ABSTRACT
The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein mediates viral entry into host cells through binding to the cell-surface receptor angiotensin-converting enzyme 2 (ACE2). It has been shown that SARS-CoV-2 RBD (RBDCoV2) has a higher binding affinity to human ACE2 than its highly homologous SARS-CoV RBD (RBDCoV), for which the mechanistic reasons still remain to be elucidated. Here, we used the multiple-replica molecular dynamics (MD) simulations, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations, and interface residue contact network (IRCN) analysis approach to explore the mechanistic origin of different ACE2 binding affinities of these two RBDs. The results demonstrate that, when compared to the RBDCoV2-ACE2 complex, the RBDCoV-ACE2 complex features the enhanced overall structural fluctuations and inter-protein positional movements and increased conformational entropy and diversity. The inter-protein electrostatic attractive interactions are a dominant force in determining the high ACE2 affinities of both RBDs, while the significantly strengthened electrostatic forces of attraction of ACE2 to RBDCoV2 determine the higher ACE2 binding affinity of RBDCoV2 than of RBDCoV. Comprehensive comparative analyses of the residue binding free energy components and IRCNs reveal that, although any RBD residue substitution involved in the charge change can significantly impact the inter-protein electrostatic interaction strength, it is the substitutions at the RBD interface that lead to the overall stronger electrostatic attractive force of RBDCoV2-ACE2, which in turn not only tightens the interface packing and suppresses the dynamics of RBDCoV2-ACE2, but also enhances the ACE2 binding affinity of RBDCoV2 compared to that of RBDCoV. Since the RBD residue substitutions involving gain/loss of the positively/negatively charged residues, in particular those near/at the binding interfaces with the potential to form hydrogen bonds and/or salt bridges with ACE2, can greatly enhance the ACE2 binding affinity, the SARS-CoV-2 variants carrying such mutations should be paid special attention to.
ABSTRACT
Objective: To research the effect of traditional Chinese medicine compound Chaijie Yiganxian (CJYGX) on the hepatic fibrosis in rats and explore the mechanism. Method: Totally 60 SD rats were randomly divided into normal group, model group, positive group (Biejia Jianwan), and low, medium and high-dose CJYGX groups. Except for the normal group, the remaining five groups were involved in establishing the hepatic fibrosis model through the intraperitoneal injection of thioacetamide (TAA). Fourth weeks after modeling, the positive group was given Biejiajian pill (1 g·kg-1·d-1), and high, medium and low-dose CJYGX groups were given CJYGX(7.96, 3.98, 1.99 g·kg-1·d-1), respectively. The model group was given normal saline once a day for 5 weeks. 24 h after the last intragastric administration with chloral hydrate, the rats were anesthetized slightly. Serum and liver tissues were collected. The liver wet weight was weighed electronically, and the liver index was calculated. Liver function indexes[alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP)] and serum markers[laminin (LN), hyaluronidase (HA), Ⅳ collagen (CⅣ), procollagen Ⅲ (PCⅢ)] of hepatic fibrosis were detected by enzyme linked immunosorbent assay (ELISA). The degree of hepatic fibrosis by Masson stain, mRNA expressions of transforming growth factor-β1 (TGF-β1), Smad2, Smad3, Smad4, Smad7 were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR), and protein expression levels of platelet derived growth factor (PDGF), TGF-β1 and α-smooth muscle actin (α-SMA) were detected by immunohistochemical or Western blot. Result: Compared with the blank group, the model group liver index was significantly increased; the contents of ALP, AST and ALT were significantly increased; the liver fibrosis indexes LN, HA, Ⅳ-C, PCⅢ and HYP were significantly increased; the degree of liver fibrosis was significantly increased in Masson tissue; the mRNA expressions of TGF-β1, Smad2, Smad3, Smad4 were significantly increased; and the Smad7 mRNA expression was significantly decreased. The protein expressions of TGF-β1, PDGF and α-SMA increased significantly (Pβ1, Smad2, Smad3, Smad4 and up-regulate mRNA expression of Smad7.Immunohistochemical and Western blot analysis show that, compared with model group, CJYGX could significantly reduce the protein expressions of TGF-β1, PDGF and α-SMA (PPConclusion: Traditional Chinese medicine compound CJYGX may protect TAA-induced hepatic fibrosis injury by interfering with TGF-β1/Smad signaling pathway.
