Zhen-wu-tang protects against myocardial fibrosis by inhibiting M1 macrophage polarization via the TLR4/NF-κB pathway.

BACKGROUND: Myocardial fibrosis is a risk factor that contributes to the increase in the incidence of cardiovascular disease and death, posing a significant threat to human health. Zhen-wu-tang (ZWT) is a classical Chinese medicinal recipe that has been extensively used to manage cardiovascular disorders throughout history. However, the fundamental processes involved in its effects were not clear. OBJECTIVE: This study examined the therapeutic effects of ZWT on myocardial fibrosis induced by isoproterenol (ISO) in mice, the effect of regulation and underlying mechanism on the polarization of M1 macrophage. METHODS: In vivo, a myocardial fibrosis mouse model was induced via intraperitoneal infusion of isoproterenol (ISO). ZWT or captopril (CAP) was administered intragastrically for 30 days. Cardiac function was evaluated by electrocardiogram (ECG) and echocardiography. By analysing myocardial fibrosis pathomorphologically and identifying fibrosis-related indicators, the protective effect of the ZWT on the heart was evaluated. A model of macrophage polarization was established in vitro by activating RAW264.7 cells with lipopolysaccharide (LPS). The regulatory effects of ZWT on macrophage polarization and the signalling pathways involved were examined by immunofluorescence staining, Western blotting (WB), quantitative real-time PCR (qRT-PCR) and siRNA transfection. RESULTS: ZWT improved cardiac function; reduced fibrotic deposition in cardiac tissues; decreased α-SMA, collagen I, and collagen III levels; and inhibited myocardial fibrosis in mice with ISO-induced myocardial fibrosis. Furthermore, the results showed that ZWT could suppress M1 macrophage polarization by downregulating the expression of CD86 and iNOS in vitro and in vivo. Finally, the results confirmed that ZWT could significantly reduce TLR4/NF-κB signalling pathway activation. CONCLUSION: ZWT showed therapeutic effects on ISO-induced myocardial fibrosis mice, and reduced M1 macrophages polarization through inhibiting TLR4/NF-κB pathway, suggesting that ZWT is a promising drug for myocardial fibrosis treatment.

Exposure to lambda-cyhalothrin and abamectin drives sublethal and transgenerational effects on the development and reproduction of Cydia pomonella.

The codling moth Cydia pomonella (Lepidoptera: Tortricidae) is a major invasive pest of pome fruits and walnuts worldwide. Lambda-cyhalothrin (LCT) and abamectin (AM) have been frequently used in C. pomonella control, but control of this pest is very difficult because shortly after hatching, larvae of this insect bore tunnels and hide inside host plant fruit. In this study, a simulated field spray bioassay method was developed against neonate larvae of C. pomonella and concentration-response bioassays were conducted to evaluate the susceptibility of the neonate larvae to LCT and AM. Exposure of neonate larvae to sublethal concentration (LC30) of LCT or AM significantly reduced the survival rate of larvae (4th and 5th instars), lowered the mean weight of larvae and pupae, and decreased the daily maximal number of eggs laid and the total number of eggs laid (fecundity) per female. The sublethal effects, including reduced body mass, mean fecundity and net reproductive rate, extended mean generation time, and shortened oviposition period, were also found in transgenerational offspring. Furthermore, the transgenerational maternal effects were more obvious for AM than LCT, in comparison to the control. Additionally, the estimated population size was decreased by exposure to LC30 of LCT and AM, and the observed reduction of fecundity and population size within and across generations was likely the result of the downregulation of the reproduction-related vitellogenin gene (CpVg) after exposure to LC30 of LCT and AM. These results provide a better understanding of the overall effects of LCT and AM on C. pomonella and the transgenerational effects which should be taken into consideration when using insecticides in order to control C. pomonella.

Metabolic functional redundancy of the CYP9A subfamily members leads to P450-mediated lambda-cyhalothrin resistance in Cydia pomonella.

BACKGROUND: The evolution of insect resistance to pesticides poses a continuing threat to sustainable pest management. While much is known about the molecular mechanisms that confer resistance in model insects and few agricultural pests, far less is known about fruit pests. Field-evolved resistance to synthetic insecticides such as lambda-cyhalothrin has been widely documented in Cydia pomonella, a major invasive pest of pome fruit worldwide, and the increased production of cytochrome P450 monooxygenases (P450s) has been linked to resistance in field-evolved resistant populations. However, the underlying molecular mechanisms of P450-mediated insecticide resistance remain largely unknown. RESULTS: Here we found that functional redundancy and preference of metabolism by P450s genes in the CYP9A subfamily confer resistance to lambda-cyhalothrin in Cydia pomonella. A total of four CYP9A genes, including CYP9A61, CYP9A120, CYP9A121, and CYP9A122, were identified from Cydia pomonella. Among these, CYP9A120, CYP9A121, and CYP9A122 were predominantly expressed in the midgut of larvae. The expression levels of these P450 genes were significantly induced by a lethal dose that would kill 10% (LD10 ) of lambda-cyhalothrin and were overexpressed in a field-evolved lambda-cyhalothrin resistant population. Knockdown of CYP9A120 and CYP9A121 by RNA-mediated interference (RNAi) increased the susceptibility of larvae to lambda-cyhalothrin. In vitro assays demonstrated that recombinant P450s expressed in Sf9 cells can metabolize lambda-cyhalothrin, but with functional redundancy and divergence through regioselectivity of metabolism. CYP9A121 preferred to convert lambda-cyhalothrin to 2'-hydroxy-lambda-cyhalothrin, whereas CYP9A122 only generated 4'-hydroxy metabolite of lambda-cyhalothrin. Although possesses a relatively low metabolic capability, CYP9A120 balanced catalytic competence to generate both 2'- and 4'-metabolites. CONCLUSION: Collectively, these results reveal that metabolic functional redundancy of three members of the CYP9A subfamily leads to P450-mediated lambda-cyhalothrin resistance in Cydia pomonella, thus representing a potential adaptive evolutionary strategy during its worldwide expansion. © 2022 Society of Chemical Industry.

Genome-wide identification, characterization, and expression profiling of ATP-binding cassette (ABC) transporter genes potentially associated with abamectin detoxification in Cydia pomonella.

The codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) is one of the most notorious pests of pome fruits and walnuts worldwide, which has developed resistance to almost all classes of insecticides, including abamectin (ABM). ATP-binding cassette (ABC) transporters are thought to play a vital roles in insecticide detoxification by reducing the toxic concentrations of insecticides in an organism tissues. Despite the tremendous progress in understanding the detoxification mechanisms at the molecular level, the physiological functions of ABC transporters in insects have been poorly investigated. In this study, we found that the ABC inhibitor verapamil synergized significantly the toxicity of ABM, suggesting a potential role of ABC in detoxification. A total of 54 ABC genes were identified in the third-instar larvae of C. pomonella after treatment with sublethal doses (LD10 and LD30) of ABM. The expression profile of these genes in ABM-treated larvae at different time points (24, 48, 72 hr) using transcriptomic analysis (RNA-seq) was also investigated. The results showed that the expression of about 30 ABC genes was significantly co-upregulated after treatment. Several specific genes were up-regulated at 48 hr after treatment of larvae with LD10 ABM. Among these up-regulated genes, we found that the relative expression level of the CPOM19553 was 29.7-fold and 16.0-fold higher when larvae were exposed to ABM at the LD10 and LD30 doses compared to control, respectively. Unlike other ABC genes, only CPOM08323 exhibited significant expression levels in the head and cuticle of the third-instar larvae of C. pomonella exposed to the two sublethal doses of ABM, with no expression was observed in the detoxification tissues such as midgut and Malpighian tubule. This study suggests that these up-regulated genes may be involved in ABM resistance in C. pomonella. Our findings will provide an additional information required for further analysis of ABC transporter genes associated with xenobiotic metabolism in C. pomonella.

Insecticide resistance in the Cydia pomonella (L): Global status, mechanisms, and research directions.

The codling moth, Cydia pomonella (Lepidoptera: Tortricidae) is a major pest of pome fruit and walnuts worldwide. Although environmentally compatible integrated control strategies, such as mating disruption, attract-kill strategy, and sterile insect technique have been conducted for management of this notorious pest, effects to control of codling moth have mainly relied on insecticides. In consequence, different levels of insecticide resistance towards organophosphates, neonicotinoids, hydrazines, benzoylureas, pyrethroids, diamides, spinosyns, avermectins, JH mimics, carbamates, oxadiazines and C. pomonella granulovirus (CpGVs) have developed in codling moth in different countries and areas. Both metabolic and target-site mechanisms conferring resistance have been revealed in the codling moth. In this review, we summarize the current global status of insecticide resistance, the biochemical and molecular mechanisms involved, and the implications for resistance management.

[Pregnane X receptor promotes programmed cell death protein 4 expression in HepG2 cells].

OBJECTIVE: To investigate the role of pregnane X receptor (PXR) in the regulation of programmed cell death proteins (PDCDs) in HepG2 cells and explore the underlying molecular mechanism. METHODS: HepG2 cells were treated with PXR agonist rifampicin (10 µmol/L) or SR12813 (1 µmol/L) for 24 h, using DMSO as the negative control. HepG2 cells were infected with constitutively activated PXR adenovirus (VP-PXR) for 36 h, with the cells infected with Mock as the negative control. The mRNA levels of PDCD2, PDCD4, PDCD5, and PDCD6 and the expression of miRNA21 were detected using qRT-PCR, and the protein level of PDCD4 was detected with Western blotting. Bioinformatic analysis was performed to predict the potential PXRresponsive elements (PXREs) motifs in the promotor region of human PDCD4. RESULTS: The expressions of PDCD5 and PDCD6 mRNA did not differ significantly between rifampicin-treated and the control cells, while PDCD4 mRNA expression increased (t=4.209, P=0.008) and PDCD2 mRNA decreased significantly (t=-2.875, P=0.017) in rifampicin-treated cells. The mRNA expressions of PDCD2, PDCD5 and PDCD6 showed no significant difference between SR12813-treated cells and the control cells, while PDCD4 mRNA expression increased obviously in SR12813-treated cells (t=4.574, P=0.006). The PXR target gene MDR1 also increased significantly in the rifampicin- and SR12813-treated cells compared with the control cells (P=0.020 and 0.01, respectively). Infection of the cells with VP-PXR adenovirus resulted in significantly increased expression of PDCD4 and MDR1 mRNA as compared with Mock group (t=3.343, P=0.000; t=3.343, P=0.024, respectively) without causing obvious changes in PDCD2 and PDCD6 mRNA expressions. The protein level of PDCD4 increased significantly in both rifampicin (t= 2.779, P=0.025) group and VP- PXR group (t=3.066, P=0.012). The expression of miRNA21, the negative regulatory factor of PDCD4, did not differ significantly between PXR agonist group and the control group. Informatic analysis revealed the presence of putative PXREs in the 5'-flanking region of PDCD4 gene. CONCLUSIONS: Our findings demonstrate that PXR agonism in HepG2 cells increases the expression of PDCD4, which is independent of miRNA21 pathway, and PDCD4 may be a target gene of PXR in HepG2 cells.

Functional characterization of a novel λ-cyhalothrin metabolizing glutathione S-transferase, CpGSTe3, from the codling moth Cydia pomonella.

BACKGROUND: Recent work has shown that two codling moth (Cydia pomonella) glutathione S-transferase genes (GSTs), CpGSTd1 and CpGSTd3, can metabolize λ-cyhalothrin, one of the recommended insecticides for C. pomonella control worldwide. However, systematical characterization of delta and epsilon GSTs, especially their potential contributions in the metabolism of λ-cyhalothrin, is currently still lacking in C. pomonella. RESULTS: In this study, a total of nine cDNA sequences were identified in C. pomonella, including four in the delta and five in the epsilon subclasses. RT-qPCR showed that seven GSTs were ubiquitously expressed at all developmental stages, and CpGSTe2, CpGSTe3, and CpGSTe4 were mainly expressed in larvae. The mRNA levels of CpGSTd2, CpGSTd4, and CpGSTe5 were significantly higher in male than in female adults. Tissue-specific expression analysis revealed that the CpGSTe2, CpGSTe3, and CpGSTe4 were highly expressed in the midgut while CpGSTd2 and CpGSTd4 were predominantly expressed in the Malpighian tubules. The transcripts of these GSTs (except CpGSTe1) were co-expressed following exposure to LD10 of λ-cyhalothrin for 3 h. Recombinant CpGSTd4, CpGSTe2, and CpGSTe3 proteins expressed in Escherichia coli displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene. In addition, λ-cyhalothrin could inhibit the activity of recombinant CpGSTd4, CpGSTe2, and CpGSTe3 enzymes, but only recombinant CpGSTe3 showed λ-cyhalothrin metabolic capacity, with 21.88 ± 1.09% of parental compound being depleted within 1 h. CONCLUSION: These data show that CpGSTe3 is a third GST gene, encoding an enzyme that metabolizes λ-cyhalothrin in C. pomonella. © 2019 Society of Chemical Industry.

Comparative Transcriptome Analysis Between Resistant and Susceptible Rice Cultivars Responding to Striped Stem Borer (SSB), Chilo suppressalis (Walker) Infestation.

The striped stem borer, Chilo suppressalis (Walker), is a notorious pest of rice that causes large losses in China. Breeding and screening of resistance rice cultivars are effective strategies for C. suppressalis management. In this study, insect-resistant traits of 47 rice cultivars were investigated by C. suppressalis artificial infestation (AI) both in field and greenhouse experiments, using the susceptible (S) cultivar 1665 as a control. Results suggest that two rice cultivars, namely 1688 and 1654, are resistant (R) and moderately resistant (MR) to C. suppressalis, respectively. Then, a comparative transcriptome (RNA-Seq) was de novo assembled and differentially expressed genes (DEGs) with altered expression levels were investigated among cultivars 1688, 1654, and 1665, with or without C. suppressalis infestation for 24 h. A total of 2569 and 1861 genes were up-regulated, and 3852 and 1861 genes were down-regulated in cultivars 1688 and 1654, respectively after artificial infestation with C. suppressalis compared to the non-infested control (CK). For the susceptible cultivar 1665, a total of 882 genes were up-regulated and 3863 genes were down-regulated after artificial infestation with C. suppressalis compared to the CK. Twenty four DEGs belong to proteinase inhibitor, lectin and chitinase gene families; plant hormone signal transduction and plant-pathogen interaction pathways were selected as candidate genes to test their possible role in C. suppressalis resistance. RT-qPCR results revealed that 13 genes were significantly up-regulated and 8 were significantly down-regulated in the resistant cultivar 1688 with C. suppressalis artificial infestation (1688AI) compared to the CK. Three genes, LTPL164, LTPL151, and LOC Os11g32100, showed more than a 10-fold higher expression in 1688AI than in 1688CK, suggesting their potential role in insect resistance. Overall, our results provide an important foundation for further understanding the insect resistance mechanisms of selected resistant varieties that will help us to breed C. suppressalis resistant rice varieties.

CD147 Promotes CXCL1 Expression and Modulates Liver Fibrogenesis.

Activated hepatic stellate cells (HSCs) release pro-inflammatory and pro-fibrogenic factors. CXC chemokine-ligand-1 (CXCL1) is expressed on HSCs. We previously found that the CD147 is overexpressed in activated HSCs. In this study, we showed an important role of CD147 in promoting liver fibrosis by activating HSCs and upregulating expression of chemokines. Specifically, we found that CD147 specific deletion in HSCs mice alleviated CCl4-induced liver fibrosis and inhibited HSCs activation. Overexpression of CD147 upregulated the secretion of CXCL1. Meanwhile, CXCL1 promoted HSCs activation through autocrine. Treating with PI3K/AKT inhibitor could effectively suppress CD147-induced CXCL1 expression. Taken together, these findings suggest that CD147 regulates CXCL1 release in HSCs by PI3K/AKT signaling. Inhibition of CD147 attenuates CCl4-induced liver fibrosis and inflammation. Therefore, administration of targeting CD147 could be a promising therapeutic strategy in liver fibrosis.

Activation of TGF-ß1-CD147 positive feedback loop in hepatic stellate cells promotes liver fibrosis.

Activation of hepatic stellate cells (HSCs) by transforming growth factor-ß1 (TGF-ß1) initiates HBV-associated fibrogenesis. The mechanism of TGF-ß1 modulating HSC activation is not fully uncovered. We hypothesized a positive feedback signaling loop of TGF-ß1-CD147 promoting liver fibrogenesis by activation of HSCs. Human HSC cell line LX-2 and spontaneous liver fibrosis model derived from HBV transgenic mice were used to evaluate the activation of molecules in the signaling loop. Wound healing and cell contraction assay were performed to detect the CD147-overexpressed HSC migration and contraction. The transcriptional regulation of CD147 by TGF-ß1/Smad4 was determined using dual-luciferase reporter assay and chromatin immunoprecipitation. We found that a positive reciprocal regulation between TGF-ß1 and CD147 mediated HSC activation. CD147 over-expression promoted HSC migration and accelerated TGF-ß1-induced cell contraction. Phosphorylation of Smad2 and Smad3 in cooperation with Smad4 mediated the TGF-ß1-regulated CD147 expression. Smad4 activated the transcription by direct interaction with CD147 promoter. Meanwhile, CD147 modulated the activated phenotype of HSCs through the ERK1/2 and Sp1 which up-regulated α-SMA, collagen I, and TGF-ß1 synthesis. These findings indicate that TGF-ß1-CD147 loop plays a key role in regulating the HSC activation and combination of TGF-ß receptor inhibitor and anti-CD147 antibody might be promised to reverse fibrogenesis.