## 3 Results
### Research on the Transmembrane Transport Mechanism of NMN  
The bilateral permeabilities of the NMN were investigated. The concentration of NMN was 0.2 mmol/L. The incubation time was up to 120 min. The apparent permeability coefficient (\(P_{app}\)) and recovery rate of NMN were calculated (Table 3). The \(P_{app}\) value of the AP-BL transport experiment in the NMN bidirectional transmembrane is (7.31 \(\pm\) 0.31) \(\times\) 10\({}^{-6}\) cm/s, and the \(P_{app}\) value of the BL-AP efflux experiment is (7.40 \(\pm\) 0.13) \(\times\) 10\({}^{-6}\) cm/s. The \(P_{app}\) values of bidirectional transmembrane transport are both on the order of 10\({}^{-6}\), indicating that the transport capacity of NMN is moderate. The recovery rate of the AP-BL transport experiment was 84.34 \(\pm\) 2.33%, and the recovery rate of the BL-AP efflux experiment was 93.41 \(\pm\) 1.62%, both within the range of 80% to 120%, indicating no metabolism. Furthermore, we calculated the ratio of \(P_{app}\) values in NMN transport and efflux experiments. The \(P_{app}\) value ratio of AP-BL/BL-AP was 0.988; the \(P_{app}\) value ratio of BL-AP/AP-BL is 1.012. The ratio of \(P_{app}\) values < 1.5 indicates that NMN is not affected by efflux in the hCMEC/D3 cell model, and the ratio of \(P_{app}\) values is approximately equal to 1. It is speculated that the transmembrane mechanism of NMN crossing the BBB of monolayer cells in vitro is passive transport, and NMN is transported across the membrane through concentration differences. No active transport was found. The time- and concentration-dependency bilateral permeabilities of the NMN were investigated. To observe the time dependency, 0.2 mmol/L of NMN was added to the donor side and was sampled from 0 to 180 min. To observe the concentration dependency, the bidirectional transport rates of NMN were measured in the concentration range of 0.05-2.00 mmol/L, and the time was set as 120 min. To check the mass balance, the recoveries of the NMN were measured at both sides of the insert and their intracellular accumulation within the hCMEC/D3 cells. The transport kinetics curve and transport percentage in the NMN time-dependency bidirectional transmembrane transport experiment are shown in Figure 5. It can be seen that, when the administration concentration is 0.2 mmol/L, the amount of NMN transported substances at the receiving end shows an increasing trend followed by a stable trend with time. This is because, as time goes on, the transport of NMN reaches saturation, and the increase in the amount of NMN transported substances is relatively small from 90 min to 180 min, and the maximum amount of transported substances is reached at 120 min, which is 56.19 \(\times\) 10\({}^{-9}\) mol. Therefore, 120 min is the optimal time for all subsequent experiments. The transport percentage result is similar to its kinetic curve and tends to stabilize with the increase in time. The growth rate of NMN transport percentage is relatively small, from 90 min to 180 min, because it has reached saturation state. Table 4 shows the recovery rate results in the NMN time-dependency bidirectional transmembrane transport experiments. It can be seen that the recovery rate of NMN in the in vitro BBB model constructed in this study was within the range of 80-120% at six time points from 30 to 180 min, indicating that NMN was not metabolized in the model constructed in this study. The transport kinetics curve and transport percentage in the NMN time-dependency bidirectional transmembrane transport experiment are shown in Figure 6. It can be seen that, when the administration time is 120 min, the amount of substance transported by NMN at the receiving and administering ends increases linearly with increasing concentration, and the higher the concentration, the higher the amount of substance transported. The amount of substance transported between the receiving end and the administration end increases linearly with the increase in the NMN concentration, and the higher the concentration,