric correction method is developed when lacking sufficient input data to perform the generalized atmospheric correction method, and an emissivity-based atmospheric correction method is presented when the emissivity is known. Consequently, an appropriate atmospheric correction method can be selected based on the available data, frequency and required accuracy. Furthermore, this study provides a method to estimate τ and Tba↑ of different frequencies using the atmospheric parameters (total water vapor content in observation direction Lwv, total cloud liquid water content Lclw and mean temperature of cloud Tclw), which is important for simultaneously determining the land surface parameters using multi-frequency passive microwave satellite data.

Treatment of the terephthalic acid-containing wastewater using a biological-aerated filter.

In this paper, the biological-aerated filter (BAF) was employed to treat the wastewater containing terephthalic acid (TA). Factors that affected the efficiency of TA and CODCr removal were evaluated experimetally, including pH, hydraulic loading, hydraulic retention time (HRT) and TA volume loading. At pH 7-8, hydraulic loading rate 0.067-0.48 m3/(m2 h), HRT more than 3.5h and TA loading 0.04-0.15g/(m3 d), the TA and CODCr removal efficiency was more than 93% and 87%, respectively. The mathematical model of matrix (TA) was obtained by Monod's relation and the experimental parameters of the model were 1.972 g/(m2d) and 9.782 mg/L.

Simultaneous positivity for anti-DNA, anti-nucleosome and anti-histone antibodies is a marker for more severe lupus nephritis.

OBJECTIVE: The purpose of this study is to examine autoantibody profile of systemic lupus erythematosus (SLE) patients with lupus nephritis (LN) and to establish the correlation between the antibody reactivity and disease activity of LN. METHODS: Autoantibodies and serological parameters were measured and analyzed in 589 SLE patients. The associations of the co-positivity of anti-dsDNA, -nucleosome and -histone antibodies (3-pos) with clinical, serological and outcome parameters were analyzed. RESULTS: At the study entry, the prevalence for anti-dsDNA (61.52 % vs. 34.11 %, P < 0.0001), anti-nucleosome (56.09 % vs. 37.21 %, P = 0.0002) and anti-histone (49.35 % vs. 33.33 %, P = 0.0013) antibodies in patients with LN were significantly higher than that in patients without LN. Patients with 3-pos had a higher proportion of proliferative renal lesions (class III + IV). The incidence of a poor renal outcome (7.14 % vs. 2.52 %, P = 0.0174) in LN patients with 3-pos was significantly higher than those without 3-pos. Moreover, the rate of remission (73.63 % vs. 82.37 %, P = 0.0245) was significantly reduced and recurrence increased (58.90 % vs. 23.44 %, P < 0.0001) in 3-pos patients as compared to that in non 3-pos within the LN group. CONCLUSION: Our data indicate a strong association between the 3-pos and renal disease activities, especially proliferative glomerulonephritis. The ability of 3-pos to predict renal flares may lead to major additional benefits in the follow-up of these patients.

Visualization of peroxynitrite in cyclophosphamide-induced oxidative stress by an activatable probe.

Oxidative stress is considered to be one of the main contributors of cyclophosphamide (CP)-induced toxicity, and the generation of free radicals will cause the interruption of multiple signal transduction pathways. Peroxynitrite (ONOO-) has strong oxidation and nitrification ability and is considered as an indirect indicator of oxidative stress. Therefore, it is necessary to design a fluorescent probe that can detect ONOO- and monitor CP-induced oxidative stress during chemotherapy. Herein, we synthesized a lipid droplet targeting fluorescent probe SX-1 based on triphenylamine-benzoindocyanine. When ONOO- is added to the probe SX-1, the CC bond in the probe is broken, thereby releasing fluorescence. The good spectral response characteristics enable SX-1 to successfully track the fluctuations of ONOO- in living cells. Most importantly, we provided the first visual evidence that the level of ONOO- in HeLa cells was up-regulated under CP induction. All results indicated that SX-1 has great potential in detecting drug-induced ONOO-, and provided a new detection tool for a deeper understanding of drug-induced organism injury.

Eucalyptol prevents bleomycin-induced pulmonary fibrosis and M2 macrophage polarization.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial pneumonia with limited therapeutic options. Eucalyptol, a terpenoid oxide isolated from eucalyptus species, reportedly exhibits various biological activities such as anti-inflammatory and antioxidant effects. In the present study, we aimed to determine whether eucalyptol could alleviate bleomycin (BLM)-induced pulmonary fibrosis and inhibit interleukin (IL)-13-induced M2 macrophage polarization. Upon treatment with eucalyptol, BLM-induced pulmonary fibrosis and lung inflammation were significantly reduced. The pulmonary neutrophil accumulation and pulmonary permeability were inhibited and the expression of hydroxyproline, alpha-smooth muscle actin, and fibronectin was significantly down-regulated. Eucalyptol also markedly inhibited the expression of arginase-1, Ym-1, IL-13, and transforming growth factor (TGF)-ß1, reduced the production of IL-13, IL-6, tumor necrosis factor (TNF)-α, and attenuated the activity of TGF-ß1 in bronchoalveolar lavage fluid (BALF). Furthermore, the in vitro assay revealed that eucalyptol disturbed M2 macrophage polarization and reduced the macrophage-mediated secretion of the profibrotic factor TGF-ß1. Eucalyptol inhibited the nuclear location of signal transducer and activator of transcription 6 (STAT6) and the phosphorylation of STAT6 and p38 mitogen-activated protein kinase (p38 MAPK), and reduced the expression of their downstream transcription factors, krupple-like factor 4 (KLF4) and peroxisome proliferator-activated receptor gamma (PPAR-γ). These findings indicated that eucalyptol alleviates BLM-induced pulmonary fibrosis by regulating M2 macrophage polarization, which, in turn, inhibits the activation of signaling molecules (e.g., STAT6 and p38 MAPK) and the expression of transcription factors (e.g., KLF4 and PPAR-γ). Thus, eucalyptol might be a potential therapeutic agent for IPF.

Differential expression of Ago2-mediated microRNA signaling in adipose tissue is associated with food-induced obesity.

Adipose tissue is a major component for the regulation of energy homeostasis by storage and release of lipids. As a core element of RNA-induced silencing complex, argonaute2 (Ago2) plays critical role in maintenance of systemic metabolic demand. Here, we show that high-fat-diet-fed mice exhibit an increase in body mass alongside systematic insulin resistance and altered rate of energy expenditure. Interestingly, Ago2 expression is associated with obesity and an increased amount of adipose tissue. Moreover, increased levels of Ago2 inhibited the expression of AMPKα by promoting its targeting by miR-148a, the most abundant microRNA in adipose tissues. Those results suggested that Ago2-miR-148a-AMPKα signaling pathway play an important function in the developing obesity and adiposity, and will further provide basic research data for the potential clinical treatment of obesity.

Programmed cell death during the formation of rhytidome and interxylary cork in roots of Astragalus membranaceus (Leguminosae).

Programmed cell death (PCD) plays a critical role throughout the lives of plants, it is regarded as a highly regulated and active process of plant cell death during the times of biotic or abiotic stress. This study aims to provide developmental anatomical characteristics of the interxylary cork formation in the roots of Astragalus. membranaceus var. mongholicus, and to subsequently show cytomorphological evidence that PCD is involved in the development of rhytidome and interxylary cork. The developmental anatomy of rhytidome and interxylary cork of the perennial fresh main root of A. membranaceus var. mongholicus was studied using light microscopy, whereas the PCD in the development of rhytidome and interxylary cork was studied using fluorescence microscopy and transmission electron microscopy. Histologically, it was observed that the parenchyma cells of secondary phloem and xylem in roots recovered their meristematic ability, and later developed into rhytidome and interxylary cork. Cytologically, ultrastructural characteristics such as nucleus malformation, vacuole disappearance, mitochondrial degeneration, and vesicle filling were observed. In roots, the nucleus of the phloem parenchyma cells were terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive from the pre-rhytidome stage to the formation of rhytidome stage and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)-negative during the mature rhytidome stage. The TUNEL assay of the xylem parenchyma cells showed positive characteristics from the early stage of interxylary cork formation to the interxylary cork formation stage, whereas DAPI-negative characteristics were observed in the mature interxylary cork. Gel electrophoresis showed that DNA cleavage was random. Our results indicated that the formation of the rhytidome and interxylary cork involved the PCD process.