Introduction: The genus Euphorbia is known to contain diterpenoids, and several isolated compounds which exhibited biological activities including significant multidrug resistance reversal effects. This work is focused on the isolation, in vitro and in silico studies of two natural bio-active flavonoids (1 & 2) isolated from Euphorbia pulcherrima bark for the very first time.Methods: The phytochemical investigation resulted in the identification of two flavonoids: 3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)-6-methoxy-4H-chromen-4-one (1) and 2--3,5,7-trihydroxy-6-methoxy-4H-chromen-4-one (2), which were isolated for the first time from Euphorbia pulcherrima.Results: The chemical structures of the two isolated compounds were confirmed by 1H NMR, 13C NMR, and ESI-HRMS spectral data. The Bioactivity activity of these compounds was evaluated; results revealed that compounds 1 & 2 exhibit promising urease inhibitory potential with IC50 values of 15.3 ± 2.13 μM and 19.0 ± 2.43 μM, respectively, whereas the positive control thiourea had an IC50 of 21.0 ± 0.23 μM. Similarly, these compounds were also evaluated against the tyrosinase enzyme; results showed that compound 1 displays significant inhibitory activity with an IC50 value of 48.7 ± 2.19 μM, whereas compound 2 exhibited a moderate effect with an IC50 value of 74.8 ± 1.79 μM, when compared with the standard . Additionally, compounds 1 and 2 also exhibited anti-glycation and phosphodiesterase inhibitory activities.Conclusion: Studies dealing with the drug like properties such as in silico screening (docking study) was also carried out to discover the structural features of both compounds 1 and 2. Results indicated that the docking scores of compounds 1 and 2 are in agreement with their IC50 values. 1 and 2) from Euphorbia pulcherrima.Isolation and characterization of two bioactive flavonoids (In silico and in vitro enzyme inhibition studies were conducted to identify the therapeutic potential of flavonoids 1 and 2.Drug-like properties were calculated to discover important pharmacophoric features. Members of this family are rich in secondary metabolites. Genus Euphorbia is the largest of this family, and regarded as a kingdom of medicinal plants in few countries of Asia such as Pakistan and India. In addition, the genus Euphorbia with various species is used in traditional medicine to cure different ailments. Some species in this family are toxic, while others can be used for things such as poinsettia (E. pulcherrima), spurges, crowns of the throne, and milk bush. However, all of these species contain latex as an abundant source of secondary metabolites + 346.0689, found 346. 0683. 1H-NMR : δ 12.26 , 9.94 , 8.07 , 7.78 , 7.74 , 7.30 , 2.76 , 2.03 .Spinacetin, compound 2.4.2.2, was isolated as an amorphous yellow powder. High-resolution ESI − MS (HRMS) exact mass m/z: calculated for C16H12O8 [M]+ 332.2617, found 332.2610. 1H-NMR : δ 12.27 , 9.95 , 8.08 , 7.79 , 7.75 , 7.32 , 2.76 , 12.39 . The spectral data of both compounds (1 and 2) were similar to those previously reported in the literature [Patuletin, compound 2.5.In vitro urease inhibitory activity of purified compounds 1 and 2 was investigated spectrophotometrically in a 96-well plate according to published procedures [Canavalia ensiformis) urease (1 U/well) and 5 µL solution of studied compound (0.5 mM) in Tris-HCl buffer (pH = 6.8) were incubated at 30 °C for 15 min in 96-well plate. The mixture was re-incubated for 15 min at 30 °C with urea 55 µL (100 mM). Afterwards, 45 µL of phenol (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 µL of alkali reagents (0.5% w/v sodium hydroxide and 0.1% sodium hypochlorite) were added, and the plates were re-incubated for another 50 min. The urease assay was monitored on regular basis with hydrolysis of urea and production of ammonia; thiourea was employed as the standard inhibitor of urease The fast change in the absorbance  was checked at 630 nm using an ELISA plate reader . The percentage inhibition was calculated according to the formula given below:2.6.p-nitrophenyl) phosphate. The enzymes activity was measured spectrophotometrically at 410 nm using a microplate reader , as reported in the literature [A mixture of 15 μL of phosphodiesterase-1 (E.C 3.1.4.1), 20 μL of magnesium acetate (30 mM), 97 μL Tris-HCl buffer (pH = 8.8), 97 μL Tris-HCl buffer (pH = 8.8), and 8 μL of test compound was placed in 96-well plates to make a final concentration of 0.000742 U/well; the mixture was incubated again in an ELISA for 30 min at 37 °C, followed by the addition of 0.33 mM bis  were incubated in such a way that each well of 96-well plate has 50 µL MGO, 50 µL BSA solution at 37 °C for 9 days in the absence and presence of various amounts of test sample (compounds). After incubation, glycation of protein was measured using fluorescence , against a blank, and using a microtiter plate spectrophotometer . Rutin was used as positive control (IC50 = 294 ± 1.50 µM ± SEM).Assessment of the anti-glycation inhibition of compounds  changes ,13. Brie2.9.1 and 2 as well as standard structures for docking. We employed Autodockvina [Tyrosinase, PDB ID: 2Y9W, protein structures  and bovine serum albumin were downloaded from (PDB) protein data bank. The targeted sequence for the other enzymes, phosphodiesterase-I (PDE-1 from snake venom) was retrieved from the UniPort database , with thdockvina  to condudockvina  was linkdockvina . Autodocdockvina ,27. LIGPdockvina  and PyMOdockvina  were emp2.10.1 and 2. Drug permeability (S + logP), distribution (S + logD), topological polar surface area (TPSA), MLogP, molecular weight (M. wt), and the total amount of nitrogen and oxygen atoms were all assessed according to literature methods [Med Chem Designer v.3 was employed to evaluate the physicochemical characteristics of compounds  methods . These m methods .3.Euphorbiaceae has long been used in traditional medicine. In this respect, the essential oil from E. hirta is used to cure asthma, because it consists of various components including triterpenes, phytosterols, tannins, polyphenols, and flavonoids, which can be used to treat different ailments. The essential oil has also been used as mosquito repellent, thus preventing malaria [E .kansui L.'s dried roots from Euphorbiaceae have been used as a herbal remedy for edoema, ascites, and cancer in Chinese traditional medicine [E. hirata L., is used for the cure of different diseases including gastrointestinal disorders, kidney stones, bronchial ailments, diabetes, and respiratory diseases [The family  malaria . Similarmedicine . Moreovediseases .1 and 2 were tested for urease, tyrosinase, and phosphodiesterase inhibition, among other enzymes. When compared to the standard thiourea (IC50 = 21.0–0.21 µM), both compounds exhibited significant inhibitory action against urease with IC50 = 15.3–2.13 and 19.0–2.43 µM, respectively.Natural products have received more attention in recent years as researchers look for new medications using cutting-edge technologies such as high-throughput screening . Compoun50 values of 48.7 and 74.8 µM, respectively. On the other hand, the standard drug showed excellent effect with IC50 =47.6 ± 0.67 µM, compound 2 demonstrated strong action with an IC50 value of 95.2 at 4.14 µM, whereas compound 1 exhibited substantial activity with an IC50 value of 148.7 at 1.09 µM. Both compounds were also evaluated for anti-glycation activity; results revealed that compound 2 exhibits strong anti-glycation activity with an IC50 value of 244.8 at 1.79 µM, whereas compound 1 was found to be inactive when compared to the standard, rutin . Listed in 1 and 2.This action might be attributed to the presence of two hydroxyl groups (–OH) on ring 3 of flavonoids, which could interact with residues near the nickel atoms in the urease active site. In summary, these two hydroxyl groups may be the active pharmacophore in both molecules. Similarly, when compared to the standard kojic acid , both compounds showed substantial to moderate inhibition against tyrosinase, with ICin vitro enzymatic activities of compounds 1 and 2. Results reveal that these two compounds exhibit significant inhibitory activities due to their distinct structural features. In addition, docking studies were conducted to evaluate the binding pattern of compounds 1 and 2 at the active sites of urease, tyrosinase, bovine serum albumin, and phosphodiesterase-I. 1 and 2 as well as the reference compounds. Based on hydrogen and hydrophobic interactions, the best-docked shape of compound 1 was investigated. Results showed that the docking energies of compound 1 are superior to those of the reference (thiourea) and other compounds. This implies that certain aspects in the structure of compound 1 are responsible for affecting the biological activity. Docked conformations of compounds 1 and 2 and thiourea are shown in Listed in 1 forms one hydrogen bond at the binding site of urease enzyme. This hydrogen bond of 3.32 Å length was found in His541 with the –OH group of compound 1 , as well as hydrophobic contacts with Thr190, Ala193, Tyr451, Lys431, Arg458, Arg196, and Leu189 as displayed in 2 . mpound 1  with commpound 2  showed fyed in 2  shows nu1 was quite interesting as depicted in 2, two of which are formed with Lys184 (3.01 Å and 3.34 Å) whereas the remaining two are formed with the residues His462 (3.16 Å) and Trp446 (3.05 Å). Likewise, four hydrophobic interactions  characteristics were estimated with the help of Med Chem Designer. Lipophilicity is defined as the logarithm value of the partition coefficient P (logP) between water and octanol (buffer), which describe how the unionized form of compound is partitioned. The total partition of both the ionized and unionized forms of the compound is described by logD. 1 and 2 have a log P value of less than 5, suggesting that they are hydrophilic. The logD values of the compounds differ from those of logP when they ionize at different pH levels, and the logD value of acidic substances is lower [P (MlogP) is a classic measure of a compound’s lipophilicity, which shows how well it penetrates lipid-rich environments from aqueous solutions. MLogP values over 4.15 indicate that the chemical is poorly absorbed [Compounds is lower . Morigucabsorbed .1 and 2 have MlogP values of −0.415 and −0.327, respectively, indicating that they should be easily absorbed. Similarly, these molecules have the potential to make H-bonding with the receptor as determined by the topological polar surface area (TPSA) score. TPSA ratings for compounds 1 and 2 are 144.52 and 137.43, respectively. Formation of additional hydrogen bonds is due to the presence of numerous nitrogen and oxygen atoms. Thus, the present investigation showed that compounds 1 and 2 are hydrophilic due to strong hydrogen bonding and high TPSA score, based on the binding affinity and adsorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics. Furthermore, analysis of the ADMET characteristics for compounds 1 and 2 shows that they have strong drug-like qualities, which opens the door for further optimization of the compounds under investigation.Compounds 1 and 2 demonstrated significant inhibitory potency against urease, tyrosinase, and phosphodiesterase, whereas for antiglycation, compound 1 was inactive. In addition, our findings showed that compounds 1 and 2 exhibit substantial urease inhibitory activity against the reference thiourea. These findings may explain why this plant is used in folk medicine for therapeutic purposes. However, more detailed studies are required to establish the safety and efficacy of these chemicals. Furthermore, research involving animals and toxicological studies on compounds 1 and 2 could also be beneficial.In summary, the isolated compounds This study was approved by the Ethical Committee of the Department of Pharmacy University Peshawar (UOS) Phrm 432, KPK, Pakistan.


The trypan blue exclusion assay demonstrated an interesting antiproliferative effect for complex 1 in ovarian carcinoma-resistant cells, A2780cis. Quantitative analysis performed by ICP-AES demonstrated a scarce ability to platinate DNA, and a significant intracellular accumulation. The investigation of the mechanism of action highlighted the ability of 1 to inhibit the relaxation of supercoiled plasmid DNA mediated by topoisomerase II and to stabilize the cleavable complex. Cytofluorimetric analyses indicated the activation of the apoptotic pathway and the mitochondrial membrane depolarization. Therefore, topoisomerase II and mitochondria could represent possible intracellular targets. The biological properties of 1 and 2 were compared to those of the related trans-dichloro(triphenylphosphino)platinum(II) complexes in order to draw structure–activity relationships useful to face the resistance phenotype.The high incidence of the resistance phenomenon represents one of the most important limitations to the clinical usefulness of cisplatin as an anticancer drug. Notwithstanding the considerable efforts to solve this problem, the circumvention of cisplatin resistance remains a challenge in the treatment of cancer. In this work, the synthesis and characterization of two  The clinical use of cisplatin, the first platinum-based anticancer drug, was approved by the FDA in 1978 for the treatment of ovarian, testicular and bladder cancer. Since then, its effectiveness against different types of cancers, including colorectal and non-small cell lung cancer, neuroblastoma and mesothelioma, was proven 2 by the suitable amines. The di-nuclear precursor was prepared from [PtCl2(NCMe)2] and triphenylarsine in acetonitrile under solvothermal conditions (130 °C). The reaction afforded crystalline cis-[PtCl2(AsPh3)(NCMe)] (2(PPh3)(NCEt)] [The synthesis of )(NCEt)] , it is wcis-[PtCl2(AsPh3)(NCMe)].In cis configuration and small deviations from ideality. Crystal data for this complex can be compared with the analogous parameter reported for cis-[PtCl2(PPh3)(NCEt)] [trans-[Pt(μ-Cl)Cl(AsPh3)]2 in a nearly quantitative yield. Complexes 1 and 2 (trans-[Pt(μ-Cl)Cl(AsPh3)]2 precursor by the suitable amines, guided by the trans effect of the Ph3As ligand, affording trans products. The complexes were recovered with very good, isolated yields . Both 1 and 2 are insoluble in water and well-soluble in DMSO, where partial isomerization from a trans to cis isomer was observed by 1H NMR analysis (NCEt)] . In part 1 and 2  were obtanalysis  in 24 h.1 and 2 was assessed using a trypan blue dye exclusion assay. Cell lines, HeLa (cervix adenocarcinoma), HT-29 (colon adenocarcinoma), A549 (lung carcinoma), A2780 (ovarian carcinoma), A2780cis (ovarian carcinoma resistant to cisplatin) and Met-5A , were incubated for 72 h in the presence of increasing concentrations of the tested complexes, and the GI50 values (concentration of complex inducing a 50% reduction in cell growth) were calculated and shown in The cytotoxicity of Cisplatin was tested as a reference drug and 3 and 4, the analogues containing the phosphorus counterpart, were used for comparison.1 and 2 induced a significant cytotoxicity, leading to GI50 values ranging mainly in the low micromolar range.After 72 h of incubation, complexes 1 appears to be more effective than 2 in all tested cell lines, underlining a relevant role for the N,N-dialkylamino side chains and suggesting a favorable contribution for the more hydrophobic residues inserted in 1. Interestingly, complex 1 shows a lower cytotoxic effect on non-tumorigenic mesothelial cells, Met-5A, with respect to ovarian carcinoma cells, A2780. Furthermore, the comparison with the antiproliferative activity observed for the corresponding triphenylphosphine complex 3 /[P]  associated with cisplatin accumulation could not be involved in the uptake of 1.In cells incubated with ll lines ,21,41. Ill lines ,42. OtheN,N-dialkylaminotriphenylphosphine Pt(II) complexes has been previously related to the ability to impair mitochondria by damaging mitochondrial membrane permeability  , AsPh3  and acetonitrile (10 mL) was heated (130 °C) in a 100 mL Carius tube until a clear yellow solution was obtained (3 h). The solution was slowly cooled at room temperature (25 °C). Pale yellow crystals formed were recovered by filtration and dried under vacuum . Single-crystal X-ray diffraction was carried out on a selected crystal and showed the cis configuration of the complex. 1H NMR: 7.19–7.72 , 1.78 . 195Pt-NMR: –3395 (cis isomer). C20H18AsCl2Pt requires: C 39.17, H 2.96, N 2.28. Experimental: C 39.35, H 3.07, N 2.15%. A solid residue was obtained eliminating the solvent from mother liquors of the preparation (0.145 g), containing a mixture of cis,trans-[PtCl2(AsPh3)(NCMe)]. Selected signals from the mixture spectra for the trans complex showed: 1H NMR: 7.18–7.77 , 2.35 ; 195Pt NMR: −3331. Crystal data can be found in the table below (A mixture of [PtClle below .2(NCMe)2] , AsPh3  and acetonitrile (15 mL) was heated (130 °C) in a 100 mL Carius tube. A clear, yellow solution was obtained, and heating was continued for 3 h. The solution was then concentrated to dryness under vacuum and the yellow residue was suspended in toluene (30 mL). The suspension was refluxed (110 °C) until the complete precipitation of a brilliant orange solid  was observed (2 h). The product  was recovered by filtration and dried under vacuum. C36H30As2Cl4Pt2 requires: C 37.78, H 2.64%. Experimental: C 37.69, H 2.83%. IR (ATR) cm 1: 3053 m, 1964 s, 1579 m, 1481 s, 1434 s, 1309 m, 1189 m, 1160 m, 1080 s, 1022 m, 998 s, 740 s, 690 s.A mixture of [PtCltrans-[Pt(µ-Cl)Cl(AsPh3)]2  was suspended in 1,2-dichloroethane (10 mL) and treated under stirring with 0.518 g of benzylbutylamine . A yellow solution was obtained almost immediately. After 1 h, the solution was concentrated under vacuum up to one fourth of the original volume and heptane was added (20 mL). The yellow solid obtained was then recovered by filtration and dried under vacuum . C29H32Cl2NAsPt·H2O requires: C 46.23, H 4.55, N 1.86%. Experimental: C 46.42, H 4.34, N 1.87%. 1H NMR: 7.68–7.37 ; 4.63 , 4.25 , 3.93 , 3.34 , 2.63 , 2.36 , 1.94 , 1.49 , 1.40 , 0.97 . 13C NMR: 135.8, 134.0, 130.4, 130.1, 130.0, 128.8, 128.5, 128.3, 57.0, 51.1, 31.1, 20.0, 13.9. 195Pt NMR: −3347.A sample of trans-[Pt(µ-Cl)Cl(AsPh3)]2  was suspended in 1,2-dichloroethane (10 mL) and treated under stirring with 55.9 μL of diethanolamine . A pale-yellow solution was obtained almost immediately. After 1 h, the solvent was eliminated under vacuum and the oily residue was treated with petroleum ether (20 mL) overnight, under stirring at room temperature. The yellow solid obtained was recovered by filtration and dried under vacuum . C22H26Cl2NO2AsPt·H2O requires: C 38.00, H 4.06, N 2.01%. Experimental: C 38.03, H 3.91, N 2.18%. 1H NMR: 7.73–7.31 ; 4.97 , 3.92 , 3.43 , 2.83 . 13C NMR: 133.8, 130.7, 129.6, 128.7, 60.4, 55.1. 195Pt NMR: −3349.A sample of 3, 10% heat-inactivated fetal bovine serum , 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B . The cell lines were cultured as monolayers in a humidified atmosphere at 37 °C and 5% CO2 and sub-cultured at 80% confluency. The resistant cell line was maintained in medium with 1 μM of cisplatin  every 2–3 passages.A2780 (human ovarian carcinoma) and A2780cis (cisplatin-resistant human ovarian carcinoma) were purchased from the European Collection of Cell Cultures, UK (ECACC). HT29 , A549 , HeLa (human cervix adenocarcinoma) and Met-5A (human mesothelium) were purchased from American Type Culture Collection, USA (ATCC). A2780, A2780cis and HT-29 were cultured in RPMI 1640 , Met-5A in RPMI 1640  supplemented with 2.38 g/L HEPES, 0.11 g/L pyruvate sodium and 2.5 g/L glucose, A549 in Nutrient Mixture F-12K  and HeLa in Nutrient Mixture F-12 Ham . All cell media were supplemented with 1.5 g/L NaHCO4) in each well and incubated with complete growth medium overnight. Then, cells were exposed to different complex concentrations and incubated for 72 h. Complexes 1 and 2 were dissolved in dimethylsulfoxide at 20 mM and diluted in medium so that the final volume of solvent was below 0.5% (v/v). Cisplatin was dissolved in 0.9% saline at a 4 mM final concentration. Following incubation, cells were washed with phosphate buffered saline , harvested, diluted in an appropriate volume of Trypan blue (0.1%) and counted by an optical microscope. The results were analyzed and the GI50 values (micromolar concentration which induces a 50% reduction in cell number with respect to the control condition) were estimated.The cytotoxicity of the tested complexes was evaluated using the Trypan blue exclusion assay. Briefly, cells were seeded in 24-well plates at the appropriate density  was determined spectrophotometrically using the molar absorption coefficient ε = 6600 Mte units . pBR322 −4 M) to achieve a DNA/complex molar ratio of 10. Exact volumes (250 μL) of such solutions were collected at fixed times (0–48 h), and the DNA was precipitated twice with 0.3 M sodium acetate and ice-cold ethanol and washed with 70% ethanol. The pellets were dissolved in milliQ® water, mineralized with concentrated HNO3 (65% v/v) for 1 h at 90 °C in a digital dry bath (Gilson), diluted in 3 volumes of 37% (v/v) HCl and added with milliQ® water up to 5 mL. The content of P and Pt was analyzed by a Spectroflame Modula sequential and simultaneous ICP-spectrometer (ICP SPECTRO Arcos with EndOnPlasma torch) equipped with a capillary cross-flow Meinhard nebulizer . Emission lines λ = 178.290 nm and λ = 214.423 nm were used to determine P and Pt, respectively. A sample undergoing the same experimental procedure and containing DNA alone was also analyzed, and confirmed the absence of Pt as a possible contaminant. Calibration was carried out by preparing 5 multi-element standard solutions containing Pt and P in the concentration range 0–2 mg L−1 for Pt and 0–10 mg L−1 for P (ppm). Standard solutions were prepared by diluting Pt (standards from Romnil) and P (Spectrascan standards from Teknolab) stock solutions of 1000 mg L−1 with HCl 2.5% v/v and HNO3 2.5% v/v, respectively.Measurement of DNA platination was performed according to a previous method . Appropr2, 5 mM DTT, 10 μg/mL albumin, 1 mM ATP, 1 U Topoisomerase IIα  and 250 ng supercoiled plasmid DNA. The reactions were performed for 1 h at 37 °C in the presence of the investigated complex at indicated concentrations. Finally, reactions were stopped by adding 4 µL of stop buffer containing 5% SDS, 0.125% bromophenol blue, 25% glycerol and 50 µg/mL proteinase K (Sigma-Aldrich) and incubating for a further 30 min at 37 °C. The samples (15 μL) were then subjected to electrophoresis on 1% agarose gel in TAE  at 100 V for 90 min. The gels were stained in TAE buffer containing 1 μg/mL of ethidium bromide (Sigma E1510) and photographed using a gel-imaging system equipped with a CCD camera coupled to a Bio-Rad Gel Doc XR apparatus .The topoisomerase II relaxation activity assay was performed in a 20 μL total volume of reaction buffer containing 5 mM TrisHCl (pH 7.5), 12.5 mM NaCl, 1 mM MgCl2, 5 mM DTT, 10 μg/mL albumin, 1 mM ATP), in the presence of the investigated complex at indicated concentrations. Reactions were terminated by adding 4 µL of stop buffer , 50 µg/mL of proteinase K  and by incubating the samples for 30 min at 37 °C. Then, 15 μL of each sample were electrophoresed at 30 V for 18 h in a 1% agarose gel prepared in TBE buffer (0.09 M TRIS-borate and 0.002 M EDTA) containing 0.5 μg/mL of ethidium bromide (Sigma E1510). Gels were de-stained in milliQ® water and photographed under UV light by a CCD camera coupled to a Bio-Rad Gel Doc XR apparatus .Supercoiled pBR322 plasmid DNA (250 ng) was incubated with 10 U Topo IIα  for 1 h at 37 °C in 20 µL of reaction buffer (5 mM TrisHCl (pH 7.5), 12.5 mM NaCl, 1 mM MgCl6 A2780cis cells were seeded into 6-well plates and allowed to grow in standard conditions for 24 h. Then, the cells were incubated with 100 μM of test complex for 60 and 120 min, washed twice with 0.9% NaCl to remove dead cells and trypsinized. The pellet was washed twice with 0.9% NaCl, mineralized with concentrated HNO3 (65%) for 1 h at 90 °C in a digital dry bath , diluted in 3 volumes of 37% (v/v) HCl and added with milliQ® water up to 5 mL. The content of P and Pt was analyzed and determined as described in To measure intracellular platinum accumulation, 2 × 105/plate into 60 mm culture cell plates and allowed to grow for 24 h. Then, the cells were incubated for a further 40 h in the presence of different concentrations of test complex and cisplatin. Following treatment, the cells were harvested and resuspended in JC-1 Working Solution, according to the manufacturer’s protocol. After incubation for 30 min at 37 °C in the dark, cells were washed in PBS, resuspended and immediately analyzed by a FACSCanto II flow cytometer .The mitochondrial transmembrane potential was evaluated according to Cossarizza et al.  and meas5 cells were seeded into 60 mm cell culture plates overnight in complete growth medium, then serial dilutions of the complex were added. Following 40 h of incubation in the presence of test complex, cells were trypsinized, and 5 × 105 cells were resuspended in 0.500 mL of binding buffer (0.01 M HEPES/NaOH (pH 7.4), 0.14 M NaCl, 2.5 mM CaCl2), Annexin V-FITC and propidium iodide (PI), as indicated by the supplier’s instructions. After incubation in the dark at room temperature for 15 min, the cytofluorimetric analysis of each sample was promptly performed by the FACSCanto II instrument .To detect phosphatidylserine translocation from the inner to the outer surface of the plasma membrane in A2780cis cells, a FITC Annexin V Apoptosis Detection Kit I (BD Pharmigen) was used. Briefly, 3 × 10trans-dichloroPt(II) complexes (1 and 2) containing the tryphenylarsine ligand were synthesized and characterized. The comparative analysis of the biological properties with the trans-dichloroPt(II) triphenylphosphine analogues  allowed to conclude that the insertion of the N-butyl,N-benzylamino side chains as well as the triphenylarsine scaffold (complex 1) induced a remarkable increase of the antiproliferative effect on ovarian carcinoma cells resistant to cisplatin.Two new 1 (triphenilarsino)Pt(II)) appears to be the most interesting complex, showing lower GI50 values compared to the related complexes on both sensitive and resistant ovarian carcinoma cells. The investigation into the mechanism of action identified topoisomerase II and mitochondria as possible intracellular targets, and a remarkable uptake ability in resistant A2780cis cells was found.Indeed, complex 1 a scaffold worthy of further development, with the aim to design more effective Pt(II) complexes.The different targets with respect to cisplatin and the significant cell accumulation, likely mediated by a passive diffusion mechanism, favored by the lipophilicity of the complex, may contribute to the overall biological effect exerted on resistant ovarian carcinoma cells and makes complex


Cocoa, being a shade loving crop cannot withstand long periods of water stress. Breeding for drought tolerance is the need of the hour due to change in climatic condition and extension of crop to non-traditional areas. Hybrids were produced by crossing four tolerant genotypes in all possible combination. The cross GV1 55 x M 13.12 didn’t yield any fruit due to cross incompatibility between these genotypes. Various biochemical parameters act as the true indicators to select tolerant and susceptible types. The major biochemical parameters considered after imposing stress included proline, nitrate reductase activity, superoxide dismutase content and glycine betaine.The drought tolerant hybrids were having high amount of proline, superoxide dismutase enzyme and glycine betaine content. Normally, plants having drought stress show low amount of nitrate reductase activity. However, in case of hybrids, the drought tolerant hybrids were having higher NR activity than the susceptible hybrids. The highest amount of NR was found in the control plants kept at fully irrigated conditions.This experiment showed the role of different biochemical enzymes and osmolytes in giving tolerance to plants during drought stress. Logistic regression analysis selected proline and nitrate reductase as the two biochemical markers for identifying efficient drought tolerant genotypes in the future breeding programmes. Cocoa is a high industrial valued crop that originated in humid tropics of rain forest and produce cocoa beans which is the only source for chocolate. Cocoa plays a significant role in economics of both producing and consuming countries. In the whole world cocoa is cultivated by around five million farmers and contributes to the subsistence of 40–50 million people . The conDrought is defined as a decrease in water inputs or precipitation in an agro/ecosystem over time that is sufficient to result in soil water deficit . DroughtMoreover, due to the increased rate of consumption of cocoa in chocolate industry, the cultivation is extending to non-traditional area. In such area acute water shortage is the most important problem to be addressed. Hence to cope up with the changing climatic scenario and cropping pattern it is essential to breed genotypes that can tolerate water stress. The screening of genotypes based on biochemical parameters helps in developing drought tolerant hybrids which can be used in further breeding programmes. The objective of the present study is to screen and identify cocoa genotypes which can tolerate drought and the role of biochemical parameters in drought stress so as to develop cocoa varieties suitable for drought prone areas.The hybridization work was conducted at Cocoa Research Centre (CRC), Kerala Agricultural University, India, using four genotypes identified as tolerant to drought in a preliminary study  as parenThis study was carried out during 2019–20 in a research greenhouse at Cocoa Research Centre, Vellanikkara located in Thrissur . Based on initial vigour, hybrids were selected and subjected to moisture stress following gravimetric method [A control was also kept at fully irrigated condition representing each of the crosses. Based on the percentage of leaves retained after the drought imposition for 2 weeks, morphological classification of these hybrids was done based on the score chart  , NitrateThe correlation coefficients were calculated to determine the degree of association of characters with percentage of leaves retained. The genotypic coefficients of correlation between character pairs were determined by using the variance and covariance components . The dirBinary logistic regression was carried out to find the relationship between the dependent biochemical parameters over the independent variable, the number of leaves retained. Improvement in selection over the base population was found by the equation,A total of 1505 hybrid seedlings were raised in the nursery. Based on initial vigour 120 hybrids were selected representing 11 crosses. After imposition of water stress for 2 weeks the percentage of leaves withered was calculated and hybrids were classified as highly tolerant (HT), tolerant (T), susceptible (S) and highly susceptible (HS) as per the score chart given. Table In all the 11 crosses, all the tolerant and highly tolerant hybrids indicated high proline content as compared to the susceptible hybrids. The control recorded minimum amount of proline under full irrigated conditions Table .In the cross M 13.12 x G I 5.9, the highest content was found in hybrid H27 (1105.64 μg/g) and the lowest value was found in H18 (167.18 μg/g). The cross M 13.12 x G II 19.5 had ten hybrids out of which H38 showed the maximum content of proline of about 2710.82 μg/g. Lowest value was found in H40 (224.46 μg/g). In cross M 13.12 x G VI 55, the highest content was found in H43 (2817.39 μg/g) which was a tolerant hybrid and lowest in H44 of only about 85.52 μg/g of proline indicating its vulnerability to drought stress. The cross G I 5.9 x M 13.12 had six hybrids where H52 had highest proline content of 536.70 μg/g and H54 (181.17 μg/g) was having lower proline content. In the cross G I 5.9 x G II 19.5, out of 13 hybrids, H63 had the highest proline content of 706.01 μg/g and the lowest was found in H66 (177.26 μg/g). The cross G I 5.9 x G VI 55 had eight hybrids, and the highest content was found in H71 (1749.05 μg/g) while the susceptible hybrid H77 (169.54 μg/g) had the lowest amount. In cross G II 19.5 x M 13.12, the highly tolerant hybrid H85 (2293.88 μg/g) was having the highest amount while the lowest values were found in H91 (139.87 μg/g) which was a susceptible hybrid. In the cross G II 19.5 x G I 5.9, high values for proline was observed in H101 (2726.81 μg/g) and the lowest in H98 (169.18 μg/g) which was classified as susceptible. In the cross G II 19.5 x G VI 55), only two hybrids were there, one of which was highly tolerant and the other one was susceptible. The H107 was having the proline content of 1984.83 μg/g and the susceptible one, H108 was having 339.69 μg/g of proline. The cross G VI 55 x G I 5.9 was having six hybrids and H113 was having the highest value of proline, 520.85 μg/g and lowest value was found in H114 (128.55 μg/g). In the cross G VI 55 x G II 19.5, highest value was observed in H118 (1354.74 μg/g) while the lowest value was observed in H116 which was susceptible to drought having only 182.50 μg/g of proline.When the progenies of all crosses were compared, it was seen that the content ranged from 85.52 μg/g in H44 (M 13.12 x G VI 55) to 2817.39 μg/g in H43 (M 13.12 x G VI 55).Tolerant hybrids had high resistance to drought stress and were able to regulate the nitrate reduction activity even with less water. In this experiment, the cross G VI 55 x G II 19.5 recorded the maximum amount of enzyme activity of about 17.06 mmol nitrate/g/hr. in H115 which is a tolerant hybrid. However, the susceptible hybrids had very low content of NRA, the lowest being in H17 (0.51 mmol nitrate/g/hr) from the cross M13.12 x G I 5.9. The control plant on the other hand, had the highest value of NRA which was kept under 100% field capacity.In the cross between M 13.12 x G I 5.9, 32 hybrids were obtained out of which the H11 showed the maximum nitrate reductase activity of about 12.59 mmol nitrate/g/hr. while the lowest value of nitrate reductase activity was observed in the susceptible hybrid H17.The cross M 13.12 x G II 19.5 had hybrids having values as high as 11.74 mmol nitrate/g/hr. in H37 to as low as 2.07 mmol nitrate/g/hr. in H36.In cross M 13.12 x G VI 55, H48 was having the highest NRA content of about 10.89 mmol nitrate/g/hr. The lowest value was found in H50 with only 1.88 mmol nitrate/g/hr. In the cross between G I 5.9 x M 13.12, highest NRA value was found in H52 (8.45 mmol nitrate/g/hr) which was a tolerant hybrid while the lowest value was found in H54 (1.81 mmol nitrate/g/hr). The cross G I 5.9 x G II 19.5 had H64 with the highest NRA value of 9.48 mmol nitrate/g/hr. while the lowest value for NRA was 1.41 mmol nitrate/g/hr. found in H59.In the cross between G I 5.9 x G VI 55, the highest NR activity was found in H76 with 10.41 mmol nitrate/g/hr. which is a highly tolerant hybrid. The lowest NR activity was found in H73 having 3.65 mmol nitrate/g/hr. which is susceptible hybrid. In cross G II 19.5 x M 13.12, H81 showed the maximum NR activity of 14.27 mmol nitrate/g/hr. and the lowest value (3.98 mmol nitrate/g/hr) was observed inthe susceptible hybrid H89.The cross G II 19.5 x G I 5.9 had hybrid having the highest value in NR activity (H97) with a value of 15.45 mmol nitrate/g/hr. and the lowest (4.76 mmol nitrate/g/hr) was reported in H98.The cross G II 19.5 x G VI 55 had two hybrids, the high NR value was observed in the tolerant hybrid H107 (12.98 mmol nitrate/g/hr) and the low value of NR activity was observed in H108 (6.65 mmol nitrate/g/hr). In the cross G VI 55 x G I 5.9, the tolerant hybrids were having the high NR activity, the highest being H111 (6.68 mmol nitrate/g/hr), and lowest in H110 (3.57 mmol nitrate/g/hr). The cross G VI 55 x G II 19.5 had values as high as 17.06 mmol nitrate/g/hr. in H115 while the lowest value was observed in H116 having 4.10 mmol nitrate/g/hr. nitrate activity.In cross M13.12 x G I 5.9, the highest value was observed in H10 (0.381 units/mg protein/g) while the lowest value was observed in H16 (0.128 units/mg protein/g). In the cross M 13.12 x G II 19.5, highest SOD value was found in H38 (0.332 units/mg protein/g) which is a highly tolerant hybrid while the lowest was H41 (0.184 units/mg protein/g). In the cross M 13.12 x G VI 55, the highest SOD value was found in H46 (0.362 units/mg protein/g). The lowest SOD value was in hybrid H45 (0.166 units/mg protein/g) which was highly susceptible. The cross G I 5.9 x M 13.12 had the highest SOD values of about 0.259 units/mg protein/g expressed in hybrid H52 while the lowest values was found in H55 (0.156 units/mg protein/g). In cross G I 5.9 x G II 19.5, the highest value was present in hybrid H68 (0.276 units/mg protein/g). The lowest value obtained was in hybrid H60 (0.162 units/mg protein/g). The cross G I 5.9 x G VI 55 had high SOD activity in hybrid H75 (0.272 units/mg protein/g) whereas lowest SOD value was found in H77 (0.191 units/mg protein/g). In cross G II 19.5 x M 13.12, SOD values was highest in H85 (0.227 units/mg protein/g). The lowest SOD value was found in H93 (0.133 units/mg protein/g). In the cross G II 19.5 x G I 5.9, hybrid H101 (0.270 units/mg protein/g) had the highest value and the lowest was found in H106 (0.154 units/mg protein/g). In the cross G II 19.5 x G VI 55, two hybrids were obtained in which the hybrid H107 was highly tolerant hybrid having high SOD content of 0.163 units/mg protein/g whereas the susceptible hybrid was having 0.106 units/mg protein/g of SOD activity. In the cross G VI 55 x G I 5.9, high SOD value was found in hybridH112 (0.221 units/mg protein/g). The low SOD value was found in hybrid H110 (0.136 units/mg protein/g). In cross G VI 55 x G II 19.5, the highest value was found in hybrid H118 (0.227 units/mg protein/g) while the lowest value was found in hybrid H116 (0.140 units/mg protein/g) which is a susceptible hybrid.H10 (M13.12 x G I 5.9) recorded the high SOD content of about 0.381 units/mg protein/g which was a tolerant hybrid and the lowest content was observed in a susceptible hybrid, H108 (G II 19.5 x G VI 55) of about 0.106 units/mg protein/g.When the hybrids of present study were analysed, the tolerant hybrid, H103 (G II 19.5 x G I 5.9) was having high amount of glycine betaine of about 13.79 μmol/g and the susceptible hybrid, H3 (4.42 μmol/g) of the cross M13.12 x G I 5.9 recorded the least. All the tolerant hybrids had high glycine betaine content as compared to the susceptible hybrids. However, the control which was kept under fully irrigated condition had least amount of glycine betaine among the crosses indicating the accumulation of glycine betaine under drought stress conditions. In cross M 13.12 x G I 5.9, the highest glycine betaine value was found in H26 (11.64 μmol/g) and the lowest value was observed in H3 (4.42 μmol/g). In cross M 13.12 x G II 19.5, the highest value was found in hybrid H34 (9.68 μmol/g) whereas the lowest value was observed in H35 (6.73 μmol/g). In cross M13.12 x G VI 55 the highest value was observed in H48 (9.87 μmol/g) and the H45 (5.29 μmol/g) recorded the lowest glycine betaine value.In cross G I 5.9 x M 13.12, high GB values were observed in H52 (8.10 μmol/g) while H55 (5.36 μmol/g) had the lowest value. In cross G I 5.9 x G II 19.5, the highest value was observed in H63 (9.52 μmol/g) to as low as 6.45 μmol/g in H70.In cross G I 5.9 x G VI 55, the highest value was observed in hybrid H71 (9.56 μmol/g). Lowest values were observed in H73 (7.07 μmol/g). In cross G II 19.5 x M 13.12, highest GB value was observed in H88 (10.08 μmol/g) while the lowest value was observed in H80 (6.78 μmol/g). In the cross between G II 19.5 x G I 5.9, H103 (13.79 μmol/g) recorded the highest value while the lowest value was observed in H99 (7.20 μmol/g). The cross G II 19.5 x G VI 55 had two hybrids, the hybrid H107 had high GB value of 9.34 μmol/g and the other hybrid, H108 which is the susceptible one, had 7.18 μmol/g of GB. In cross G VI 55 x G I 5.9, H112 was having the highest GB content of about 10.61 μmol/g while the lowest value was observed in H109 (6.93 μmol/g). In the cross G VI 55 x G II 19.5, the highest value was observed in hybrid H119 (9.23 μmol/g) while the lowest value was observed in H116 (4.42 μmol/g).All the biochemical parameters estimated showed positive corelation with dependant variable (percentage of leaves retained). Among the biochemical characters, proline showed maximum correlation with the dependent variable, percentage of leaves retained (0.777) Table .Table 4CHigh positive effect on the percentage of leaves retained in path analysis result is a direct measure of plant’s tolerance to drought. It was expressed by proline (0.386) and low direct effect was shown by nitrate reductase activity 0.166)  and positive correlation indicated that proline content and NRA content had a positive correlation with drought tolerance and also these characters expressed a significant value of less than 0.05 which is the constant indicating the 95% accuracy with the results.Based on the Exp (B) value from regression model, expressed percentage for drought tolerance over the base population was calculated and it was found that if selection is based on proline, a new population from the base population will express 51.55% improvement regarding the tolerance. In case of NRA, a new population will show 87.48% improvementover the base population  accumulation during stress greatly depends on the balance between ROS production and ROS scavenging mechanism . When pl2- radicals to H2 O and O2. Hence, the amount ofSOD increases with increase in stress conditions indicating it’sdirect relation to drought stress [Curtilobumsolanum and Solanumtuberosum due to over production of SOD in chloroplasts were also reported [In the experiment when plants were subjected to analysis, the tolerant and highly tolerant hybrids showed more amount of superoxide dismutase enzyme as compared to the susceptible hybrids and the control which was kept under fully irrigated condition had the least amount of SOD in it. This indicated that SOD will get accumulated under drought stress conditions and forms a defence system against the stress . Superoxt stress , 38. Simreported  which suArabidopsis thaliana [Brassica napus [Persimmon [Many plants accumulate compounds, termed compatible solutes, to cope with stress conditions. One of the most extensively studied compatible solutes is glycine betaine  Not onlythaliana , Brassicca napus  Persimmoersimmon  and riceersimmon . The metersimmon . When thersimmon , 50.Hence, these biochemical parameters were reliable source to detect drought stress in cocoa. However, the correlation and path analysis revealed that proline and the nitrate reductase are the two important parameter that contribute to drought tolerance. To find out the actual relationship between the dependent variables  and the independent variable (the number of leaves retained, a binary regression analysis was carried out. The results of present study indicated that the proline and nitrate reductase can be used as biochemical indicators in cocoa drought breeding programme Fig. .Fig. 2BiThe parameters proline and glycine betaine represented the osmolyte group whereas nitrate reductase and superoxide dismutase represented the enzyme group. In all the crosses, the content of proline was high in highly tolerant and tolerant hybrids as compared to susceptible hybrids. The control which was fully irrigated condition was having the least amount of proline. The glycine betaine also followed the same trend, as tolerant and highly tolerant hybrids were having more amount of glycine betaine as compared to susceptible hybrids and the control indicating that these two osmolytes accumulated only during water stress. In case of superoxide dismutase, the highly tolerant and tolerant hybrids were having high amount of superoxide dismutase as compared to the susceptible hybrids whereas the control was having the least amount of superoxide dismutase enzyme. In case of nitrate reductase activity, the highly tolerant and tolerant hybrids were having high amount of enzyme as compared to the susceptible hybrids. The control kept at fully irrigated condition was having the highest amount of nitrate reductase enzyme. The hybrids having high amount of nitrate reductase were more tolerant because generally, this enzyme reduces under drought stress. Based on corelation, path analysis and binary logistic regression analysis, proline and nitrate reductase can be used as reliable parameters in drought breeding programmes in the future.


Furthermore, PC4 positively correlated with SOX9 in multiple cancers. PC4 was an upstream regulator of SOX9 in lung adenocarcinoma. Furthermore, PC4 mediated lung adenocarcinoma cell sensitivity to the HIF-PH inhibitor DMOG and the mTOR inhibitor rapamycin, and PC4 mediated the synergistic effect of DMOG and cisplatin. Finally, PC4 destabilized HIF-1α upon cisplatin treatment. Our research showed that PC4 participates in mediating lung adenocarcinoma cell sensitivity to multiple drugs. Mechanistically, PC4 governs multiple downstream pathways associated with chemotherapy resistance, including the SOX9 and HIF-1α pathways. Thus, PC4 is a promising chemotherapeutic target in lung adenocarcinoma.Reduced sensitivity to chemotherapeutic drugs is almost inevitable in lung adenocarcinoma patients. Thus, understanding the relevant mechanisms is urgent. Positive cofactor 4 (PC4) was at first revealed to be a coactivator of basal transcription. Previous research has shown that PC4 participates in various cellular processes in normal and malignant cells. However, it is still unknown whether PC4 participates in altering the lung adenocarcinoma cell sensitivity to chemotherapy, and the relevant mechanisms remain to be explained. In this study, we discovered that PC4 was overexpressed in cisplatin-resistant lung adenocarcinoma cells. PC4 decreased cisplatin's cytotoxic effects on lung adenocarcinoma  Reduced sensitivity to chemotherapeutic drugs is almost inevitable in lung adenocarcinoma (LUAD) therapy. Recently, many genes were discovered as targets to predict the progression or therapeutic response of lung adenocarcinoma . AlthougPositive cofactor 4 (PC4), or SUB1, was at first discovered to be a coactivator of the basal transcription , 7. AccoSRY-box transcription factor 9 (SOX9) is a SOX transcription factor and is located on chromosome 17 , 16. SOXin vivo and in vitro. Mechanically, SOX9 is a downstream factor of PC4. PC4 overexpression upregulates while PC4 knockdown inhibits the expression of SOX9. PC4 also decreases lung adenocarcinoma cell sensitivity to the HIF-PH inhibitor DMOG and the mTOR inhibitor rapamycin. DMOG exerts a synergistic enhancive effect on cisplatin, which is weakened by PC4. Moreover, PC4-decreased cell sensitivity to cisplatin is accompanied by destabilization of HIF-1α.In this study, PC4 is demonstrated to promote cell migration in lung adenocarcinoma cells without affecting cell growth and apoptosis. PC4 decreases lung adenocarcinoma sensitivity to cisplatin HEK293 T cells and human lung adenocarcinoma cell lines (H1299 and PC-9) were obtained and maintained in our laboratory as previously described . The medCisplatin  was freshly weighed and dissolved in PBS. A-485, DMOG, LY294002, MG132, MHY1485, and rapamycin  were dissolved in DMSO.The pLVX-shRNA2 and pLVX-IRES-ZsGreen1 plasmids  were selected as the lentiviral vectors for PC4 knockdown and overexpression, respectively. The target sequences for PC4 knockdown were PC4 sh1 GAAGGAACAGATTTCTGACAT; PC4 sh2 GACAGGTGAGACTTCGAGA; and PC4 sh3 GCAAAGTGCTAATTGATAT. The PC4-knockdown negative control (PC4 NC) sequence was designed online from the Invivogen website and was GCTGGATACGGATAATTAACA. The sequences were cloned into pLVX-shRNA2 in accordance with previous research . The pLVThe production of PC4-knockdown and PC4-overexpression lentivirus was conducted by cotransfecting HEK293 T cells with the pLVX-shRNA2 vector (or pLVX-IRES-ZsGreen1), the psPAX2, and the pMD2.G  plasmids as previously described , 23. 48 Blank, ODControl, ODExperiment). The cell viability was calculated as previously described [combinational therapy/cell viability cisplatin × 100%. The enhancive effect of drug on cisplatin is calculated as follows:  × 100%. Each result is representative of 6 replicate samples.The CCK-8 test was accomplished using the Cell Counting Kit-8 (CCK-8)  as we previously described . The OD escribed . The relThe Annexin V-FITC/PI assay was applied to evaluate cell apoptosis. Cells were seeded in advance in a 6-well plate. 48 hours later, drugs were administered. Cell apoptosis was analyzed 24 hours after drug administration. The flow cytometer  was used to detect cell apoptosis through the Annexin V-FITC/PI apoptosis kit .α .RIPA lysis containing protease inhibitor  was used for protein extractions. Equal proteins were loaded for SDS-PAGE and blotted as reported by previous research . The ant4 lung adenocarcinoma cells were suspended in 200 µl non-FBS RPMI and planted in the upper chamber . 600 µl RPMI including 20% FBS was placed in the lower chamber. After 24-hour incubation, the cells underwent paraformaldehyde fixing and crystal violet staining. Then microscopic quantification was performed for data analysis. The results represent three independent experiments.Transwell assay was used to evaluate the cell migration. About 3 × 10Cells were seeded in 96-well plates for 24 hours, followed by wash with PBS and fixation with 4% formaldehyde. BSA (5%) containing 0.3% Triton was used to block the nonspecific proteins, and SOX9 primary antibodies  were added at a dilution of 1 : 100 and incubated overnight. The secondary antibody tagged with Alexa Fluor 546  was added and incubated for 1 hour. DAPI  was used to counterstain the nuclei. A Nikon Eclipse Ti fluorescence microscope was applied to capture the images.Six-week nude mice were obtained from Daping Hospital Laboratory Animal Center. The nude mice underwent subcutaneous implantation of H1299 vec and H1299 PC4 cells. The cisplatin treatment and tumor size calculation were in accordance with our previous research . The anip value was less than 0.05. The GraphPad Prism 5  software was applied to produce the figures.The SPSS 18.0  software was applied to finish the statistical analyses. The data was considered significant when the p = 0.002).First, we searched the GEO database for bioinformatic data on cisplatin resistance in lung cancer. The GSE108214 series was analyzed. GSM2892607, GSM2892612, GSM2892615, and GSM2892622 were included in the A549 cisplatin-sensitive group (A549 sens). GSM2892609, GSM2892614, GSM2892617, and GSM2892623 were included in the A549 cisplatin-resistant group (A549 res). p > 0.05) and the PC4-overexpressing H1299 cells  were not statistically significant. Furthermore, CCK-8 assays demonstrated no significant changes in cell growth in the PC-9 sh1 cells in comparison with the PC-9 NC cells (p < 0.001) and significantly increased in the H1299 PC4 cells in contrast to the H1299 vec cells (p < 0.001).First, PC4 was knocked down in PC-9 cells and overexpressed in H1299 cells through lentivirus infection Figures . As showNC cells . SimilarNC cells . InteresNC cells  showed tμM cisplatin for 24 hours were measured by western blot. According to the results, the caspase 3 cleavage and PARP cleavage were obviously activated in the PC-9 sh1 cells and obviously inhibited in the H1299 PC4 cells compared to their respective controls (p = 0.058). But the cell viability was significantly reduced in the PC-9 sh1 cells in comparison with the PC-9 NC cells at 48, 72, and 96 hours after cisplatin treatment  (p < 0.05) , but cisplatin-induced apoptosis significantly increased cell apoptosis in the PC-9 sh1 cells in comparison with the PC-9 NC cells . Similarly, cell apoptosis was not significantly different between the untreated H1299 vec cells and H1299 PC4 cells  but was obviously reduced in the H1299 PC4 cells treated with cisplatin than in the H1299 vec cells .Next, we assessed the impact of PC4 on cell sensitivity to cisplatin. First, the levels of apoptotic proteins in PC4-knockdown and PC-overexpression cells treated with 20 controls . Accordi < 0.05) . Similar < 0.05) . Next, fIn vivo research was conducted through subcutaneous injection of H1299 vec and H1299 PC4 cells into nude mice followed by treatment with or without cisplatin. Representative images of the nude mice are listed in p < 0.05). Interestingly, the tumor weights were also significantly reduced in the untreated H1299 vec group in comparison with the untreated H1299 PC4 group (p < 0.05). To more accurately reveal the impact of PC4 in decreasing LUAD cell sensitivity to CDDP, we further calculated the ratio of tumor weights. As shown in p < 0.05).p = 0.028) (p < 0.001) (http://timer.comp-genomics.org). As shown in r = 0.383, p < 0.001), kidney renal clear cell carcinoma (KIRC) , liver hepatocellular carcinoma (LIHC) , and rectum adenocarcinoma (READ) . Next, we wondered whether SOX9 acted as a downstream target of PC4. According to the qRT-PCR assay (p = 0.004) in H1299 cells, while knockdown of PC4 inhibited SOX9 mRNA expression (p = 0.037) in PC-9 cells. Furthermore, the immunofluorescence results  . Further< 0.001) . Next, tCR assay , overexp results  showed tp > 0.05) after A-485 treatment, 84.67 ± 5.53% vs. 76.30 ± 5.83% (p < 0.05) after DMOG treatment, 68.71 ± 4.95% vs. 50.41 ± 5.29% (p < 0.05) after LY294002 treatment, 92.02 ± 10.40 vs. 99.60 ± 8.34% (p > 0.05) after MG132 treatment, 97.14 ± 3.03% vs. 85.27 ± 4.05% (p < 0.05) after MHY1485 treatment, and 99.02 ± 3.75% vs. 92.06 ± 4.78% (p < 0.05) after rapamycin treatment. The cell viability of the H1299 vec and H1299 PC4 cells was 62.18 ± 8.19% vs. 74.07 ± 5.61% (p < 0.05) after A-485 treatment, 39.60 ± 2.32% vs. 51.87 ± 6.16% (p < 0.05) after DMOG treatment, 59.48 ± 3.30% vs. 54.02 ± 9.51% (p > 0.05) after LY294002 treatment, 87.17 ± 9.95% vs. 79.95 ± 6.60% (p > 0.05) after MG132 treatment, 82.42 ± 5.09% vs. 80.95 ± 9.64% (p > 0.05) after MHY1485 treatment, and 73.10 ± 8.84% vs. 92.47 ± 8.56% (p < 0.05) after rapamycin treatment. These results showed that LUAD cell sensitivity to DMOG and rapamycin was significantly increased in the PC4-knockdown cells and decreased in the PC4-overexpression cells.Next, we evaluated whether PC4 participated in mediating lung adenocarcinoma cell sensitivity to other therapeutic drugs. The p300/CBP inhibitor A-485, the HIF-PH inhibitor DMOG, the PI3K/Akt pathway inhibitor LY294002, the proteasome inhibitor MG132, the mTOR activator MHY1485, and the mTOR inhibitor rapamycin were used in our research Figures . As the p < 0.05). The enhancive effect of LY294002 on cisplatin was 51.88 ± 3.84% in PC-9 NC cells and only 43.00 ± 6.01% in PC-9 sh1 cells (p < 0.05). Likewise, A-485, MG132, MHY1485, and rapamycin possessed no significant differences in enhancing cisplatin's cytotoxic effect between the H1299 vec and H1299 PC4 cells (p > 0.05). The effect of DMOG on enhancing the effect of cisplatin was 40.33 ± 10.03% in the H1299 vec cells and 10.94 ± 11.63% in the H1299 PC4 cells (p < 0.05). The effect of LY294002 on enhancing the effect of cisplatin was 64.82 ± 4.04% in the H1299 vec cells and 46.62 ± 8.59% in H1299 PC4 cells (p < 0.05). Thus, overexpression of PC4 decreased the synergistic effect of DMOG and cisplatin while knockdown of PC4 increased it. Interestingly, the synergistic effect of LY294002 and cisplatin was inhibited in both the PC4-knockdown and PC4-overexpression LUAD cells.Furthermore, we sought to evaluate the impact of PC4 in mediating the synergetic effects of cisplatin combined with other drugs. Thus, the cell viability ratios were calculated to evaluate the enhancive effect of each drug with cisplatin Figures . A-485, α stabilization, we presume that the effect of PC4 in decreasing synergistic effect of DMOG and cisplatin is accompanied by changes in the expression of HIF-1α. First, the expression of HIF-1α in PC4-knockdown and PC4-overexpression cells was detected. However, as shown in α in lung adenocarcinoma cells. Next, we assessed the impact of PC4 on the stability of HIF-1α upon cisplatin treatment. Increasing concentrations of cisplatin were administered to PC4-knockdown and PC4-overexpression cells for 24 hours. As shown in α in both H1299 vec and PC-9 NC cells. Overexpression of PC4 decreased the stability of HIF-1α upon cisplatin treatment, while knockdown of PC4 further increased it.As DMOG is an inhibitor of HIF-PH, which results in HIF-1PC4 has been reported to participate in many cellular activities in cancers , 25, butin vitro study revealed the aggressive phenotype PC4 conferred to lung adenocarcinoma cells migration, which is consistent with previous research in other cancer cell types [in vivo and in vitro, as shown by a decreased cell viability, increased cell apoptosis, and lower tumor weight in PC4-knockdown group upon cisplatin treatment.Through lentivirus infection, PC4 was stably inhibited in PC-9 cells and overexpressed in H1299 cells. The ll types , 14. NexSOX9 is well studied in cancer chemotherapy, and its high expression and activation have been proven to promote cell resistance to chemotherapy in multiple types of cancers, including lung cancer , 26–28. Previous CCK-8 assays revealed that PC4 knockdown or overexpression had no impact on the growth of PC-9 and H1299 cells. However, treatment with cisplatin induced significant changes in the OD values, which was due to the changes in PC4 expression and was a direct reflection of the impact of PC4 in decreasing LUAD cell sensitivity to cisplatin. Based on this, CCK-8 tests were used to detect the cell response to more therapeutic drugs and their synergistic effect with cisplatin to determine whether PC4 also mediated cell sensitivity. As a universal solvent, DMSO is widely used to dissolve multiple small molecules. However, previous research demonstrated that DMSO interacted with platinum complexes, decreasing their cytotoxic effects [combinational therapy/cell viability cisplatin × 100%). Then, the effect of each drug on enhancing the effect of cisplatin was calculated as  × 100%. The results showed that DMOG exerts synergistic enhancive effects on cisplatin, which were altered by PC4 knockdown and overexpression. DMOG synergistically enhanced cytotoxic effect of cisplatin in PC-9 and H1299 cells, and this effect was enhanced upon PC4 knockdown and reduced upon PC4 overexpression. As DMOG is an inhibitor of HIF-PH, which results in HIF-1α stabilization and accumulation, we further investigated whether PC4 impacted the expression of HIF-1α. Surprisingly, HIF-1α was not altered by PC4. Next, we evaluated the stability of HIF-1α upon cisplatin treatment and found that cisplatin stabilized HIF-1α in both PC-9 and H1299 cells, and overexpression of PC4 decreased the stability of HIF-1α upon cisplatin treatment, while knockdown of PC4 increased it. These results suggested that the PC4-induced decrease in lung adenocarcinoma cell sensitivity to cisplatin is associated with a decrease in HIF-1α stability.To accurately evaluate the synergistic effect of other drugs on cisplatin, we first calculated the ratio of cell viability (cell viability α pathways. Thus, PC4 is a promising chemotherapeutic target in LUAD.In summary, our research showed that PC4 participates in mediating LUAD cell sensitivity to cisplatin, DMOG, and rapamycin and enhances the synergistic effect of DMOG and cisplatin. Mechanistically, PC4 governs multiple downstream pathways associated with chemotherapy resistance, including the SOX9 and HIF-1


Melatonin has attracted substantial attention because of its excellent prospects for both medical applications and crop improvement. The microbial production of melatonin is a safer and more promising alternative to chemical synthesis approaches. Researchers have failed to produce high yields of melatonin in common heterologous hosts due to either the insolubility or low enzyme activity of proteins encoded by gene clusters related to melatonin biosynthesis.Escherichia coli by combining the physostigmine biosynthetic genes from Streptomyces albulus and gene encoding phenylalanine 4-hydroxylase (P4H) from Xanthomonas campestris and caffeic acid 3-O-methyltransferase (COMT) from Oryza sativa. A threefold improvement of melatonin production was achieved by balancing the expression of heterologous proteins and adding 3% glycerol. Further protein engineering and metabolic engineering were conducted to improve the conversion of N-acetylserotonin (NAS) to melatonin. Construction of COMT variant containing C303F and V321T mutations increased the production of melatonin by fivefold. Moreover, the deletion of speD gene increased the supply of S-adenosylmethionine (SAM), an indispensable cofactor of COMT, which doubled the yield of melatonin. In the final engineered strain EcMEL8, the production of NAS and melatonin reached 879.38 ± 71.42 mg/L and 136.17 ± 1.33 mg/L in a shake flask. Finally, in a 2-L bioreactor, EcMEL8 produced 1.06 ± 0.07 g/L NAS and 0.65 ± 0.11 g/L melatonin with tryptophan supplementation.Here, a combinatorial gene pathway for melatonin production was successfully established in E. coli and provided alternative strategies for improvement of melatonin production.This study established a novel combinatorial pathway for melatonin biosynthesis in The online version contains supplementary material available at 10.1186/s12934-021-01662-8. Melatonin is an ancient and ubiquitous molecule , widely N-acetylserotonin (NAS) intermediates. Accordingly, four enzymes, including tryptophan-5-hydroxylase (TPH), tryptophan decarboxylase (TDC), serotonin N-acetyltransferase (SNAT), and N-acetylserotonin methyltransferase (ASMT), are involved in the catalytic process [N-acetylserotonin to melatonin. The catalytic efficiency (Vmax/Km) for COMT activity was 709-fold higher than for ASMT in Arabidopsis thaliana, indicating a pivotal role of COMT in the synthesis of melatonin [O-methylated and the resulting 5-MT is N-acetylated, was recently proposed, adding further complexity to the pathway of melatonin biosynthesis [The biosynthetic pathway of melatonin was first elucidated in animals . In anim process . In plan process . First,  process , 14. Sec process . Third, elatonin . Therefoelatonin . In addiynthesis , 17. Comynthesis , 17. TheHomo sapiens or Schistosoma mansoni. Moreover, the genes encoding TDC and ASMT were cloned from H. sapiens, and the gene encoding SNAT was cloned from Bos taurus. Recently, Luo et al. established a recombinant melatonin biosynthetic pathway in E. coli. The genes encoding TPH and ASMT were cloned from H. sapiens and the genes encoding TDC and SNAT were cloned from Candidatus Koribacter versatilis and Streptomyces griseofuscus respectively [E. coli, genes encoding SNAT and ASMT were cloned from several animals and plants. When these enzymes derived from mammals and plants are heterologous expressed in E. coli, the expression level is extremely low or the expression product is inactive, which limits the high production of melatonin in prokaryotic cells [Therefore, almost all the genes necessary to establish the melatonin biosynthetic pathway in genetically engineered bacteria were cloned from animals and plants –20. Germectively . In otheic cells .Streptomyces harbors the first three genes that encode enzymes to produce NAS, which is the immediate precursor of melatonin [Streptomyces griseofuscus and Streptomyces pseudogriseolus can produce physostigmine [psmA-H responsible for the biosynthesis of physostigmine in Streptomyces griseofuscus. Of these, PsmH and PsmF are required for the NAS biosynthesis starting from 5-HTP, which is shared with the melatonin pathway. Based on the physostigmine biosynthetic pathway, in this paper, a novel biosynthetic pathway for melatonin production was established in E. coli. In addition, metabolic engineering and enzyme engineering strategies were employed to optimize the biosynthetic pathway and the production of melatonin was effectively improved by 11-fold compared to the first generation strain.To date, the gene cluster involved in melatonin biosynthesis in microorganisms has not been identified. However, a gene cluster responsible for physostigmine biosynthesis in elatonin . Physostelatonin , 25. Aftstigmine  identifiE. coli Trans1-T1  was used as the host strain for plasmid construction and propagation. E. coli BW25113∆tnaA was used for protein expression and in vivo hydroxylation of L-tryptophan to melatonin. For efficient biosynthesis of melatonin, genomic modification of the E. coli BW25113∆tnaA strain was performed using λ-red recombination [2HPO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 3.5 g/L KH2PO4, 246.5 mg/L MgSO4·7H2O, 14.7 mg/L CaCl2·2H2O, 27.8 mg/L FeSO4·7H2O, and 2 g/L sodium citrate dihydrate. The fed-batch medium contained (per liter) 10 g of glucose, 8 g of (NH4)2HPO4, 13.3 g of KH2PO4, 1.2 g of MgSO4 · 7H2O, 1.7 g of citric acid, and 10 mL of a trace metal solution that contained (per liter of 5 M HCl) 10 g of FeSO4·7H2O, 2.25 g of ZnSO4 · 7H2O, 1 g of CuSO4·5H2O, 0.5 g of MnSO4·5H2O, 0.23 g of Na2B4O7·10H2O, 2 g of CaCl2 · 2H2O, and 0.1 g of (NH4)6MO7O24. When necessary, the medium was supplemented with 50 ug/mL kanamycin, 100ug/mL ampicillin, and 17 ug/mL chloramphenicol.All bacterial strains used in this study are listed in Table bination . Luria–Bbination . M9Y meddcm and speD genes were performed by λ-red recombination. The target sequences were also assembled by the ClonExpress MultiS One Step Cloning Kit. To replace the promoters of the Mtn, luxS, metF, and metK genes, PCR was performed to obtain the upstream region, the kan resistance gene containing FRT at both ends, the promoter and RBS and the beginning of the gene’s ORF as the downstream region. Then, the PCR products described above were assembled to obtain the target genes. According to the λ-red recombination protocol, various E. coli BW25113∆tnaA derivatives were constructed. Knockout of dcm and speD were performed in essentially the same manner as that described above. The target genes used for the deletion of the dcm and speD genes were made by ligating the upstream region, the kan resistance gene containing FRT at both ends and the downstream region in order.All plasmids used in this study are listed in Table 600 reached 0.6. The enzymes were induced with 0.1% l-arabinose. After incubation at 30 °C and 200 rpm for 8 h, 200 OD cells were harvested by centrifugation at 4 °C and 5000×g for 10 min. This induced bacterial pellet was suspended into 20 mL M9Y media (50-mL flask) containing 2 g/L tryptophan, 4 g/L methionine (and 3% glycerin when necessary) and grown at 30 °C and 200 rpm. Samples were collected at 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, and the concentrations of 5-HTP, NAS and melatonin were analyzed by HPLC. Acetyl Coenzyme A kit  was used to detect the concentration of acetyl coenzyme A. Bioscreen C  was used to measure the grow curve of E. coli BW25113.The melatonin-producing strains were cultured in 100 mL LB media (500-mL shaking flask) at 37 °C and 200 rpm until the ODE. coli BW25113 ∆tnaA was transformed with the pBAD-Sa5HTP, pBAD-Xc5HTP, pBAD-Tf5HTP, pBAD-He5HTP and pBAD-Ct5HTP plasmids. The culture methods were the same as described above (production of melatonin), and 200 OD-induced cultures were harvested and suspended into 20 mL of M9Y medium containing 2 g/L tryptophan and grown at 30 °C and 200 rpm. Samples were collected at the time points described above and analyzed by HPLC.4OH, and the temperature was set at 37 ℃. The dissolved oxygen concentration was maintained above 20% air saturation by supplying air at 1 vvm (air volume/working volume/minute) and by automatically controlling the agitation speed up to 700 rpm. When the initial 10 g/L glucose was consumed, a feeding solution containing 500 g of glucose and 10 g of MgSO4 · 7H2O per liter was periodically added. When the OD600 reached 20, the culture was induced by adding L-arabinose to a final concentration of 1 g/L, the temperature was set at 30℃, and a solution of 4 g/L tryptophan, 6 g/L methionine and 3% (v/v) glycerol was added to the media. Samples were collected to measure the biomass concentrations (OD600) and glucose concentrations ) and for HPLC analysis. For optimized conditions, the culture was induced when OD600 reached 40 and after 12 h induction, a solution of 6 g/L tryptophan, 10 g/L methionine and 3% (v/v) glycerol was added to the media.A single colony of Ec-MEL8 from a cell plate was inoculated into 5 mL LB media and cultured at 37 °C and 200 rpm overnight. Then, 1% (v/v) of the culture was transferred into 100 ml LB media (500-mL flask) and grown at 37 °C and 200 rpm for approximately 8–10 h. 5% (v/v) of the cultures were transferred to a 2-L bioreactor  with 1 L fed-batch media. The pH was controlled at 6.8 by automatic feeding of 30% (v/v) NHl-tryptophan from Sigma , 5-HTP from Aladdin , NAS from Sigma and melatonin from Aladdin  were used as the standards. The standards and the supernatant concentration of tryptophan, 5-HTP and NAS were measured after dilution of the samples with methanol/water (15:85 v/v). For analysis of melatonin, standard and supernatant of cultures were diluted to a final concentration of methanol/water (40:60 v/v). The samples were filtered through a 0.22-μm nylon filter and then analyzed by HPLC  using an Agilent ZORBAX Eclipse Plus C18 column . Tryptophan, 5-HTP and NAS were quantified under 275 nm UV detection with methanol/water (15:85 v/v) as the mobile phase. Melatonin was quantified under 290 nm UV detection with methanol/water (40:60 v/v) as the mobile phase. NAS and melatonin were identified by QTRAP 6500 and AB SCIEX using Gemini 3 µm NX-C18 110 Å (50 × 2 mm) with 95% methanol (containing 0.1% formic acid) and 5% water (containing 0.1% formic acid) (v/v).Streptomyces albulus are shared by melatonin biosynthesis. Enlightened by the physostigmine biosynthetic pathway, a novel biosynthetic pathway for melatonin production was designed . It was reported that some bacterial P4Hs exhibit activity towards both phenylalanine and tryptophan. Moreover, the substitution of a small number of residues  causes the preferred substrate of P4H to change from phenylalanine to tryptophan [Xanthomonas campestris [Halomonas elongata, Thermomonas fusca, and Cupriavidus taiwanensis [, were also selected to catalyze the first step of melatonin biosynthesis. The genes encoding homologs of PsmH and PsmF were selected from S. albulus to establish the second and third catalytic steps of melatonin biosynthesis. For the final step of melatonin biosynthesis, two kinds of enzymes: ASMT and COMT have been identified to exhibit the catalytic activity. ASMT, as a methyltransferase initially identified in animals and plants, had low enzyme activities, indicating its role as a rate-limiting enzyme in melatonin synthesis [Oryza sativa (OsCOMT) was the preferred enzyme, and this enzyme exhibits the highest activity for converting NAS to melatonin as ever reported [S. albulus, BLASTP analysis revealed a putative gene encoding COMT, which shared the highest sequence identity with OsCOMT. The resulting SaCOMT and OsCOMT were then selected to establish the final catalytic step of melatonin biosynthesis.When searching tryptophan derivatives in prokaryotes, we found three intermediates of physostigmine biosynthesis  in ned Fig. . In the yptophan . Therefompestris , Halomonwanensis , were alynthesis . Besidesynthesis , 30. Comynthesis , also O-ynthesis , 32. Thereported . In S. aE. coli based on the physostigmine biosynthetic pathway. All four proteins from S. albulus, namely, SaP4H, SaCOMT, SaPsmF and SaPsmH, were expressed well as soluble recombinant proteins in E. coli. Among these genes, the genes encoding SaP4H and SaCOMT were optimized according to the E. coli codon preference, while the genes encoding SaPsmF and SaPsmH were not optimized. The 5-HTP biosynthetic module and COMT were involved in the first and final steps of melatonin biosynthesis. According to previous reports, these two steps were proposed to be the rate-limiting of melatonin production and were tested first [Above all, we designed a new melatonin biosynthetic pathway in ed first , 20.P. aeruginosa and FolM from E. coli were cloned and inserted downstream of the genes encoding the W179F mutants of P4Hs in the pBAD plasmid , in which the tnaA gene encoding tryptophanase was deleted to block the degradation of tryptophan and 5-HTP, was generated  were assembled. Besides, another two strains, namely EcMEL3 and EcMEL4, were made by placing the genes encoding SaPsmH, SaPsmF and COMT on another plasmid (pZS), with the 5-HTP biosynthetic module on the pBAD plasmid. Unexpectedly, melatonin was not produced in all these four strains, while significant NAS accumulation was observed . The low conversion efficiency of 5-HT to NAS might indicate the lack of acetyl-CoA. Glucose, acetate and glycerol were reported to be able to provide energy and the acetyl group for acetyltransferase in E. coli , 34. 10 S-adenosylmethionine (SAM), the other important cofactor, plays a key role in the process of COMT catalysis. SAM is a common methyl donor in vivo that participates in the rate-limiting steps of the biosynthesis of multiple compounds and undergoes many genome modifications to remove inhibition [E. coli, a series of genes related to SAM recycling were deleted or overexpressed  in melatonin production  significantly enhanced the catalytic activity of COMT from Arabidopsis thaliana (AtCOMT) [speD, the EcMEL8 strain was obtained. EcMEL8 produced 136.17 ± 1.33 mg/L melatonin and 879 ± 71.42 mg/L NAS in a shake flask  . The corask Fig. c. Figureask Fig. e. The re600 = 38). Melatonin yield reached its highest level in the medium of 0.32 ± 0.03 g/L at 71 h with the synthesis efficiency of melatonin produced via per g of dry cell weight (DCW) at 35 mg/g DCW. NAS production continued to increase even after 83 h and reached 3.0 ± 0.26 g/L , SDS-PAGE analysis of pBAD-5HTPs. The red arrow indicates the expression of P4Hs. (b), SDS-PAGE analysis of EcSaCOMT and EcOsCOMT. Supplementary Fig. 2. Construction of plasmids for expression of melatonin-related proteins. Supplementary Fig 3. SDS-PAGE analysis of EcMEL1, EcMEL2, EcMEL3, and EcMEL4. Supplementary Fig 4. SDS-PAGE analysis of EcMEL5, EcMEL6, EcMEL7, EcMELCX and EcMELCXPM. Supplementary Fig. 5. Comparison of the melatonin production by EcMEL7, EcMELCS, EcMEL-CtacS and EcMEL-CT7S. Supplementary Fig 6. Comparison of adding glucose, acetate and glycerol in M9Y medium for biosynthesis of NAS and melatonin. Supplementary Fig. 7. The concentration of Acetyl Coenzyme A in EcMEL7 and EcMEL7+5% glycerol during the whole-cell biocatalysis. Supplementary Fig. 8. The grow curve of BW25113ΔtnaA and BW25113ΔtnaA ΔspeD. Table S1. Primers used in DNA manipulation. Table S2. The OD600 of Melatonin Producing Strains in M9Y medium. Table S3. The OD600 of Melatonin Producing Strains after adding glucose, glycerol, and acetate.


In vitro and in vivo models of MI were established by hypoxia/reoxygenation (H/R)-treatment of H9C2 cells and through the ligation of the left anterior descending coronary artery of rats, respectively. CCK-8 and EdU assays were performed to measure cell viability and proliferation, respectively. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed to determine the apoptotic rate of cells. Western blot was used to determine protein expression. The MI area was analyzed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. NGR1 promoted viability and proliferation, and inhibited the apoptotic rate of H/R-treated H9C2 cells. In addition, NGR1 downregulated the protein expression of caspase-3 and Bax, and upregulated Bcl-2 expression in H/R-treated H9C2 cells. The JAK2/STAT3 signaling pathway was activated following NGR1 treatment in vivo and in vitro, and inhibition of the JAK2/STAT3 signaling pathway reversed the effects of NGR1 on H/R-treated H9C2 cells. Finally, NGR1 reduced the area of MI. NGR1 relieved MI in vivo and in vitro by activating the JAK2/STAT3 signaling pathway.Myocardial infarction (MI), caused by continuous ischemia and hypoxia of the coronary artery, is one of the major causes of human mortality. This study aimed to investigate the role of notoginsenoside R1 (NGR1) in MI therapy.   Myocardial infarction (MI), caused by persistent ischemia and hypoxia of the coronary artery, is a major cause of human fatality. Typically, MI results in myocardial ischemia and necrosis, frequently complicated by manifestations such as malignant arrhythmia, heart failure, and shock . MI has Panax notoginseng, a popular traditional Chinese medicine, is commonly used to prevent and treat cardiovascular diseases in China. Notoginsenoside R1 (NGR1) is a principal active component of P. notoginseng, which has anti-inflammatory, antioxidant, and anti-apoptotic properties [operties . Recent operties , nervousoperties , organ doperties , and tumoperties . In addioperties  confirmein vitro MI model using hypoxia/reoxygenation (H/R)-treated H9C2 cells in the present study. In addition, we established an in vivo model by ligating the left anterior descending coronary artery. We aimed to analyze the in vivo and in vitro effects of NGR1 on MI using these models. We hypothesized that NGR1 promotes proliferation and inhibits the apoptosis of H/R-treated H9C2 cells, as well as relieves MI in vivo by activating the JAK2/STAT3 signaling pathway.Therefore, we established an 2 at 37°C. Cells were used for further experiments once they reached logarithmic growth phase (growth to approximately 80% fusion state). The cells were then separated into control (CON), H/R, H/R + 20 μM NGR1 (L-NGR1), H/R + 40 μM NGR1 (H-NGR1), and H/R + H-NGR1 + 50 μM JAK2 inhibitor (AG490) (H-NGR1 + AG490) groups. In the CON group, H9C2 cells were cultured in DMEM supplemented with 10% FBS in a 5% CO2 incubator at 37°C. In the H/R, L-NGR1, H-NGR1, and H-NGR1 + AG490 groups, H9C2 cells were cultured in DMEM without FBS in a incubator with 37°C, 5% CO2, 94% N2, and 1% O2 for 24 h, and then cultured in DMEM with 10% FBS in a constant-temperature incubator at 37°C with 5% CO2 and 95% air for 6 h. The cultured cells were treated with NGR1 (20 μM or 40 μM) and 50 μM AG490 in the same medium for 24 h.The rat H9C2 cell line was procured from the Cell Bank of the Shanghai Chinese Academy of Sciences . H9C2 cells were seeded in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum (FBS) and cultured in an incubator with 5% COtreatment group/A control group.Cell viability was analyzed using the CCK-8 assay. First, the cells in each group were collected and placed in 96-well plates. Next, 10 μL of CCK-8 solution was added, and the cells were cultured for 72 h. A microplate reader was used to read the absorbance (A) at 450 nm. Cell viability was calculated using the following formula: Cell viability = A 4 cells/well). The proliferation ability was detected using a Cell-Light™ EdU DNA Cell Proliferation Kit , following the manufacturer’s instructions.The cells in each group were collected and plated in a 96-well plate (2 × 106 cells/well) were plated in 24-well plates. After washes and resuspension, 5 μL of Annexin V-FITC and 10 μL of PI were added to the cells. Finally, apoptotic cells were measured using a flow cytometer .The apoptotic rate of cells was assessed using an annexin V-FITC kit. Briefly, cells  were obtained from the Animal Center of Nanjing Medical University. The groups were as follows: control group , model group , model group + 20 mg/kg NGR1 , model group + 40 mg/kg NGR1 . The Following four weeks of test drug/saline administration, the myocardial tissue was dissected and cleaned with precooled 0.9% sodium chloride solution. Then, the myocardial tissue slices were immersed in 1% TTC phosphate buffer solution at 37°C for 30 min and fixed in 4% paraformaldehyde for 24 h. The infarcted tissue appeared white to light in color, whereas the non-infarcted tissue was dark red. The size of the MI area and the proportion of infarcted tissue areas were measured and calculated.Apoptosis in cells and tissues were analyzed using TUNEL staining. The cells in each group were cultured in 6-well plates and then fixed with 4% formaldehyde. After washing twice with phosphate buffer solution, the cells were treated with 0.5% Triton X-100 for 20 min. Finally, the TUNEL working solution was added, followed by incubation at 37°C for 1 h. Apoptotic cells were analyzed using fluorescence microscopy.Protein was extracted from cells and tissues using radioimmunoprecipitation assay (RIPA) buffer , and the concentration was measured using the BCA Protein Assay Kit (Beyotime). The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene fluoride membrane (Millipore). Next, the PVDF membrane was incubated with the primary antibody including Bax , Cleaved Caspase 3 , Bcl-2 , GAPDH , pJAK , JAK , pSTAT3  and STAT3  at 4°C overnight. The membrane was further incubated with a secondary antibody  for 1.5 h. Finally, the target proteins on the membrane were visualized with Pierce™ ECL Western blot substrate. Quantitative detection of the Western blots was performed using ImageJ .Enzyme linked immunosorbent assay (ELISA)The plasma samples of the rats in different groups were collected, and the levels of brain natriuretic peptide (BNP) and N-terminal pro-B type natriuretic peptide (NT-proBNP) were determined by ELISA kits  based on the information provided by the manufacturer.Statistical analysis was performed using SPSS . Data are expressed as the mean ± standard deviation. One-way ANOVA was used for comparison between groups. Statistical significance was set at P < 0.05.Myocardial infarction (MI), caused by continuous ischemia and hypoxia of the coronary artery, is one of the major causes of human mortality. This study aimed to investigate the role of notoginsenoside R1 (NGR1) in MI therapy. Our study found that NGR1 promoted viability and proliferation, and inhibited the apoptotic rate of H/R-treated H9C2 cells. In addition, NGR1 relieved MI in vivo and in vitro by activating the JAK2/STAT3 signaling pathway.###P < 0.001, *P < 0.05, **P < 0.01)  (The molecular structure formula of NGR1 was shown in  < 0.01)  and prol< 0.001)  of H/R-t###P < 0.001, *P < 0.05, **P < 0.01)    ; this fi < 0.01) . Further < 0.01) .Figure ###P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001). Additionally, following AG490 treatment, the phosphorylation of JAK2 and STAT3 was decreased compared to that in the NGR1 group  (As shown in  < 0.01) .Figure ###P < 0.001, **P < 0.01, &P < 0.05, &&P < 0.01)    , whereas < 0.01) . Further < 0.01) .Figure ##P < 0.01, **P < 0.01,)      . In addi < 0.01) . Similar < 0.01) . Moreove < 0.01) . Further < 0.01) .Figure in vivo. Thus, our findings indicate that NGR1 may be a potential target drug for MI treatment.Herein, we confirmed that NGR1 promotes proliferation and suppresses apoptosis in H/R-treated H9C2 cells by regulating the JAK2/STAT3 signaling pathway. In addition, NGR1 relieved the progression of MI in vivo and in vitro and promotes the growth of H/R-treated H9C2 cells. Likewise, NGR1 was found to inhibit apoptosis in diabetic retinopathy [in vivo and in vitro. Zhang et al. [MI is a major cardiovascular disease that is closely associated with cardiac insufficiency and sudden cardiac death. Evidence suggests that MI-associated mortality has continued to grow in recent years. MI has been reported to lead to adverse cardiac remodeling and apoptosis in cardiac cells during chronic progression . Since ainopathy , atherosinopathy , and burinopathy . These fg et al.  demonstrg et al.  confirmein vivo and in vitro. We hypothesized that NGR1 may regulate the proliferation and apoptosis of H/R-treated H9C2 cells by activating the JAK2/STAT3 signaling pathway. Accordingly, we inhibited the JAK2/STAT3 signaling pathway using the JAK inhibitor, AG490. Interestingly, AG490 treatment reversed the effects of NGRI on cell proliferation and apoptosis, as well as the protein expression of caspase-3, Bax, and Bcl-2, further indicating that NGR1 attenuates MI by activating the JAK/STAT3 signaling pathway.The JAK/STAT pathway is mainly composed of the JAK as well as the STAT protein familes. JAK is a common upstream kinase of STAT, and JAK/STAT is widely associated with diverse biological effects such as cell stress, proliferation, differentiation, and apoptosis ,22,23. Tin vivo and in vitro. Accordingly, the present study provides a theoretical basis for the application of NGR1 and confirms that NGR1 represents a novel therapeutic drug for MI treatment.In conclusion, this study is the first to examine the role of NGR1 in the treatment of MI. We demonstrated that NGR1 relieves MI by activating the JAK/STAT3 signaling pathway


Lactiplantibacillus plantarum is a genetically and phenotypically diverse species of lactic acid bacteria. We announce the hybrid de novo assembly of Oxford Nanopore Technologies and Illumina DNA sequence reads, producing a closed circular chromosome of 3,206,992 bp and six plasmids of the inulin-utilizing L. plantarum strain Lp900. Lactiplantibacillus plantarum is a genetically and phenotypically diverse lactic acid bacterium species. We announce the hybrid de novo assembly of Oxford Nanopore Technologies and Illumina DNA sequence reads, producing a closed circular chromosome of 3,206,992 bp and six plasmids of the inulin-utilizing L. plantarum strain Lp900. Lactiplantibacillus plantarum is one of the most versatile species of lactic acid bacteria (LAB) found in fermented and unfermented foods and the human gastrointestinal tract, displaying remarkable phenotypic and genomic diversity  broth overnight at 37°C and passaged in fresh medium, and cells were harvested during exponential growth, flash frozen, reheated at 65°C for 10 min, resuspended in THMS  containing 20 mg/ml lysozyme and 300 U mutanolysin, and incubated for 1 h at 37°C. Subsequently 1× TAE was added , and the sample was treated with SDS  at 65°C for 20 min. Afterwards, samples were incubated with RNase (0.1 mg/ml) and proteinase K (0.35 mg/ml) for 1 h and 30 min, respectively, at 37°C. Genomic DNA was extracted with phenol-chloroform, precipitated with absolute ethanol and sodium acetate , and dissolved overnight in sterile water (le water .https://github.com/rrwick/Filtlong) down to 300 MB of data with predicted genome coverage of ∼90×. Extracted FASTA files from Illumina and Nanopore sequencing data were integrated in a hybrid genome assembly using Unicycler v0.4.8-beta  and sequenced using 125-bp paired-end libraries on an Illumina HiSeq 2500 system (Illumina) with a total number of 3,677,745 reads, with target coverage of ∼138×. Illumina sequencing was performed at BaseClear . Nanopore libraries were prepared using the SQK-LSK109 1D genomic DNA ligation kit  with 1 μg of genomic DNA as the input and sequenced on a MinION Mk1 instrument using an R9.4.1 flow cell (Oxford Nanopore Technologies), which delivered >1 Gb of raw data consisting of 128,396 1D reads with a mean length of 8,057 kb. Base calling was performed on the MinIT device running Guppy v2.0.10. Raw data were filtered using Filtlong v0.2.0 (4.8-beta , produci v1.13.3 .L. plantarum Lp900 were deposited in GenBank under BioProject number PRJNA630372 and BioSample number SAMN14833145. Raw sequences of both Illumina and Nanopore reads were submitted to the NCBI SRA under BioProject number PRJNA630372.The complete genome sequence and plasmids of