In this study, we distinguish R septa from P septa and PS fibrosis through the collagen quantitative method by SHG/TPEF technology. Besides, we found the appearance of delicate and dense R septa indicating fibrosis regression in advanced fibrosis MASH patients. We further demonstrated the phenomenon and possible mechanism of lower fibrosis levels in patients with R septa by noninvasive tests and transcriptomics.
This study is the continuation of our previous work on fibrosis regression. In Sun et al’s study, a new histological evaluation system “Beijing classification” was proposed. In this system, the PIR score was used to evaluate the dynamic changes in the quality of fibrosis in CHB patients with advanced fibrosis stage: predominantly progressive (thick/broad/loose/pale septa with inflammation); predominately regressive (delicate/thin/dense/splitting septa); and indeterminate, which displayed an overall balance[12]. This new staging system highlighted the quality of fibrosis and can infer the dynamic direction of fibrosis [21]. Although the “Beijing classification” was created in CHB patients, based on the concept of “hepatic repair complex (HRC)”, the morphological changes of fibrosis regression may be common in chronic liver diseases of different etiologies. We confirmed this theory in secondary liver biopsies of partially successful weight loss patients in our MAFLD cohort.
One strength of this study is that we could find the regression tendency of MASH fibrosis by one biopsy which continues the idea of “Beijing classification”. The main finding of our study is that this delicate, thin, dense, splitting septa in MASH also indicates fibrosis regression. This is an expansion of the “Beijing classification” pathological evaluation system, from viral hepatitis to other liver diseases, from a liver disease characterized by portal fibrosis to perisinusoidal fibrosis. In this study, the percentage of R septa did not reach the criteria of “predominantly regressive” of the PIR system (50%)[12]. The main reason we believe is the lack of effective drugs for MASH, so it is very difficult for MASH patients to achieve fibrosis regression as much as CHB patients. Even so, we found that the appearance of R septa, although did not reach the percentage of 50%, can still show the tendency of the good direction of MASH fibrosis. We believe our conclusion can be used to help determine the efficacy of new MASH drug development, and can more sensitively detect the regression of fibrosis, which predicts the improvement of outcome in MASH patients.
The other strength of this study is that we could precisely identify the delicate R septa from PS fibrosis. MASH fibrosis is characterized by PS fibrosis, which is a key pathological feature different from viral hepatitis[22]. The R septa and PS fibrosis were both thin and delicate which was hard to distinguish from each other. Identifying the R septa is especially important for judging whether fibrosis is in a regressive or progressive stage according to the theory of “Beijing classification”. To solve this problem, we applied the SHG/TPEF technology and compared the detailed collagen parameters between R septa and PS fibrosis. The SHG/TPEF can quantitatively show the structure and characteristics of collagen without traditional staining. Although the R septa looked as thin as PS fibrosis on regular collagen staining, through quantitative analysis, the R septa had a significant difference with PS fibrosis. Further, by comparing the distance between fibers, we found that the fibers in R septa were more compact, and the fibers in PS fibrosis were more diffuse, which was also a key difference between the two structures.
The analysis of DEGs provides insights into why the presence of R septa suggests fibrosis regression. In our study, most DEGs were concentrated on the ECM function and related pathways. Fibrogenic genes including Col3A1, Col4A1, Col4A2, BGN, THBS2, and THBS1 were down-regulated in patients with R septa and up-regulated in patients without R septa. The function of these genes reveals the underlying mechanism of fibrosis regression in patients with R septa.
The Col3A1 gene encodes the pro-alpha1 chains of type III collagen (COL III). The COL III formation biomarker ProC3 has been reported to be associated with lobular inflammation, ballooning, and elevated fibrosis stage in MASH[23]. The Col4A1 and Col4A2 are both key genes that encode type IV collagen (COL IV), which is the major structural component of basement membranes[24]. Accordingly, the overexpression of COL IV in the space of Disse may serve as perisinusoidal basement membrane formation, which is associated with capillarization of the hepatic sinusoid and liver fibrosis[24, 25]. BGN encodes a member of the small leucine-rich proteoglycan (SLRP) family of proteins, which play a role in collagen fibril assembly in multiple tissues[26]. Downregulation of BGN could reduce collagen deposition and inhibit the TGF-β1/Smad pathway to reduce apoptosis, inflammation, and DNA damage in fibrotic livers[27]. The protein encoded by THBS2 belongs to the thrombospondin family. It is a disulfide-linked homotrimeric glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. A recent study showed that THBS2 positively correlated with inflammation and ballooning according to MAFLD activity score, and had a good diagnosing ability to identify MASH and advanced fibrosis patients[28]. Another multi-transcriptome analysis revealed that THBS2 and COL4A2 were positively associated with each other in liver fibrosis progression[29].
This study has a few limitations. First, this is a cross-sectional study, and whether patients with R septa have a better prognosis requires a long-term follow-up. Although our team has carried out follow-ups of advanced MASH fibrosis patients with different types of septa, the endpoints of MASH appear slowly and require a longer observation time. We believe that the emergence of R septa can have a beneficial impact on MASH patients, but long-term observation is still needed to confirm. Second, the sample size of this study is limited, especially for subjects with R septa. With the successful development of new MASH drugs, we believe there will be more patients with MASH fibrosis regression in the future, and a larger sample size will be available to further verify our theory. Finally, the key genes that were related to the emergence of R septa in our study need more experiments to explore the specific mechanism.
In conclusion, the appearance of R septa is a sign of regression in MASH patients with advanced fibrosis. The R septa resemble perisinusoidal fibrosis but can be differentiated by quantitative means of new technology.