Construction and expression of Mycobacterium tuberculosis fusion protein SHR3 and its immunogenicity analysis in combination with various adjuvants.

Tuberculosis (TB) today remains the leading cause of global deaths due to infectious bacterial pathogens. The Bacillus Calmette-Guérin (BCG) vaccine is the only vaccine clinically used to prevent TB. However, its limitations in preventing latent infection and TB reactivation mean that it does not provide comprehensive protection. In this study, we successfully constructed and expressed the multistage fusion protein, SHR3, and used whole blood IFN-γ release assay (WBIA) with flow cytometry to detect antigen specificity, further confirmed by enzyme-linked immunosorbent assay (ELISA). SHR3 and its subfractional proteins stimulated the level of IFN-γ production by lymphocytes from M. tb-infected patients, inducing the production of single-positive and double-positive CD4+ and CD8+ T cells with IFN-γ and IL-2, at levels significantly higher than those of healthy controls. The fusion protein and complex adjuvant group (SHR3/DMT) induced mice to produce significantly higher levels of IgG antibodies and their subclasses, with IgG2a/IgG1 results showing a convergent Th1-type response; mice in the BCG + SHR3/DMT group induced secretion of the highest levels of IL-2, and TNF-α, irrespective of stimulation with purified protein derivative or SHR3. These findings suggest that SHR3/DMT could be a potential subunit vaccine candidate that may serve as an effective booster vaccine after BCG primary immunization.

Long-Term Immunogenicity and In Vitro Prophylactic Protective Efficacy of M. tuberculosis Fusion Protein DR2 Combined with Liposomal Adjuvant DIMQ as a Boosting Vaccine for BCG.

The resuscitation of dormant Mycobacterium tuberculosis is an important cause of adult tuberculosis (TB) transmission. According to the interaction mechanism between M. tuberculosis and the host, the latency antigen Rv0572c and region of difference 9 (RD9) antigen Rv3621c were selected in this study to prepare the fusion protein DR2. Stimulating clinically diagnosed active tuberculosis infections (i.e., TB patients), latent tuberculosis infections, and healthy controls confirmed that T lymphocytes could recognize DR2 protein in the peripheral blood of TB-infected individuals more than subcomponent protein. The DR2 protein was then emulsified in the liposome adjuvant dimethyl dioctadecyl ammonium bromide, and imiquimod (DIMQ) was administered to C57BL/6 mice immunized with Bacillus Calmette-Guérin (BCG) vaccine to evaluate their immunogenicity. Studies have shown that DR2/DIMQ, a booster vaccine for BCG primary immunization, can elicit robust CD4+ Th1 cell immune response and predominant IFN-γ+ CD4+ effector memory T cells (TEM) subsets. Furthermore, the serum antibody level and the expression of related cytokines increased significantly with the extension of immunization time, with IL2+, CD4+, or CD8+ central memory T cells (TCM) subsets predominant in the long term. This immunization strategy showed matched prophylactic protective efficacy by performing in vitro challenge experiment. This result provides robust evidence that the novel subunit vaccine prepared by fusion protein DR2 combined with liposomal adjuvant DIMQ is a promising TB vaccine candidate for further preclinical trials as a booster vaccine for BCG.

Corrigendum: Identification of an autophagy-related 12-lncRNA signature and evaluation of NFYC-AS1 as a pro-cancer factor in lung adenocarcinoma.

[This corrects the article DOI: 10.3389/fgene.2022.834935.].

Construction and expression of Mycobacterium tuberculosis fusion protein AR2 and its immunogenicity in combination with various adjuvants to form vaccine.

Tuberculosis (TB) is recognized as a highly infectious disease worldwide, and Bacille Calmette-Guerin (BCG) remains the only TB vaccine licensed for clinical use. As there is little evidence that BCG is effective in adults, there is an urgent need for a safe and effective vaccine to control TB in adults. In this study, we tested the immunomodulatory efficiency of the fusion protein AR2. whole blood IFN-γ release assay (WBIA) was used to detect antigen specificity. The immunogenicity of the vaccine was tested in C57BL/6 mice, and confirmed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and qRT-PCR. The fusion protein AR2 was successfully constructed and expressed. The level of IFN-γ in the peripheral blood of subjects stimulated by AR2 was significantly higher than in those induced by all subcomponent proteins. AR2-specific IgG and the Th1 cytokines IFN-γ, TNF-α, and iNOS were significantly increased in the group treated with the fusion protein and compound adjuvant (AR2+DMC). Likewise, the number of IFN-γ+ CD4+, IFN-γ+CD8+, and IL-4+ CD8+ T lymphocytes increased significantly. The combination of the fusion protein and the compound adjuvant (AR2+DMC) may be a suitable candidate for an enhanced TB vaccine. This study provides theoretical and experimental support for future research to enhance the effectiveness of TB vaccines and provides an experimental basis for evaluating the influence of different adjuvants on vaccine efficacy.

Identification of an autophagy-related 12-lncRNA signature and evaluation of NFYC-AS1 as a pro-cancer factor in lung adenocarcinoma.

Objective: To develop an autophagy-related lncRNA-based risk signature and corresponding nomogram to predict overall survival (OS) for LUAD patients and investigate the possible meaning of screened factors. Methods: Differentially expressed lncRNAs and autophagy genes were screened between normal and LUAD tumor samples from the TCGA LUAD dataset. Univariate and multivariate Cox regression analyses were performed to construct the lncRNA-based risk signature and nomogram incorporating clinical information. Then, the accuracy and sensitivity were confirmed by the AUC of ROC curves in both training and validation cohorts. qPCR, immunoblot, shRNA, and ectopic expression were used to verify the positive regulation of NFYC-AS1 on BIRC6. CCK-8, immunofluorescence, and flow cytometry were used to confirm the influence of NFYC-AS1 on cell proliferation, autophagy, and apoptosis via BIRC6. Results: A 12-lncRNA risk signature and a nomogram combining related clinical information were constructed. Furthermore, the abnormal increase of NFYC-AS1 may promote LUAD progression through the autophagy-related gene BIRC6. Conclusion: 12-lncRNA signature may function as a predictive marker for LUAD patients, and NFYC-AS1 along with BIRC6 may function as carcinogenic factors in a combinatorial manner.

Use of DosR and Rpf antigens from Mycobacterium tuberculosis to screen for latent and relapse tuberculosis infection in a tuberculosis endemic community of Huainan City.

The dormancy survival regulator (DosR) antigens upgraded during latency and resuscitation-promoting factors (Rpfs) expressed over the reactivation from dormant Mycobacterium tuberculosis (M. tuberculosis) could be used to diagnose tuberculosis (TB) at different stages. We performed a retrospective cohort study based on four groups, including healthy controls (HCs), active tuberculosis infections (ATBs), latent tuberculosis infections (LTBIs), and relapse tuberculosis infections (RTBs) enrolled between November 2020 and June 2021. Compared to the fusion protein E6-C10, combined with early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate of 10 kDa (CFP-10), the DosR- or Rpf-encoded antigens could not elicit significant IFN-γ concentration for the diagnosis of ATB. Of note, the DosR antigens produce significantly more antigen-specific IFN-γ in LTBIs than Rpfs, and the levels of antigen-specific IFN-γ elicited in RTBs stimulated by Rpfs were higher than the DosR antigens. Among the DosR antigens, Rv2003c was the most immunogenic in diagnosing LTBIs, followed by Rv2007c and Rv2005c. As far as Rpfs are concerned, Rv0867c was the best antigen to identify RTBs, followed by Rv2389c and Rv1009. Both Rv2450c and Rv1884c showed relatively limited IFN-γ concentration in RTBs. Besides, the selected DosR antigens and Rpfs showed ideal specificity and inadequate sensitivity, which could have been enhanced by the fusion antigens prepared by the DosR antigens or Rpfs, respectively. The results of this study can provide more accurate detection methods for LTBIs and RTBs and could be used for screening the dormant M. tuberculosis throughout reactivation.

Enhanced immunogenicity of the tuberculosis subunit Rv0572c vaccine delivered in DMT liposome adjuvant as a BCG-booster.

COVID-19 has affected the progress made in the prevention and treatment of tuberculosis (TB); hence, the mortality of tuberculosis has risen. Different strategies-based novel TB vaccine candidates have been developed. This study identifies strategies to overcome the limitations of Bacille Calmette-Guérin (BCG) in preventing latent infection and reactivation of TB. The latency antigen Rv0572c was selected based on the mechanism of interaction between Mycobacterium tuberculosis and its host. The rRv0572c protein was used to stimulate whole blood samples derived from patients with clinically diagnosed active TB (ATBs) or latent TB infections (LTBIs) and healthy control (HCs) donors, confirming that this protein can be recognized by T cells in patients with TB, especially LTBIs. C57BL/6 mice were used to investigate the immunogenicity of the rRv0572c protein emulsified in the liposome adjuvant dimethyldioctadecylammonium [DDA], monophosphoryl lipid A [MPLA], trehalose-6, 6'-dibehenate [TDB] (DMT). The results demonstrated that rRv0572c/DMT could boost BCG-primed mice to induce antigen-specific CD4+ T cell production and generate functional T cells dominated by antigen-specific CD8+ T cells. The rRv0572c/DMT vaccine could also trigger limited Th2 humoral immune responses. These findings suggest that rRv0572c/DMT is a potential subunit vaccine candidate that can be used as a booster vaccine for BCG.

MiR-1193 was sponged by LINC00963 and inhibited cutaneous squamous cell carcinoma progression by targeting SOX4.

Cutaneous squamous cell carcinoma (CSCC), a class of skin tumor derived from epidermal keratinocyte, is reputed as one of the most malignant tumors globally. MicroRNAs (miRNAs) are increasingly identified as essential players in CSCC. Current study aimed to uncover the impact and mechanism of miR-1193 in CSCC. We identified the low expression of miR-1193 in CSCC cell lines. Gain- and loss-of-function assays showed that miR-1193 acted as an inhibitor of proliferation and migration in CSCC cells. Furthermore, we illustrated that miR-1193 targeted and inhibited SRY-box 4 (SOX4), and that long intergenic non-protein coding RNA 963 (LINC00963) sponged miR-1193 to upregulate SOX4 expression. Rescue assays showed that LINC00963 regulated CSCC progression through miR-1193/SOX4 axis. In conclusion, our study firstly revealed the LINC00963/miR-1193/SOX4 axis in CSCC, indicating miR-1193 as a promising biological target in CSCC progression.

Dietary Patterns and Overweight/Obesity: A Review Article.

BACKGROUND: Dietary patterns analysis may provide insights into the influence of overall diet on overweight/obesity. In the past two decades, the relation between dietary patterns and overweight/obesity has been a research focus and a number of results were reported in the research field. METHODS: An electronic literature search was conducted in PubMed and Web of Science, to identify human studies published by Mar 2015 and written in English. The following keywords or phrases were involved: dietary patterns, dietary pattern, factor analysis, principal component analysis, diet, obesity, adiposity, overweight and BMI. All the studies were retrieved and prudent/healthy (n=17) and western/unhealthy (n=18) dietary patterns were identified. RESULTS: When compared with the lowest categories of a prudent/healthy dietary pattern, a reduced overweight/obesity risk was shown in the highest (OR=0.64; 95% CI: 0.52, 0.78; P<0.0001). While there was an increased overweight/obesity risk in the highest when compared with the lowest categories of a western/unhealthy dietary pattern (OR=1.65; 95% CI: 1.45, 1.87; P<0.0001). CONCLUSION: A prudent/healthy dietary pattern and limit intake of western/unhealthy dietary pattern should be followed, which helps to keep a healthy body mass.

Combined effects of NQO1 Pro187Ser or SULT1A1 Arg213His polymorphism and smoking on bladder cancer risk: Two meta-analyses.

OBJECTIVES: Objectives: Cigarette smoking is the major risk factor of bladder cancer via exposure to chemical carcinogens. Nicotinamide adenine dinucleotide phosphate (NADP+): quinine oxidoreductase 1 (NQO1) and sulfotransferase 1A1 (SULT1A1) have been reported to involve in the metabolism of polycyclic aromatic hydrocarbons (PAHs) and aromatic amines. Therefore, the risk of bladder cancer (BC) may be influenced by polymorphisms in the genes that modulate metabolic detoxification in particular by interacting with cigarette smoking. Considering the limited power by the individual studies with a relatively small sample size, especially when analyzing the combined effect of polymorphisms in NQO1 and SULT1A1 genes and smoking, these 2 meta-analyses have aimed to clarify the combined effects of them on BC risk by integrating related studies. MATERIAL AND METHODS: Two meta-analyses included 1341 cases and 1346 controls concerning NQO1 Pro187Ser and smoking, and 1921 cases and 1882 controls on SULT1A1 Arg213His and smoking were performed. Odds ratios (OR) and 95% confidence intervals (CI) were used for assessing the strength of the association. RESULTS: The result has demonstrated that smokers with NQO1 Pro/Ser or Ser/Ser genotypes have a prominent association with the risk of BC as compared with non-smokers with NQO1 Pro/Pro genotype, with OR equal to 3.71 (95% CI: 2.87-4.78, pheterogeneity = 0.376). Besides, smokers carrying SULT1A1 Arg/Arg genotypes were observed to confer 2.38 fold increased risk of BC (95% CI: 1.44-3.93, pheterogeneity = 0.001) when compared with non-smokers with SULT1A1 Arg/Arg or His/His genotypes. CONCLUSIONS: These findings have suggested that the NQO1 Pro187Ser or SULT1A1 Arg213His polymorphism combination with smoking significantly confer susceptibility to BC. Int J Occup Med Environ Health 2017;30(5):791-802.

PAX4 promotes PDX1-induced differentiation of mesenchymal stem cells into insulin-secreting cells.

A shortage of postmortem pancreatic tissue for islet isolation impedes the application of cell replacement therapy in patients with diabetes. As an alternative for islet cell transplantation, transcription factors, including PDX1, PAX4, and neurogenin-3, that aid in the formation of insulin-producing ß cells during development have been investigated. The present study evaluated the effects of PAX4 and PDX1 on the differentiation of mesenchymal stem cells (MSCs) into insulin-producing ß-like cells in vitro using recombinant adenoviruses carrying PDX1 or PDX1 plus PAX4. RT-PCR, Western blot, and immunofluorescence assays were used to detect the expression levels of relevant genes and proteins, and enzyme-linked immunosorbent assays were used to determine the amount of insulin and C-peptide secreted by the virus-infected cells following stimulation with high glucose. The results showed that PAX4 markedly enhanced the propensity of PDX1-positive MSCs to form mature islet-like clusters and functional insulin-producing ß-like cells. Our findings provide a novel foundation for generating ß-like cells from MSCs with PAX4 and PDX1 for future clinical application.

STUDY OF ACAROID MITES POLLUTION IN STORED FRUIT-DERIVED CHINESE MEDICINAL MATERIALS.

OBJECTIVE: to investigate the species and breeding density of acaroid mites in stored fruit-derived Chinese medicinal materials in Anhui province. METHODS: samples of stored fruit-derived Chinese medicinal materials were collected from 30 herb stores and storehouses in 17 Anhui cities, where the breeding acaroids mites were detected. RESULTS: 20 species of acaroids mites were found in 33 samples, belonging to 15 genus, 5 families of the acaridae respectively, among which T. putrescentiae, A. farinae, C. lactis, and C. berlesei are predominant species. CONCLUSION: stored fruit-derived Chinese medicinal materials in Anhui areas suffer from serious acaroid mites pollution. Therefore, proactive measures should be taken to control acaroid mites from breeding in an effort to reduce the harm on medicinal materials.

Objetivo: investigar las especies y la densidad de reproducción de ácaros en productos medicinales chinos almacenados derivados de la fruta en la provincia de Anhui. Métodos: muestras de productos medicinales chinos almacenados derivados de la fruta fueron recogidos a partir de 30 herbolarios y almacenes en 17 ciudades de Anhui, donde se detectó la reproducción de ácaros. Resultados: se detectaron 20 especies de ácaros en 33 muestras, pertenecientes a 15 géneros, 5 familias de ácaros respectivamente, entre los cuales T. putrescentiae, A. farinae, C. lactis y C. berlesei son las especies predominantes. Conclusión: los productos medicinales chinos almacenados derivados de la fruta en la zona de Anhui sufren una grave contaminación por ácaros. Por lo tanto, se deben tomar medidas dinámicas para controlar la reproducción de ácaros en un esfuerzo por reducir los daños en los productos medicinales.

Study of acaroid mites pollution in stored fruit-derived Chinese medicinal materials / Estudio de la contaminación por ácaros en productos medicinales chinos almacenados derivados de la fruta

Objective: to investigate the species and breeding density of acaroid mites in stored fruit-derived Chinese medicinal materials in Anhui province. Methods: samples of stored fruit-derived Chinese medicinal materials were collected from 30 herb stores and storehouses in 17 Anhui cities, where the breeding acaroids mites were detected. Results: 20 species of acaroids mites were found in 33 samples, belonging to 15 genus, 5 families of the acaridae respectively, among which T. putrescentiae, A. farinae, C. lactis, and C. berlesei are predominant species. Conclusion: stored fruit-derived Chinese medicinal materials in Anhui areas suffer from serious acaroid mites pollution. Therefore, proactive measures should be taken to control acaroid mites from breeding in an effort to reduce the harm on medicinal materials (AU)

Objetivo: investigar las especies y la densidad de reproducción de ácaros en productos medicinales chinos almacenados derivados de la fruta en la provincia de Anhui. Métodos: muestras de productos medicinales chinos almacenados derivados de la fruta fueron recogidos a partir de 30 herbolarios y almacenes en 17 ciudades de Anhui, donde se detectó la reproducción de ácaros. Resultados: se detectaron 20 especies de ácaros en 33 muestras, pertenecientes a 15 géneros, 5 familias de ácaros respectivamente, entre los cuales T. putrescentiae, A. farinae, C. lactis y C. berlesei son las especies predominantes. Conclusión: los productos medicinales chinos almacenados derivados de la fruta en la zona de Anhui sufren una grave contaminación por ácaros. Por lo tanto, se deben tomar medidas dinámicas para controlar la reproducción de ácaros en un esfuerzo por reducir los daños en los productos medicinales (AU)

Improved antifungal activity of amphotericin B-loaded TPGS-b-(PCL-ran-PGA) nanoparticles.

To develop amphotericin B-loaded biodegradable TPGS-b-(PCL-ran-PGA) nanoparticles (PLGA-TPGS-AMB NPs) for fungal infection treatment, PLGA-TPGS NPs and PLGA NPs were synthesized by a modified double emulsion method and characterized in terms of size and size distribution, morphology and zeta potential. Drug encapsulation efficiency, in vitro drug release, and in vitro/vivo tests against Candida glabrata were completed. The data showed that both of the two AMB-loaded NPs (PLGA-AMB NPs, PLGA-TPGS-AMB NPs) achieved significantly higher level of antifungal effects than water suspended AMB. In comparison with PLGA-AMB NPs, PLGA-TPGS-AMB NPs had a stronger protective effect against candidiasis and gained an advantage of prolonged antifungal efficacy. In conclusion, PLGA-TPGS-AMB NPs system significantly improves AMB bioavailability by increasing the aqueous dispersibility and improving the antifungal activity. And this would be an excellent choice for the antifungal treatment of the entrapped drug because of its low toxicity and higher effectiveness.

Autophagy-targeted vaccine of LC3-LpqH DNA and its protective immunity in a murine model of tuberculosis.

The development of more effective antituberculosis vaccines would contribute to the control of the global problem of infection with Mycobacterium tuberculosis (MTB). Recently, the highlighted importance of autophagy in the host immune response against MTB has attracted the attention of researchers. However, the vaccines targeted at autophagy remain to be developed. In this study, we report on an autophagy-targeted vaccine of 19kDa MTB lipoprotein (LpqH) DNA that harbors another gene coding microtubule-associated protein light chain-3(LC3), which transports LpqH to autophagosomes and displays enhanced protective efficacy against MTB. After the transfection of pCMV-LpqH DNA, a significant increase LC3 II was detected in RAW264.7 cells, which was similar to that observed with treatment with rapamycin, a reagent used to induce autophagy. To target autophagy, the gene coding LC3, as a marked protein of autophagosome, was linked to the lpqH gene to express an LC3-LpqH fused protein. Interestingly, LC3-LpqH fused protein was determined to be transported to an autophagosome, which was demonstrated by the co-localization of GFP-LC3 with LC3-LpqH at autophagosomes. Notably, the mice immunized with LC3-LpqH/Ag85B displayed decreased mycobacterial loads in the lungs and spleen when challenged with virulent MTB by intravenous infection, which was consistent with increased IgG2a in serum and IFN-γ and IL-2 produced by splenocyte. In conclusion, our study demonstrates that an LC3-LpqH DNA vaccine could have autophagy as its target, which contributes to the enhancement of the Th1 immune response and vaccine protective efficacy.

A method for the establishment of a cell line with stable expression of the GFP-LC3 reporter protein.

As the function of autophagy becomes evident in a number of diseases, including cancer and infection, it is crucial to construct macrophage cell lines with stable expression of the microtubule-associated protein light chain 3 (GFP-LC3). In this study, a mouse LC3 open-reading frame was amplified by RT-PCR, and cloned into the pEGFP-C1 plasmid for expression of the GFP-LC3 fusion protein. The recombinant plasmid was transfected into RAW264.7 cells using Lipofectamine 2000 reagent and stably transfected clones were selected by G418 screening. Autophagic puncta formation was observed by fluorescense microscopy. Additionally, we found that starvation treatment induced a significant increase in the number of autophagosomes, while wortmannin treatment significantly repressed the formation of autophagosomes. This study indicated that the RAW264.7 cell line stably expressing GFP-LC3 is available for use in a GFP-LC3 puncta formation assay, and may contribute to basic investigations of autophagic function or drug screening targeted at autophagy.

Vitamin E reduces hepatic fibrosis in mice with Schistosoma japonicum infection.

To investigate whether vitamin E protects against hepatic fibrosis in mice with Schistosoma japonicum infection, 24 pathogen-free Kunming mice were selected and randomly divided into four groups: control (uninfected, untreated), model (infected, untreated), low-dose intervention (infected, vitamin E-treated, 30 mg/g bodyweight/day) and high-dose intervention (infected, vitamin E-treated, 60 mg/g bodyweight/day). Mice were infected with Schistosoma japonicum by inoculating abdominal skin with snail hosts. The activities of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were detected in hepatic tissue by colorimetry. The expression levels of laminin (LN), hyaluronic acid (HA), procollagen type Ⅲ (PC-III) and type Ⅳ collagen (IV-C) were detected in the serum by radioimmunoassay. Finally, areas and numbers of granulomas were assessed through histopathology 42 days following treatment. The results revealed that mean areas of granulomas were smaller in the low- and high-dose intervention groups compared to those in the model group. Furthermore, the higher dose of vitamin E resulted in smaller granulomas than the low dose. The levels of LN, HA, PC-III and IV-C in the serum were lower following vitamin E treatment than in the model group. By contrast, activity of SOD, GPx and CAT in hepatic tissue was higher following vitamin E treatment compared to the model group. The activity of MDA was lower in hepatic tissue following vitamin E treatment compared to the model group, but was higher compared to controls. In general, the higher dose of vitamin E affected measurements to a greater extent than the lower dose. In conclusion, vitamin E treatment may reduce the growth of granulomas, slowing the process of hepatic fibrosis, and this effect may be the result of the altered activity of the oxidation-reduction enzyme system.

[Study on the expression of immune function in cases infected by Cyclospora cayetanensis].

In Cyclospora cayetanensis oocyst-positive patients, T cell subsets in peripheral mononuclear cell and membrane interleukin-2 receptor (mIL-2R) were detected with the method of biotin-streptavidin (BSA) and soluble interleukin-2 receptor (sIL-2R) as well as special IgG, IgM in serum was detected by ELISA. Results showed that there is a significant difference between the infected and uninfected individuals.

Extraction of protoporphyrin disodium and its inhibitory effects on HBV-DNA.

AIM: To explore an ideal method for extracting protoporphyrin disodium (PPN) from unanticoagulated animal blood, and to study the inhibitory effects of PPN on HBV-DNA duplication and its cytotoxicity to 2.2.15 cell strain. METHODS: Protoporphyrin methyl ester and other intermediate products were prepared with protoheme separated from protein hydrolysates of coagulated animal blood, which were finally made into PPN and detected quantitatively with an ultraviolet fluorescent analyzer. Ten microg/ml, 20 microg/ml, 40 microg/ml, 80 microg/ml and 160 microg/ml of PPN-aqueous solution were added into culture medium for 2.2.15 cells respectively. Eight days later, the drug concentration in supernatant from the culture medium was detected when inhibition rate of HBeAg, cell survival rate when inhibition rate of HBeAg was 50% (ID50), and when survival cells in experimental group were 50% of those in control group (CD50), and the therapeutic index (TI) was also detected. PPN with different concentration of 10 microg/ml, 20 microg/ml, 40 microg/ml, 80 microg/ml and 160 microg/ml was respectively mixed and cultivated with HepG2 2.2.15 cell suspension, and then the inhibition of PPN against HBV-DNA was judged by PCR. RESULTS: The extract of henna crystal was identified to be PPN. When the concentrations of PPN were 160 microg/ml and 80 microg/ml, the inhibition rates of HBeAg were 89.8% and 82.4%, and the cell survival rates were 98.7% and 99.2%. CONCLUSION: It is suggested that PPN can be extracted from unanticoagulated animal blood. PPN can inhibit HBV-DNA expression and duplication in vitro, and has no cytotoxicity to liver cells. Further study and application of PPN are warranted.