The Role And Mechanism Of Trypsin(PRSS1)-trypsin Inhibitor(SPINK1) In Human Pancreatitis

Background:The stochiometric relationship between the amount of trypsin(encoded by the PRSS1 gene)and the amount of pancreatic secretory trypsin inhibitor(encoded by the SPINK1 gene)determines the destructive capacity of prematurely activated trypsin in the pancreas.However,the interaction between these two mutant genes has not been fully studied.Aim:To explore the extent to which gene mutations alter the expression of trypsinogen as well as secretory trypsin inhibitors,the interplay between PRSS1-PRSS2 rs10273639_C risk haplotype and the SPINK1 rs 17107315_C risk haplotype,and the impact of their imbalanced ratio on the risk and pathogenesis of pancreatitis.Methods:1.Total RNA sequencing(RNA-Seq)was performed using human pancreatic tissue(n=15)to assess relative differences in wild-type and risk haplotype expression.2.North American Pancreatitis Study 2(NAPS2)RAP/CP cases(n=1341)and controls(n=5691)were assessed using genome-wide association analysis(GWAS)for SPINK1 risk Haplotypes and PRSS1-PRSS2 risk haplotypes were co-occurred in the population.3.Co-expression of PRSS1 and SPINK1 transcripts in human pancreatic cell subpopulations from pancreatitis patients was analyzed using single cell sequencing(scRNA-Seq)and further subgroup analysis and transcription factor and pathway analysis was performed.Results:1.At the tissue and organ level,total RNA-Seq from 3 SPINK1 heterozygous and 12 PRSS1 heterozygous patients revealed that the high-risk allele PRSS1 rs10273639_C increased pancreatic protease expression at the transcriptional level compared to PRSS1 rs10273639_T(p=0.021).PRSS1 rs6667_C variant,with a moderate LD of PRSS1-PRSS2 rs10273639_C resulted in a significant decrease in PRSS1 expression.In contrast,SPINK1 rs17107315_C tended to reduce pancreatic secretory trypsin inhibitor expression at the transcriptional level(p<0.001).2.At the population level,the results were determined by examining the NAPS2 cohort of 1341 chronic and recurrent acute patients with pancreatitis and 5691 controls,GWAS revealed that SPINK1 rs17107315_C did not significantly increase the risk of pancreatitis in the absence of PRSS1 rs10273639_C(OR=1.621,p=0.390),but in the presence of 1 or 2 PRSS1 rs10273639_C mutations(TC or CC),SPINK1 rs17107315_C increased the risk of pancreatitis(OR=2.160,2.197;p=0.013,0.042,respectively).The PRSS1-PRSS2 rs10273639_TT genotype confers protection against the risk of SPINK1 rs17107315_TC pancreatitis.3.pancreatic tissue samples from four pancreatitis patients by scRNA-Seq revealed mismatched expression of SPINK1 and PRSS1 in an undifferentiated cell population with pancreatic cell characteristics and a low SPINK1/PRSS1 ratio,limiting the protective effect of secretory trypsin inhibitors against trypsin activation.Further subpopulation analysis of undifferentiated cells revealed a very high expression of inflammatory stress-related genes in the subpopulation of cells with the lowest SPINK1/PRSS1 ratio,which is consistent with intracellular trypsin-related damage.PRSS1-PRSS2 risk haplotypes have little effect on PRSS1 expression or interaction with SPINK1,which may lead to increased CP risk through TRB complex gene mutations(mainly TRBV29-1)and T cell immunity-related pathways(mainly Th17/IL-17).Conclusions:The PRSS1 rs10273639_C mutation increases trypsinogen expression and the SPINK1 rs17107315_C mutation decreases secretory trypsin inhibitor expression.Co-presentation of mutations in both genes increased the risk of recurrent acute pancreatitis and chronic pancreatitis due to trypsin and trypsin inhibitor mismatches.the PRSS1-PRSS2 risk haplotype had little effect on PRSS1 expression or interaction with SPINK1,suggesting that the strong risk of(alcoholic)CP may be through a mutated TRB complex and an altered immune response.Mismatches between trypsin and trypsin inhibitor cell populations are also present in the pancreas of patients with pancreatitis,especially in undifferentiated cells,suggesting that dedifferentiation may be a potential pathogenesis of chronic pancreatitis,as well as pancreatitis-pancreatic cancer transformation.