Ling-Gui-Qi-Hua formula alleviates left ventricular myocardial fibrosis in rats with heart failure with preserved ejection fraction by blocking the transforming growth factor-ß1 /Smads signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ling-Qui-Qi-Hua (LGQH) decoction, composed of Poria cocos (Schw.) Wolf, Cinnamomum cassia (L.) J. Presl, Paeonia veitchii Lynch, and Atractylodes macrocephala Koidz., is a compound formula derived from Ling-Gui-Zhu-Gan decoction recorded in the Treatise on Febrile and Miscellaneous. It has shown cardioprotective effects on patients or rats with heart failure with preserved ejection fraction (HFpEF). Nevertheless, the active ingredients of LGQH and its anti-fibrotic mechanism remain unknown. AIM OF THE STUDY: To determine the active ingredients in LGQH decoction and verify that LGQH decoction may inhibit left ventricular (LV) myocardial fibrosis in HFpEF rats by blocking the transforming growth factor-ß1 (TGF-ß1)/Smads signaling pathway from the perspective of animal experiments. MATERIALS AND METHODS: First, liquid chromatography-mass spectrometry (LC-MS) technology was used to identify active components in the LGQH decoction. Secondly, a rat model of the metabolic syndrome-associated HFpEF phenotype was established and subsequently received LGQH intervention. The mRNA and protein expression of targets in the TGF-ß1/Smads pathway were detected by quantitative real-time polymerase chain reaction and western blot analysis. Finally, molecular docking was conducted to examine the interactions between the active ingredients in the LGQH decoction and key proteins of the TGF-ß1/Smads pathways. RESULTS: According to LC-MS analysis, the LGQH decoction contained 13 active ingredients. In animal experiments, LGQH attenuated LV hypertrophy, enlargement, and diastolic function in HEpEF rats. Mechanically, LGQH not only down-regulated TGF-ß1, Smad2, Smad3, Smad4, α-SMA, Coll I, and Coll III mRNA expressions and TGF-ß1, Smad2, Smad3, P-Smad2/Smad3, Smad4, α-SMA, and Coll I protein expressions, but also up-regulated Smad7 mRNA and protein expressions, which ultimately led to myocardial fibrosis. Furthermore, molecular docking confirmed that 13 active ingredients in the LGQH decoction have excellent binding activities to the critical targets of the TGF-ß1/Smads pathway. CONCLUSION: LGQH is a modified herbal formulation with multiple active ingredients. It might alleviate LV remodeling and diastolic dysfunction and inhibit LV myocardial fibrosis by blocking TGF-ß1/Smads pathways in HFpEF rats.

Effects and mechanism of Compound Qidan Formula on rats with HFpEF induced by hypertension and diabetes mellitus based on Ang â ¡/TGF-ß1/Smads signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound Qidan Formula is composed of traditional Chinese herbs and has a good curative effect in the clinical application of cardiovascular diseases such as heart failure. However, its potential molecular mechanisms of action remain highly unknown. AIM OF THE STUDY: To observe the effect of Compound Qidan Formula on cardiac function in rats with HFpEF induced by hypertension and diabetes mellitus, and to explore its mechanism from Ang Ⅱ/TGF-ß1/Smads signaling pathway. MATERIALS AND METHODS: A total of 50 SPF-grade spontaneously hypertensive rats (SHR) aged 14 weeks, fed with a high-fat and high-sucrose diet for 16 weeks, and after 2 weeks of a high-fat and high-sucrose diet, 1% streptozotocin (25 mg/kg body weight)was injected intraperitoneally to establish a rat model of HFpEF induced by hypertension and diabetes mellitus. After 8 weeks of intragastric administration, the changes in cardiac morphology and function were evaluated by echocardiography after anesthesia; the heart tissue was taken and embedded in paraffin for Masson staining, and the pathomorphological changes of left atrial tissue were observed under the optical microscope; the mRNA transcription levels of Ang Ⅱ, AT1R, TGF-ß1, Smad2, Smad3, MMP-9 and TIMP-1in left atrial tissue of rats were detected by RT-PCR; and the protein expressions were detected by Western blot. RESULTS: Compared with the SHR-DM group, the QD-Low and QD-High groups significantly decreased the left atrial (LA) anteroposterior diameter and interventricular septal thickness (IVST) and improved the peak velocity of mitral valve blood flow in early diastolic period (E), maximum mitral valve blood flow in systolic period (A), mitral ring myocardial movement velocity in early diastolic period (e') and E/e' ratio; the QD-High group significantly improved the E/A ratio, left atrial ejection fraction (LAEF) and left ventricular ejection fraction(LVEF). Masson staining showed that compared with the WKY group, the SHR-DM group had obvious myocardial histomorphological lesions. Compared with the SHR-DM group, the Compound Qidan Formula groups significantly improved cardiomyocyte hypertrophy and disordered arrangement and inhibited myocardial fibrosis; the mRNA expression levels of Ang Ⅱ, AT1R, TGF-ß1, Smad2, Smad3, and MMP-9 in myocardial tissue of Compound Qidan Formula groups were significantly decreased, and the mRNA expression level of TIMP-1 was significantly increased. The protein expression levels of Ang Ⅱ, TGF-ß1, P-Smad2/3, and MMP-9 were significantly decreased. CONCLUSION: Compound Qidan Formula, composed of traditional Chinese herbs, can significantly improve cardiac function, improve atrial and ventricular remodeling, and prevent myocardial fibrosis and hypertrophy in rats with HFpEF induced by hypertension and diabetes mellitus. The mechanism may be related to regulating the Ang Ⅱ/TGF-ß1/Smad2/3 signaling pathway.

Linggui Qihua Decoction Inhibits Atrial Fibrosis by Regulating TGF-ß1/Smad2/3 Signal Pathway.

Myocardial fibrosis is a critical factor in the development of heart failure with preserved ejection fraction (HFpEF). Linggui Qihua decoction (LGQHD) is an experienced formula, which has been proven to be effective on HFpEF in clinical and in experiments. Objective. This study aimed to observe the effect of LGQHD on HFpEF and its underlying mechanism. Methods. Spontaneously hypertensive rats (SHR) were induced with high-glucose and high-fat to establish HFpEF models and were treated with LGQHD for 8 weeks. The heart structure was detected by echocardiography, and the histopathological changes of the myocardium were observed by hematoxylin-eosin (HE) and Masson staining. Reverse transcription PCR (RT-PCR) and western blot were used to detect mRNA and protein expression of the target gene in rat myocardium. Results. In this study, LGQHD improved cardiac morphology and atrial fibrosis in HfpEF rats, decreased tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression, up-regulated matrix metalloproteinase-9 (MMP-9) mRNA expression, and inhibited the expression of angiotensin II (Ang II), angiotensin II type 1 receptor (AT1), transforming growth factor ß1 (TGF-ß1), Smad2/3 mRNA, and protein in myocardial tissue of HFpEF rats. Conclusion. LGQHD can suppress atrial fibrosis in HFpEF by modulating the TGF-ß1/Smad2/3 pathway.

A cellular mechanism underlying the restoration of thermo/photoperiod-sensitive genic male sterility.

During anther development, the transformation of the microspore into mature pollen occurs under the protection of first the tetrad wall and later the pollen wall. Mutations in genes involved in this wall transition often lead to microspore rupture and male sterility; some such mutants, such as the reversible male sterile (rvms) mutant, are thermo/photoperiod-sensitive genic male sterile (P/TGMS) lines. Previous studies have shown that slow development is a general mechanism of P/TGMS fertility restoration. In this study, we identified restorer of rvms-2 (res2), which is an allele of QUARTET 3 (QRT3) encoding a polygalacturonase that shows delayed degradation of the tetrad pectin wall. We found that MS188, a tapetum-specific transcription factor essential for pollen wall formation, can activate QRT3 expression for pectin wall degradation, indicating a non-cell-autonomous pathway involved in the regulation of the cell wall transition. Further assays showed that a delay in degradation of the tetrad pectin wall is responsible for the fertility restoration of rvms and other P/TGMS lines, whereas early expression of QRT3 eliminates low temperature restoration of rvms-2 fertility. Taken together, these results suggest a likely cellular mechanism of fertility restoration in P/TGMS lines, that is, slow development during the cell wall transition of P/TGMS microspores may reduce the requirement for their wall protection and thus support their development into functional pollens, leading to restored fertility.

MS1, a direct target of MS188, regulates the expression of key sporophytic pollen coat protein genes in Arabidopsis.

Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen-stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP-oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1.

The temporal regulation of TEK contributes to pollen wall exine patterning.

Pollen wall consists of several complex layers which form elaborate species-specific patterns. In Arabidopsis, the transcription factor ABORTED MICROSPORE (AMS) is a master regulator of exine formation, and another transcription factor, TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK (TEK), specifies formation of the nexine layer. However, knowledge regarding the temporal regulatory roles of TEK in pollen wall development is limited. Here, TEK-GFP driven by the AMS promoter was prematurely expressed in the tapetal nuclei, leading to complete male sterility in the pAMS:TEK-GFP (pat) transgenic lines with the wild-type background. Cytological observations in the pat anthers showed impaired callose synthesis and aberrant exine patterning. CALLOSE SYNTHASE5 (CalS5) is required for callose synthesis, and expression of CalS5 in pat plants was significantly reduced. We demonstrated that TEK negatively regulates CalS5 expression after the tetrad stage in wild-type anthers and further discovered that premature TEK-GFP in pat directly represses CalS5 expression through histone modification. Our findings show that TEK flexibly mediates its different functions via different temporal regulation, revealing that the temporal regulation of TEK is essential for exine patterning. Moreover, the result that the repression of CalS5 by TEK after the tetrad stage coincides with the timing of callose wall dissolution suggests that tapetum utilizes temporal regulation of genes to stop callose wall synthesis, which, together with the activation of callase activity, achieves microspore release and pollen wall patterning.

The Regulation of Sporopollenin Biosynthesis Genes for Rapid Pollen Wall Formation.

Sporopollenin is the major component of the outer pollen wall (sexine). It is synthesized using a pathway of approximately eight genes in Arabidopsis (Arabidopsis thaliana). MALE STERILITY188 (MS188) and its direct upstream regulator ABORTED MICROSPORES (AMS) are two transcription factors essential for tapetum development. Here, we show that all the sporopollenin biosynthesis proteins are specifically expressed in the tapetum and are secreted into anther locules. MS188, a MYB transcription factor expressed in the tapetum, directly regulates the expression of POLYKETIDE SYNTHASE A (PKSA), PKSB, MALE STERILE2 (MS2), and a CYTOCHROME P450 gene (CYP703A2). By contrast, the expression of CYP704B1, ACYL-COA SYNTHETASE5 (ACOS5), TETRAKETIDE a-PYRONE REDUCTASE1 (TKPR1) and TKPR2 are significantly reduced in ams mutants but not affected in ms188 mutants. However, MS188 but not AMS can activate the expression of CYP704B1, ACOS5, and TKPR1 In ms188, dominant suppression of MS188 homologs reduced the expression of these genes, suggesting that MS188 and other MYB family members play redundant roles in activating their expression. The expression of some sporopollenin synthesis genes (PKSA, PKSB, TKPR2, CYP704B1, and ACOS5) was rescued when MS188 was expressed in ams Therefore, MS188 is a key regulator for activation of sporopollenin synthesis, and AMS and MS188 may form a feed-forward loop that activates the expression of the sporopollenin biosynthesis pathway for rapid pollen wall formation.

The transcription factors MS188 and AMS form a complex to activate the expression of CYP703A2 for sporopollenin biosynthesis in Arabidopsis thaliana.

The sexine layer of pollen grain is mainly composed of sporopollenins. The sporophytic secretory tapetum is required for the biosynthesis of sporopollenin. Although several enzymes involved in sporopollenin biosynthesis have been reported, the regulatory mechanism of these enzymes in tapetal layer remains elusive. ABORTED MICROSPORES (AMS) and MALE STERILE 188/MYB103/MYB80 (MS188/MYB103/MYB80) are two tapetal cell-specific transcription factors required for pollen wall formation. AMS functions upstream of MS188. Here we report that AMS and MS188 target the CYP703A2 gene, which is involved in sporopollenin biosynthesis. We found that AMS and MS188 were localized in tapetum while CYP703A2 was localized in both tapetum and locule. Chromatin immunoprecipitation (ChIP) showed that MS188 directly bound to the promoter of CYP703A2 and luciferase-inducible assay showed that MS188 activated the expression of CYP703A2. Yeast two-hybrid and electrophoretic mobility shift assays (EMSAs) further demonstrated that MS188 complexed with AMS. The expression of CYP703A2 could be partially restored by the elevated levels of MS188 in the ams mutant. Therefore, our data reveal that MS188 coordinates with AMS to activate CYP703A2 in sporopollenin biosynthesis of plant tapetum.