Progress in Research on Inhibitors Targeting SARS-CoV-2 Main Protease (Mpro).

Since 2019, the novel coronavirus (SARS-CoV-2) has caused significant morbidity and millions of deaths worldwide. The Coronavirus Disease 2019 (COVID-19), caused by SARS-CoV-2 and its variants, has further highlighted the urgent need for the development of effective therapeutic agents. Currently, the highly conserved and broad-spectrum nature of main proteases (Mpro) renders them of great importance in the field of inhibitor study. In this study, we categorize inhibitors targeting Mpro into three major groups: mimetic, nonmimetic, and natural inhibitors. We then present the research progress of these inhibitors in detail, including their mechanism of action, antiviral activity, pharmacokinetic properties, animal experiments, and clinical studies. This review aims to provide valuable insights and potential avenues for the development of more effective antiviral drugs against SARS-CoV-2.

Aptamer-Functionalized Nanoparticles in Targeted Delivery and Cancer Therapy.

Using nanoparticles to carry and delivery anticancer drugs holds much promise in cancer therapy, but nanoparticles per se are lacking specificity. Active targeting, that is, using specific ligands to functionalize nanoparticles, is attracting much attention in recent years. Aptamers, with their several favorable features like high specificity and affinity, small size, very low immunogenicity, relatively low cost for production, and easiness to store, are one of the best candidates for the specific ligands of nanoparticle functionalization. This review discusses the benefits and challenges of using aptamers to functionalize nanoparticles for active targeting and especially presents nearly all of the published works that address the topic of using aptamers to functionalize nanoparticles for targeted drug delivery and cancer therapy.

Aptamers, the Nucleic Acid Antibodies, in Cancer Therapy.

The arrival of the monoclonal antibody (mAb) technology in the 1970s brought with it the hope of conquering cancers to the medical community. However, mAbs, on the whole, did not achieve the expected wonder in cancer therapy although they do have demonstrated successfulness in the treatment of a few types of cancers. In 1990, another technology of making biomolecules capable of specific binding appeared. This technique, systematic evolution of ligands by exponential enrichment (SELEX), can make aptamers, single-stranded DNAs or RNAs that bind targets with high specificity and affinity. Aptamers have some advantages over mAbs in therapeutic uses particularly because they have little or no immunogenicity, which means the feasibility of repeated use and fewer side effects. In this review, the general properties of the aptamer, the advantages and limitations of aptamers, the principle and procedure of aptamer production with SELEX, particularly the undergoing studies in aptamers for cancer therapy, and selected anticancer aptamers that have entered clinical trials or are under active investigations are summarized.

TLR9 agonist enhances radiofrequency ablation-induced CTL responses, leading to the potent inhibition of primary tumor growth and lung metastasis.

Radiofrequency ablation (RFA) is the most common approach to thermal ablation for cancer therapy. Unfortunately, its efficacy is limited by incomplete ablation, and further optimization of RFA is required. Here, we demonstrate that incubation at 65 °C triggers more EG7 tumor cell death by necrosis than treatment at 45 °C, and the 65 °C-treated cells are more effective at inducing antigen-specific CD8+ cytotoxic T lymphocyte (CTL) responses after injection in mice than the 45 °C-treated ones. Dendritic cells (DCs) that phagocytose 65 °C-treated EG7 cells become mature with upregulated MHCII and CD80 expression and are capable of efficiently inducing effector CTLs in mouse tumor models. RFA (65 °C) therapy of EG7 tumors induces large areas of tumor necrosis and stimulates CTL responses. This leads to complete regression of small (~100 mm3) tumors but fails to suppress the growth of larger (~350 mm3) tumors. The administration of the Toll-like receptor-9 (TLR9) agonist unmethylated cytosine-phosphorothioate-guanine oligonucleotide (CpG) to DCs phagocytosing 65 °C-treated EG7 cells enhances the expression of MHCII and CD40 on DCs as well as DC-induced stimulation of CTL responses. Importantly, the intratumoral administration of CpG following RFA also increases the frequencies of tumor-associated immunogenic CD11b-CD11c+CD103+ DC2 and CD11b+F4/80+MHCII+ M1 macrophages and increases CD4+ and CD8+ T-cell tumor infiltration, leading to enhanced CD4+ T cell-dependent CTL responses and potent inhibition of primary RFA-treated or distant untreated tumor growth as well as tumor lung metastasis in mice bearing larger tumors. Overall, our data indicate that CpG administration, which enhances RFA-induced CTL responses and ultimately potentiates the inhibition of primary tumor growth and lung metastasis, is a promising strategy for improving RFA treatment, which may assist in optimizing this important cancer therapy.

Roles of Host Immunity in Viral Myocarditis and Dilated Cardiomyopathy.

The pathogenesis of viral myocarditis includes both the direct damage mediated by viral infection and the indirect lesion resulted from host immune responses. Myocarditis can progress into dilated cardiomyopathy that is also associated with immunopathogenesis. T cell-mediated autoimmunity, antibody-mediated autoimmunity (autoantibodies), and innate immunity, working together, contribute to the development of myocarditis and dilated cardiomyopathy.

Intact Arabidopsis RPB1 functions in stem cell niches maintenance and cell cycling control.

Plant meristem activity depends on accurate execution of transcriptional networks required for establishing optimum functioning of stem cell niches. An Arabidopsis mutant card1-1 (constitutive auxin response with DR5:GFP) that encodes a truncated RPB1 (RNA Polymerase II's largest subunit) with shortened C-terminal domain (CTD) was identified. Phosphorylation of the CTD repeats of RPB1 is coupled to transcription in eukaryotes. Here we uncover that the truncated CTD of RPB1 disturbed cell cycling and enlarged the size of shoot and root meristem. The defects in patterning of root stem cell niche in card1-1 indicates that intact CTD of RPB1 is necessary for fine-tuning the specific expression of genes responsible for cell-fate determination. The gene-edited plants with different CTD length of RPB1, created by CRISPR-CAS9 technology, confirmed that both the full length and the DK-rich tail of RPB1's CTD play roles in the accurate transcription of CYCB1;1 encoding a cell-cycle marker protein in root meristem and hence participate in maintaining root meristem size. Our experiment proves that the intact RPB1 CTD is necessary for stem cell niche maintenance, which is mediated by transcriptional regulation of cell cycling genes.

[Chinese herbal medicine Euphorbia esula extract induces apoptosis and inhibits the proliferation, migration and invasion of multidrug resistant gastric carcinoma cells].

This paper aims to study the effects of traditional Chinese medicine Euphorbia esula on multidrug resistant human gastric cancer cells in the cell proliferation, migration, invasion and apoptosis, and to study the apoptosis-inducing pathway. Different dilutions of Euphorbia esula extract were used to process human multidrug resistant gastric cancer SGC7901/ADR cells. Cell proliferation inhibition phenomenon was determined by MTT experiment. Nuclear morphological changes of apoptotic cells and apoptotic indexes were observed and determined by Hochest33528 staining followed with fluorescence microscope observing. Flow cytometry was used to detect cell apoptosis rate. Cell migration and invasion ability were observed and determined by Transwell method. Spectrophotometry was used to detect caspase-3 and caspase-9 enzyme activity. Western blotting was used to detect subcellular distribution of cytochrome c. The results showed that Euphorbia esula extract had obvious inhibition effect on proliferation of gastric cancer multidrug resistant SGC7901/ADR cells, which was time- and concentration-dependent. After processing multidrug resistant gastric cancer SGC7901/ADR cells with Euphorbia esula extract, the apoptotic index and apoptosis rate were significantly increased than those in the control group, which showed a time- and dose-dependent mode; but if a caspase inhibitor was added, apoptosis index was not obviously increased. Transwell method showed that migration and invasion ability of the Euphorbia esula extract-processed SGC7901/ADR cells dropped significantly. Spectrophotometry showed that in Euphorbia esula extract-processed SGC7901/ADR cells, caspase-3 and caspase-9 expression were increased, which had significant differences with the control group. Western blotting test showed that the distribution of cytochrome c decreased in mitochondria, while increased in the cytoplasm (i.e., cytochrome c escaped from mitochondria to the cytoplasm). In conclusion, Euphorbia esula extract could inhibit the proliferation, migration and invasion, and induce apoptosis in human gastric cancer multidrug resistant SGC7901/ADR cells; and cytochrome c, caspase-9 and caspase-3 might be involved in cell apoptosis induced by Euphorbia esula extract, suggesting endogenous or mitochondrial apoptotic pathway.

Euphorbia lunulata extract acts on multidrug resistant gastric cancer cells to inhibit cell proliferation, migration and invasion, arrest cell cycle progression, and induce apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: The milky sap or the aboveground part of the plant Euphorbia lunulata has long been used by Chinese people to treat noncancerous growths and cancerous ailments but the specific mode of action and the action mechanism remain to be elucidated. AIM OF THE STUDY: To investigate the effects of Euphorbia lunulata extract on cell proliferation, migration, invasion, cell cycle progression, and apoptosis of multidrug resistant human gastric cancer cells; To study the mechanism of apoptosis induction by Euphorbia lunulata extract in multidrug resistant human gastric cancer cells. MATERIALS AND METHODS: The aboveground part of fresh Euphorbia lunulata plant was extracted first with ethanol and then with n-hexane. The aseptic extract at varying concentrations was used to treat the multidrug resistant human gastric cancer SGC7901/ADR cells. After treatment, the inhibition of cell proliferation was examined by MTT assay. The inhibitions of cell migration and invasion were detected by Transwell method. The alteration of cell cycle progression was studied by flow cytometry. The morphological changes of cell nuclei were observed with fluorescence microscopy following Hoechst 33258 staining and the apoptotic indexes were calculated. The activation of caspase enzymes was analyzed by spectrophotometry. The sub-cellular distribution of cytochrome complex and the expression of Bax and Bcl-2 proteins were determined by Western blot. RESULTS: The proliferation, migration, and invasion of SGC7901/ADR cells were significantly inhibited by Euphorbia lunulata extract, which showed time- and dose-dependent manners. Cell cycle was arrested in G2/M phase. Significant apoptotic morphological changes were observed in the nuclei of the treated cells, and apoptotic indexes were increased significantly; these changes were diminished when Z-VAD-FMK, a caspase inhibitor, was also presented. The activities of caspase-3, caspase-8, and caspase-9 were increased. The sub-cellular distribution of cytochrome complex was altered----reduced in the mitochondria and increased in the cytoplasm. The expression of Bax was upregulated, while that of Bcl-2 was downregulated. CONCLUSION: Euphorbia lunulata extract inhibited the proliferation, migration, and invasion of SGC7901/ADR cells, arrested cell cycle progression, and induced cell apoptosis; the mechanism of apoptosis induction involved both the extrinsic and the intrinsic pathways.

Apoptosis of human gastric carcinoma cells induced by Euphorbia esula latex.

AIM: To investigate the effect of Euphorbia esula (E. esula) extract in inhibiting proliferation and inducing apoptosis in SGC-7901 cells. METHODS: E. esula extract at different concentrations was used to inhibit proliferation and induce apoptosis of human gastric carcinoma SGC-7901 cells. Inhibition of proliferation was detected with thiazolyl blue assay, and apoptosis was detected with fluorescence microscopy, transmission electron microscopy, and flow cytometry. The mechanisms were studied by measurement of caspase-3 and caspase-8 activities and Bax and Bcl2 mRNA expression. RESULTS: The thiazolyl blue assay showed that SGC-7901 cell viability and proliferation were inhibited significantly by E. esula extract in a time- and concentration-dependent manner. Fluorescence microscopy revealed that the cell nuclei showed the characteristic changes of apoptosis, such as uneven staining and chromatin marginalization. Some key features of apoptosis were also observed under transmission electron microscopy, which included cellular shrinkage and the foaming or bubbling phenomenon. When the cells were analyzed by flow cytometry, a sub-G1 peak could be seen clearly. Spectrophotometric assay of caspase-3 and caspase-8 activities in the treated cells showed an approximately two-fold increase. Reverse transcription polymerase chain reaction showed that Bax mRNA expression was upregulated, while Bcl2 mRNA expression was downregulated. CONCLUSION: E. esula extract inhibited proliferation and induced apoptosis in SGC-7901 cells, in a caspase-dependent manner, involving upregulation of Bax and downregulation of Bcl2.

[The increased level of IL-17 in stomach and up-regulated splenic Th17 cell frequency and IL-17 mRNA level in rats infected with Helicobacter pylori].

OBJECTIVE: To investigate the variation of Th17 cell frequency and IL-17 level in rats infected with Helicobacter pylori (Hp). METHODS: A total of 30 rats were used to be infected with Hp by intragastric administration of bacterial suspension. Every 10 rats were sacrificed after 3, 6 and 9 weeks, respectively. The gastric tissues of rats were pathologically examined by HE staining. The level of IL-17 in the gastric tissues of rats was detected by ELISA. The flow cytometry was performed to determine Th17 cell frequency and reverse transcription PCR was to detect the level of IL-17 mRNA in single-cell suspensions of rat spleen. RESULTS: Hp infection induced the obvious pathological changes in the rat gastric mucosa. The level of IL-17 in the gastric tissues increased with the prolonging of the Hp infection. Both Th17 cell frequency and IL-17 mRNA level in rat spleen cells infected with Hp at 3, 6 and 9 weeks were significantly higher than those in control rats, and they were significantly different when compared among different time points. CONCLUSION: Hp infection could induce the damage of rat gastric mucosa and increase IL-17 level in the stomach. Furthermore, Hp infection could up-regulate the Th17 cell frequency and IL-17 mRNA level in rat spleen cells.

Calcitonin is expressed in the submaxillary glands of rats.

Calcitonin is usually produced by the parafollicular cells of the thyroid. However in an immunohistochemistry experiment we observed that the cells of the serous acini of rat submaxillary gland tissue were stained positive with calcitonin antibodies. We further used immunocytochemistry and nucleic acid hybridization to localize the distribution of calcitonin protein and calcitonin mRNA respectively in cultivated cells of rat submaxillary glands. The results showed that the cytoplasm of the epithelial cells of the submaxillary glands had positive staining in immunocytochemistry using calcitonin monoclonal antibody and positive reaction in nucleic acid hybridization using calcitonin mRNA complementary DNA probe. For the first time we found that the cells of the submaxillary glands of rats can produce the hormone calcitonin.

The Euphorbia lunulata Bge extract inhibits proliferation of human hepatoma HepG2 cells and induces apoptosis.

PURPOSE: The purpose of this study was to determine the effect of Euphorbia lunulata Bge extract on the proliferation of human hepatoma HepG2 cell line. METHODS: Different dilutions of Euphorbia lunulata Bge extract were used to treat human hepatoma HepG2 cells. Hoechst 33258 and PI-staining fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect the rates of apoptosis and apoptotic peaks. Western blotting was performed to analyze the subcellular distribution of cytochrome C. RESULTS: The Euphorbia lunulata Bge extract was found to inhibit the proliferation of human hepatoma HepG2 cells via a time and concentration-dependent manner. CONCLUSION: Altogether, the results suggest that the Euphorbia lunulata Bge extract is effective in inhibiting the proliferation of human hepatoma HepG2 cells and inducing cell apoptosis. The mechanism may be related to the mitochondrial pathways or cellular apoptosis pathways.

[Effect of compound gardenia oil and jujube seed oil on learning and memory in ovariectomized rats].

OBJECTIVE: To observe the effect of compound of gardenia oil and jujube seed oil learning and memory in ovariectomized rats and its mechanism. METHODS: Animals were randomly divided into six groups: sham group, model group, estrogen group, low dose group, middle dose group and high dose group. The ovariectomized rat models were established by resection of the lateral ovaries. The effect of compound of gardenia oil and jujube seed oil on learning and memory in ovariectomized rats was observed by means of Morris water maze. Acetylcholinesterase (AchE) and nitric oxide synthase (NOS) activities in rat brain were determined. RESULTS: The compound of gardenia oil and jujube seed oil could shorten the incubation period of appearance in castration rats and increase the number passing through Yuan Ping table in ovariectomized rats. As the training time extended, the incubation period of appearance was gradually shortened. The compound of gardenia oil and jujube seed oil could increase NOS activity, and decrease AChE activity in brain of ovariectomized rats. CONCLUSION: The compound of jujube seed oil and gardenia oil could promote the learning and memory in ovariectomized rats. This effect may be related with the increase in activities of NOS, AchE in rat brain.

Semen Ziziphi Spinosae and Fructus Gardeniae extracts synergistically improve learning and memory of a mouse model.

Semen Ziziphi Spinosae (SZS) and Fructus Gardeniae (FG) are two herbs commonly used in traditional Chinese medicine. Previous studies have suggested that Fructus Gardeniae as well as Semen Ziziphi Spinosae are able to regulate the function of the central nervous system. However, their effect on learning and memory has yet to be elucidated. In this study, we examined the effect of SZS and FG on the learning and memory of mice using the methods of step-through and -down passive avoidance tasks and Morris water maze tasks. The results showed that SZS and FG extracts have certain effects on improving the performance of the learning and memory-impaired mouse model. Of note, compound extracts of SZS and FG have a synergistic effect on the learning and memory of mice.