S of TGF-1, Smad2, COL1A1 and MMP2 mRNA had been detected, -actin was utilised as an internal reference gene. Every sample was repeated at least 3 occasions. The 2-Ct process was utilised to calculate the relative quantitative expression in the target gene in every sample. The primer sequences are shown in Table two.Table two. Primers for qPCR. Gene Name TGF-1 Smad2 COL1A1 MMP2 -actin Primer Sequences F five -CCGATGAGTATTGGGCCAAAGAGC-3 R five -GACACGTTGAACACGAAGAAGATGC-3 F five -TGTCATCCATTCTGCCATTCACTCC-3 R 5 -CACCACTTCTCCTCTTGCCCATTC-3 F five -TGGATTCTCGGTTACTGCTGTTGATAG-3 R five -TTCGGGTTTCCACACATCCTTATCG-3 F 5 -CAACAGAAGGCAGGACAGATGGATAC-3 R five -GGAAGATGAAGGGGAATACACAAGGAG-3 F five -CATCTATGAAGGCTACGC-3 R five -GGCTGTGGTGGTGAAG-2.10. ELISA Detection A single hundred milligrams of lung tissue was added to 100 of precooled PBS (pH 7.four) to prepare a tissue homogenate making use of a low-temperature homogenizer. The homogenate was centrifuged at 3000 rpm for 20 min, along with the supernatant was collected for detection. There have been 5 replicates per group, and every sample had three repetitions. ELISAs were performed as outlined by the directions with the Chicken transforming growth element 1 (TGF-1) ELISA Kit, Chicken Smad2 (Smad2) ELISA Kit, Chicken collagen alpha-1(I) chain (COL1A1) ELISA Kit, and Chicken matrix metalloproteinase 2 (MMP2) ELISA Kit (Enzyme-linked (Mlbio) Biotechnology Co., Ltd., Shanghai, China). The double antibody sandwich ELISA method was employed to detect the protein levels of TGF-1, Smad2, COL1A1 and MMP2. two.11. Statistical Evaluation Data are expressed as the mean SEM. SPSS 23.0 statistical software program was applied for oneway ANOVA followed by the LSD post hoc test. Variations had been regarded as statistically significant in the degree of p 0.05. three. Final results 3.1. Identification with the Components in QLJP making use of UHPLC-MS The components of QLJP had been analyzed applying UHPLC S. The total ion chromatogram of QLJP was obtained in negative and positive ion modes (Figure 1). Comparison with the reference compounds inside the database revealed 27 classes of constituent compounds of QLJP, including carboxylic acids and derivatives, organooxygen compounds, flavonoids, steroids and steroid derivatives, macrolides and analogs, peptidomimetics, phenols, linear 1,3-diarylpropanoids, benzopyrans, indoles and derivatives, organonitrogen compounds, pyrans, oxepanes, coumarins and derivatives, isoflavonoids, pyridines and derivatives, cinnamic acids and derivatives, diazanaphthalenes, lactones, macrolactams, aurone flavonoids, benzofurans, naphthalenes and others.ADAM12, Human (HEK293, His) Flavonoid compounds were identified because the principal medicinal components of QLJP and incorporated rutin, kaempferol-3-O-rutinoside, for-Animals 2023, 13,with all the reference compounds in the database revealed 27 classes of constituent compounds of QLJP, including carboxylic acids and derivatives, organooxygen compounds, flavonoids, steroids and steroid derivatives, macrolides and analogs, peptidomimetics, phenols, linear 1,3-diarylpropanoids, benzopyrans, indoles and derivatives, organonitrogen compounds, pyrans, oxepanes, coumarins and derivatives, isoflavonoids, pyridines six of 16 and derivatives, cinnamic acids and derivatives, diazanaphthalenes, lactones, macrolactams, aurone flavonoids, benzofurans, naphthalenes and others.CRHBP, Human (HEK293, His) Flavonoid compounds have been identified as the major medicinal elements of QLJP and included rutin, mononetin, isoquercitrin, quercetin, kaempferol, and kaempferol-3-O-glucoside (Figure 1, kaempferol-3-O-rutinoside, formononetin, isoquercitrin.PMID:24576999